HHH Syndrome (Hyperornithinaemia-Hyperammonaemia-Homocitrullinuria), Cystinuria and PREPL Deficiency (2p21 Deletions)

Two amino acid transporter disorders: the mitochondrial ornithine transporter ORNT1 (SLC25A15) in the urea cycle and the apical cystine and dibasic amino acid transporter b0,+ (SLC3A1 and SLC7A9) in the proximal tubule and intestine

Literature and regulatory information updated: 31 October 2026.

0. Initial Reference Table

1. Introduction

HHH syndrome and cystinuria belong to the same functional family, that of amino acid transporter defects, but occupy opposite places in physiology. In HHH syndrome a transporter of the inner mitochondrial membrane of the hepatocyte fails and no longer brings ornithine into the matrix, and the consequence is systemic: the urea cycle loses its starting substrate and ammonia accumulates. In cystinuria a transporter of the apical membrane of the proximal tubule and enterocyte fails; filtered cystine is not reabsorbed and precipitates in the urine, without systemic toxicity, but with recurrent stones and kidney damage [1, 34]. The two entities share a biochemical meeting point, ornithine, which is transported by both systems, and therefore appear together in the differential diagnosis of dibasic aminoacidurias and lysinuric protein intolerance [42].

1.1 HHH syndrome

In 1969, Vivian Shih, Mary Efron and Hugo Moser described a three-year-old boy with cognitive impairment and myoclonic seizures in whom intermittent hyperammonaemia was associated with raised plasma ornithine and homocitrulline in the urine; they called it hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome and proposed a "block in the metabolic pathway of ornithine", because the findings did not match those of a proven hepatic ornithine transcarbamylase (OTC) deficiency [1, 9, 43]. The parents described episodes of sudden jumps "as if he had been pricked by a pin", with head drop, and the symptoms varied with protein intake [44]. In the following years, studies with carbon-14-labelled ornithine in fibroblasts showed that ornithine did not enter the mitochondrion, that ornithine was low in the hepatic mitochondria of one patient and that HHH fibroblasts complemented those from gyrate atrophy, which separated the two hyperornithinaemias [44].

In 1999, Camacho and colleagues used the sequences of the Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 genes, which encode mitochondrial ornithine transporters, to identify the human ORNT1 gene at 13q14; its expression restored ornithine metabolism in patient fibroblasts, and three alleles (F188Δ, E180K and a 13q14 microdeletion; historical numbering, = NM_014252.4:c.562_564del p.(Phe188del) and NM_014252.4:c.538G>A p.(Glu180Lys) [12, 45]) accounted for 21 of the 22 alleles of 11 probands [46]. The designation "ORNT1 deficiency" came into use as a synonym, alongside "ornithine transporter deficiency" and "ornithine translocase deficiency"; the gene is now called SLC25A15 and the protein is also designated ORC1 [1, 9]. HHH syndrome is classified among the urea cycle disorders (UCDs), alongside the deficiencies of the six cycle enzymes and citrin deficiency [24]; general management of hyperammonaemia follows that of the other UCDs, and neonatal encephalopathy of genetic cause enters the differential diagnosis.

1.2 Cystinuria

Cystinuria is one of the oldest hereditary aminoacidurias in the medical literature. William Hyde Wollaston described in 1810 a stone he called "cystic oxide", and Archibald Garrod included cystinuria, at the beginning of the twentieth century, among his inborn errors of metabolism [47]. In 1966, Rosenberg and colleagues divided it into three types according to the urinary excretion of presumed heterozygotes: in type I heterozygotes excreted normal amounts of cystine and dibasic amino acids, in types II and III they excreted high or moderate amounts [3]. Molecular biology came to replace this classification. Calonge and colleagues demonstrated in 1994 that variants of the rBAT gene (SLC3A1) segregated with cystinuria and that the most frequent variant in the series, M467T, almost completely abolished rBAT-induced transport in Xenopus oocytes [48]. In 1999, the International Cystinuria Consortium identified at the 19q12–13.1 locus the SLC7A9 gene, which encodes the light chain b0,+AT, and showed that its variants caused "non-type I" cystinuria, including the V170M founder variant of Libyan Jews [49].

In 2002, Dello Strologo and colleagues proposed the genetic classification used today: type A (variants in SLC3A1), type B (variants in SLC7A9) and type AB (variants in both genes) [3]. The 2021 European recommendation adds the genotype notations AA and BB for biallelic cases, A0 and B0 for patients with a single identified variant, and AAB, ABB or BBB for combinations with more than two variants [4]. Large homozygous deletions at 2p21 that remove SLC3A1 and neighbouring genes give syndromes with cystinuria and extrarenal manifestations, the first of which was described by Parvari in 2001 in a Bedouin family and the most frequent delineated by Jaeken in 2006 as hypotonia-cystinuria syndrome, attributed to the combined loss of SLC3A1 and PREPL [5, 33]. Cystinuria should not be confused with cystinosis (lysosomal accumulation of cystine due to cystinosin deficiency) or with homocystinuria.

1.3 PREPL deficiency

The third entity in this article arose from the dissection of a deletion syndrome. In 2001, Ruti Parvari and colleagues described in a Bedouin family a 179-kilobase homozygous deletion at 2p that removed SLC3A1 and neighbouring genes, with cystinuria and mitochondrial disease, and identified among the lost genes one of then unknown function [11]. This gene, cloned in 1997 from a brain complementary DNA library under the designation KIAA0436, was named prolyl endopeptidase-like (PREPL) because of its structural similarity to prolyl endopeptidase [11]. In 2006, Jaak Jaeken and colleagues delineated, in 11 patients from nine families, a recessive hypotonia-cystinuria syndrome caused by deletions of 23.8 to 75.5 kilobases that removed SLC3A1 and PREPL, and, finding that the flanking genes C2orf34 and PPM1B retained normal expression, attributed to the loss of PREPL everything in the clinical picture that was not cystinuria [5, 33].

Definitive proof came in 2014, when Luc Régal, Andrew Engel and colleagues studied an infant girl with myasthenic symptoms from birth, a response to edrophonium and no cystinuria: she was compound heterozygous for a premature termination variant inherited from her father and for the SLC3A1 and PREPL deletion inherited from her mother, so that she retained one functional SLC3A1 allele and the phenotype was determined solely by biallelic loss of PREPL [7, 8]. Endplate studies in the same patient showed a neuromuscular transmission defect, and the entity came to be designated congenital myasthenic syndrome 22, the twenty-second in the series [8, 25]. In 2018, the same team delineated the phenotype in ten patients and developed a functional blood assay that distinguishes PREPL deficiency from Prader-Willi syndrome, its main clinical mimic [6]. Meanwhile, the biochemistry of the protein was revised: in 2021 PREPL was shown to be a (thio)esterase, and not a peptidase, with a long mitochondrial isoform, and in 2024 it was shown that missense variants can cause the disease without abolishing catalytic activity [30, 50].

2. Molecular Genetics and Pathophysiology

2.1 SLC25A15 and the mitochondrial ornithine transporter

The SLC25A15 gene lies at 13q14.11, spans about 23 kb and has seven exons; the reading frame is in exons 2 to 7 and exon 1 contains part of the 5' untranslated region [9]. It encodes a 301-amino-acid protein of the mitochondrial carrier family (SLC25 family), with six helices spanning the inner membrane and a tripartite structure of three homologous domains [9, 44]. The MANE reference transcript is NM_014252.4 (protein NP_055067.1); GeneReviews still uses NM_014252.3, with identical numbering for the variants cited in this article [1, 12]. Expression is high in the liver and pancreas and also in the lung, testis and brain, mainly in astrocytes [9, 46].

The ORNT1 protein exchanges, in a one-to-one ratio, the L-isomers of ornithine, citrulline, lysine and arginine, and, less efficiently, a basic amino acid for a proton [9]. In the periportal hepatocyte, where the urea cycle enzymes are located, it exchanges cytosolic ornithine for mitochondrial citrulline and links the two halves of the cycle: ornithine produced by arginase in the cytosol must enter the matrix for OTC to combine it with carbamoyl phosphate and form citrulline [1, 9]. Residues E77, R179 and E180 take part in substrate binding, N74 and N78 form part of the pocket, and W224 and R275 appear to trigger the conformational change of translocation [9].

When the transporter fails, ornithine is trapped in the cytosol. Five consequences follow [1, 9, 51]. The first is hyperammonaemia: without intramitochondrial ornithine, OTC becomes functionally deficient and the cycle slows in periportal hepatocytes. The second is hyperornithinaemia, because cytosolic ornithine does not reach ornithine aminotransferase (OAT) in the matrix, which is the degradation pathway. The third is the production of homocitrulline and orotic acid: excess carbamoyl phosphate reacts with lysine (the "homo-ornithine"), forming homocitrulline and homoarginine, or is diverted to pyrimidine synthesis. The fourth is the increase in polyamines (spermine, spermidine) from cytosolic ornithine, with effects on mitochondrial calcium, apoptosis and autophagy. The fifth is secondary creatine deficiency: excess ornithine inhibits arginine:glycine amidinotransferase (AGAT), the first step of creatine synthesis, and available arginine decreases; in two patients, urinary creatine excretion normalised with citrulline or arginine, but not with ornithine [9]. HHH syndrome is thus a disorder of metabolic compartmentalisation, with simultaneous functional deficiency of OTC and OAT [51].

The relatively milder severity of the hyperammonaemia, compared with the neonatal enzyme deficiencies of the cycle, is explained in part by transporter redundancy. The SLC25A2 gene (ORNT2, or ORC2), intronless and resulting from retrotransposition, has 87% nucleotide identity and 88% protein identity with SLC25A15, also transports citrulline, histidine, homocitrulline and D-isomers, and its overexpression restores ornithine metabolism in HHH fibroblasts; the SLC25A29 gene (ORNT3) has the same capacity [1, 9, 44]. Fibroblasts with null alleles retain about 20–25% of ornithine transport [9]. In two Mexican families with a mild phenotype, the T32R variant in ORNT1 coexisted with a gain-of-function variant (glycine 181) in ORNT2, an observation reported in a congress abstract and cited in Nyhan's Atlas [44].

The mechanism of the progressive neurological manifestations is unclear. Hypotheses under study include subclinical hyperammonaemia in astrocytes (which is associated with cognitive decline, but not with the pyramidal syndrome), cerebral creatine deficiency, excess polyamines, similar to that of argininaemia, and oxidative stress induced by ornithine and homocitrulline, demonstrated in rat brain [1, 9]. Spastic paraparesis is common to HHH syndrome, arginase 1 deficiency and P5CS deficiency, three diseases in which delivery of ornithine to the mitochondrion or the arginine/ornithine balance is altered [52].

2.2 Spectrum of SLC25A15 variants

Between 1999 and 2014, 35 variants were described: 18 missense, 7 small insertions, 2 small deletions, 4 nonsense, one large deletion, one micro-rearrangement and one intronic rearrangement, all in the coding region [9]. Most substitutions affect residues whose side chains project into the inner pore of the protein, which suggests direct interference with translocation; some reduce activity (NM_014252.4: c.95C>G p.(Thr32Arg), c.562_564del p.(Phe188del), c.569G>A p.(Gly190Asp), c.818T>A p.(Met273Lys), c.815C>T p.(Thr272Ile), c.337G>T p.(Gly113Cys), c.212T>A p.(Leu71Gln), c.847C>T p.(Leu283Phe)) and others abolish it (c.658G>A p.(Gly220Arg), c.535C>T p.(Arg179Ter), c.79G>A p.(Gly27Arg), c.824G>A p.(Arg275Gln), c.823C>T p.(Arg275Ter)); in ClinVar, p.(Met273Lys) and p.(Leu283Phe) are classified as of uncertain significance and p.(Gly190Asp) has no classification assigned [45] [9]. Sequencing detects 99.3% of pathogenic variants; an exon–intron deletion of about 4.5 kb has been described [1]. There are five unprocessed pseudogenes of SLC25A15 (at Yq11.13, 21p11.2, 22q11.1, 13q12.3 and 13q12.11), which require care in probe design and messenger RNA analysis, together with the similarity to SLC25A2 [1].

Saudubray refers to the Japanese variant as "R197X"; reading of the primary sources and validation in VariantValidator confirm NM_014252.4:c.535C>T p.(Arg179Ter) [1, 12, 51]. The percentages of 28% and 16% (GeneReviews diagnosis section) and of 29% and 14% (variant table of the same chapter) correspond to different denominators of the same series [1].

Genotype does not predict phenotype. Among 16 patients from Quebec, 15 homozygous for p.Phe188del, the same range of severity described in other patients in the literature was observed; intellectual disability and lower-limb spasticity appeared together or in isolation, unrelated to age at diagnosis or adherence to treatment [53]. Nor is there a correlation between residual transport capacity in fibroblasts and genotype or phenotype [9], and some patients with two null alleles did not have neonatal hyperammonaemia [9]. Heterozygotes are asymptomatic and have no detectable biochemical abnormalities [1, 51]. No other allelic disorders of SLC25A15 are known [1].

2.3 The b0,+ system: SLC3A1 and SLC7A9

Cystine, a dimer of two cysteines joined by a disulphide bond, and the dibasic amino acids lysine, arginine and ornithine enter the epithelium of the proximal tubule and small intestine through the b0,+ system, which exchanges them for neutral amino acids [34]. The system is a disulphide-linked heterodimer: the light chain b0,+AT, encoded by SLC7A9 (19q13.11; MANE NM_014270.5), carries out transport, and the heavy chain rBAT, a glycoprotein encoded by SLC3A1 (2p21; MANE NM_000341.4), takes the light chain to the membrane [10, 12, 49]. The dibasic amino acids then exit into the interstitium through the basolateral transporter y+L (y+LAT1, encoded by SLC7A7), whose defect causes lysinuric protein intolerance; in cystinuria the defect is in the luminal membrane, in lysinuric protein intolerance in the antiluminal membrane [34, 42].

Under normal conditions, 99% of filtered cystine is reabsorbed; homozygotes typically excrete more than 400 mg (1.6 mmol) per day, usually 600–1400 mg (2.5–6 mmol), compared with less than 50 mg in normal individuals [4, 34]. Ornithine, lysine and arginine are soluble and are lost in the urine without known clinical consequence; cystine, by contrast, has low solubility: below 250 mg/L (1.05 mmol/L) at pH below 6 and about 500 mg/L (2.1 mmol/L) at pH above 7.5 [4, 47]. Supersaturated urine precipitates hexagonal crystals in the distal tubular lumen, which grow to form stones; nephron obstruction by crystals, with inflammation, contributes to kidney damage [10, 34]. No signs of cystine deficiency have been described [34]. High sodium intake increases cystine excretion, possibly by increasing the intracellular load of sodium and neutral amino acids and slowing apical reabsorption [4].

2.4 Classifications and inheritance patterns of cystinuria

The genetic classification of cystinuria and its translation into inheritance patterns are summarised in the following table, which brings together the notations of the European recommendation and of GeneReviews.

The two inheritance patterns coexist and are both documented: OMIM records autosomal recessive and autosomal dominant inheritance for both genes, and Orphanet classifies cystinuria as recessive and semidominant [3, 19]. The practical relevance lies in SLC7A9 heterozygotes, who may form stones over their lifetime, especially with additional factors such as dehydration [10]. The French HAS protocol (2019) is more restrictive regarding digenic inheritance: patients previously classified as AB were, in most cases, reclassified as AAB or BBA when a third variant was identified, and there are relatives of patients with AB genotype who are completely asymptomatic; the same document acknowledges that some authors describe type B as autosomal dominant with incomplete penetrance, but stresses that the vast majority of B heterozygotes never form cystine stones [32]. The old classification into types I, II and III, based on excretion by obligate heterozygotes, corresponded approximately to type A (type I) and type B (non-type I), but was abandoned because the three phenotypes result from only two genes [3, 34].

2.5 Spectrum of SLC3A1 and SLC7A9 variants

At least 152 variants in SLC3A1 and 104 in SLC7A9 have been described, with a highly variable spectrum between ethnic groups [34]. In 2026, an analysis of pathogenic variants in the 2022 HGMD database counted 116 new variants in SLC3A1 and 76 in SLC7A9 compared with 2016 [28]. The French protocol records, in the professional version of HGMD from January 2019, 241 pathogenic or likely pathogenic variants in SLC3A1 and 159 in SLC7A9 [32]. SLC3A1 variants account for 50–70% of cystinurias and SLC7A9 variants for 25–45%; in 5–7% no genetic explanation is found [10]. The proportion between the two types varies with the population: European studies described a similar distribution of AA and BB genotypes, but in a French cohort of 112 patients, 74 of the 99 with a classified genotype had type A cystinuria (70 AA, 3 AAA and 1 AAB) and 22 type B [32]. Sequencing detects 80–85% of variants and deletion and duplication analysis the remaining 15–20%, in both genes; large rearrangements account for 17–21% of the variants found and, in SLC3A1, frequently involve the neighbouring genes PREPL and CAMKMT [4, 10]. The French protocol gives a published range of 11–33% for large deletions and insertions and 18% of mutated alleles in the French cohort of 112 patients [32]. Hence the rule of always including multiplex ligation-dependent probe amplification (MLPA) or an equivalent method [10].

There is no clinically useful genotype–phenotype correlation: neither the genotypic classification nor the old phenotypic classification predicts age at onset, the number of stone passages or the total number of stone events [4, 34]. SLC3A1 expression matures between mid-gestation and about four and a half years of age, which may explain the transient cystinuria observed in screened newborns who later "lose" the phenotype; it is not clear whether these infants were heterozygotes [10].

2.6 Contiguous deletions at 2p21 and PREPL deficiency

Homozygous deletions at 2p21 that include SLC3A1 give type A cystinuria with an extrarenal phenotype determined by the neighbouring genes. When the deletion involves SLC3A1 and PREPL, the gene for a cytosolic oligopeptidase, hypotonia-cystinuria syndrome results; Jaeken and colleagues identified deletions of 23.8 to 75.5 kb in 11 patients from 9 Flemish and French families, with normal expression of the flanking genes C2orf34 and PPM1B, and attributed the phenotype beyond cystinuria to PREPL deficiency [5, 33]. A deletion that also includes CAMKMT (formerly C2orf34) gives atypical HCS, with mild to moderate intellectual disability; when it also includes PPM1B, it gives 2p21 deletion syndrome, described in a Bedouin family with a 179 kb deletion [5, 10]. Isolated PREPL deficiency, without cystinuria, also exists and can be confirmed by a lymphocyte assay showing absence of the protein and of its reactivity; PREPL is normal in Prader-Willi syndrome, which is the main clinical differential [6]. Recessive contiguous deletions at 2p16 combine cystinuria and mitochondrial disease [34].

2.7 PREPL: gene, protein and molecular mechanism

PREPL lies at 2p21, immediately adjacent to SLC3A1 and on the opposite strand, with overlap of the 3' ends of the two genes, an arrangement that explains why most pathogenic deletions involve both [11, 29]. The gene has 14 coding exons and produces nine splice variants that give rise to four proteins; the long transcript NM_006036.4 encodes 727 amino acids and is the one used for numbering in the clinical literature, whereas the MANE Select transcript is the shorter NM_001171613.2, which requires positions to be converted between the two systems [11, 12]. The protein has a beta-propeller domain in the amino-terminal half and a catalytic domain with a serine triad in the carboxy-terminal half; replacement of the catalytic residues by alanine abolishes reactivity with a serine hydrolase-specific probe, and, unlike prolyl oligopeptidase and oligopeptidase B, PREPL activity depends only on the carboxy-terminal domain [11, 33]. Expression is ubiquitous, highest in the brain, muscle and kidney, in that order [11].

The function has been reclassified twice. No peptide substrate was ever identified, and in 2021 Karen Rosier and colleagues demonstrated (thio)esterase activity, inhibitable by palmostatin M, compatible with a depalmitoylation function, determined the crystal structure of the protein and showed that the long isoform is located in the mitochondrion, whereas the short one remains in the cytosol; in Prepl-null mice, mitochondrial complex activity was reduced and mitochondrial gene expression dysregulated, and mitochondrial ultrastructure was altered both in the mice and in a patient [50]. The second function, independent of catalysis, is the interaction with the mu-1A subunit of the clathrin-associated adaptor protein complex 1 (AP-1): PREPL promotes dissociation of the complex from target membranes and thus regulates AP-1-mediated transport in the trans-Golgi network [8, 57]. Because trafficking of the vesicular acetylcholine transporter between the vesicle membrane and the cytosol depends on AP-1, the absence of PREPL offers an explanation for the deficient filling of synaptic vesicles [8].

This mechanism translates into a distinctive electrophysiological signature. In endplate studies of the anconeus muscle of the 2014 patient, the miniature endplate potential amplitude and the miniature current were reduced, as was the quantal content of the endplate potential, owing to a decrease in the number of vesicles available for release with normal release probability, whereas the number of acetylcholine receptors per endplate, receptor kinetics and endplate geometry were normal and synaptic vesicles were abundant near the active zones; the conclusion was a defect of the late stages of exocytosis, with reduced acetylcholine content per vesicle [7, 8]. The endplate was enlarged, as occurs in Lambert-Eaton syndrome, and in two regions there were degenerative signs [8]. The 2026 review by Ricardo Maselli groups PREPL with MYO9A and RPH3A among the congenital myasthenic syndromes linked to organelle homeostasis and G-protein signalling, and likens the PREPL pattern to that of the forms with defective acetylcholine synthesis, in which it is quantal size that decreases [29, 57].

The known variants comprise deletions, premature termination variants, frameshifts, splice-site variants and missense variants. The following table brings together the variants published in patients with isolated PREPL deficiency, with the MANE nomenclature confirmed in VariantValidator alongside the long-transcript nomenclature used in the articles.

ClinVar contains, for PREPL, numerous records of termination and frameshift variants classified as pathogenic in MANE nomenclature, including NM_001171613.2:c.872C>G p.(Ser291Ter), c.993C>G p.(Tyr331Ter), c.1414G>T p.(Gly472Ter) and c.370del p.(Asp124fs), alongside records of copy-number variations covering both PREPL and SLC3A1; the count of pathogenic entries for the gene should not be read as a burden of point variants, because a substantial part corresponds to multigene deletions [45]. Missense variants require additional caution, because they retain hydrolytic activity: structural analysis placed them in regions of intraprotein or protein–protein interaction, with reduced binding that differed for each known interactor, and cell lines with the catalytically inactive variant p.Ser559Ala (long-transcript numbering) showed that hydrolytic activity is required for normal mitochondrial function, but not for regulation of AP-1-dependent transport [30]. ClinGen assigned the gene the haploinsufficiency score reserved for recessive phenotypes and no evidence of triplosensitivity [30].

Two genetic mechanisms deserve note. The first is uniparental disomy: in a Chinese infant girl with severe neonatal hypotonia, an apparently homozygous frameshift variant turned out to be maternal, and trio analysis showed 96.7% of chromosome 2 variants homozygous and of maternal origin, with complete uniparental disomy and no copy-number variations [14]. The second is the coexistence of different molecular mechanisms on the two alleles and between siblings of the same family [59]. The link to Prader-Willi syndrome gained a molecular basis in 2024: Prepl is underexpressed in the brain of neonatal mouse models of the syndrome, the hypothalamic–pituitary axis is affected similarly in both models, with defective orexigenic signalling and growth retardation, and fibroblasts from patients with PREPL deficiency and with Prader-Willi syndrome have deficient mitochondrial bioenergetics, correctable by coenzyme Q10 in PREPL-deficient cells and by PREPL overexpression in Prader-Willi fibroblasts [60].

3. Clinical Spectrum and System-Specific Manifestations

3.1 HHH syndrome: sources and limits of the data

Clinical data on HHH syndrome come from retrospective literature series and not from prospective cohorts. The systematic review by Martinelli and colleagues brought together 111 patients (109 published in 61 articles and two unpublished from Rome), and GeneReviews added six later reports and three unpublished patients, for a total of 122 [1, 9]. Denominators vary from sign to sign, because not all reports describe all signs [1]. The largest homogeneous cohort is that of 16 French-Canadian patients from Quebec, 15 homozygous for p.Phe188del [53]. The sex ratio was about two males to every female among 97 patients with this information recorded [9].

3.2 HHH syndrome: presentation by age

Age at onset and age at diagnosis do not coincide. Among 54 patients with known age at onset, 22% had symptom onset in the neonatal period, 24% in the first year, 44% in childhood and 9% in adolescence or adulthood; among 105 patients with known age at diagnosis, at least a quarter were diagnosed in adulthood, one-third of patients with neonatal onset were diagnosed only later, and the mean diagnostic delay was 6.3 ± 10.1 years (0–37) [9]. In the GeneReviews series of 116, the distribution by age at diagnosis was 8% (9/116) in the neonatal period, about 10% (12/116) between one month and one year, almost half (56/116) between one and 12 years and about one-third (39/116) after 12 years [1].

The acute presentation combines hyperammonaemia with tachypnoea, respiratory alkalosis, vomiting, ataxia, lethargy, confusion and coma; about one-third of patients had overt coma, and coma or lethargy at presentation occurred in about 70% of patients with early onset, becoming less frequent with later age at onset [9]. Acute episodes may include dysphasia, gait disturbances, sudden falls and behavioural changes [9]. Mild encephalopathy with disorientation, irritability and episodic confusion may resolve spontaneously or with glucose infusions, which makes it easy to miss [1]. The patient described by Nyhan went into coma after receiving 8 g/kg/day of protein during treatment of a burn and was initially diagnosed with Reye syndrome [44].

The chronic presentation is characterised by aversion to protein-rich foods, progressive encephalopathy with regression and motor dysfunction; patients with late onset present mainly with intellectual disability, recurrent vomiting, ataxia or seizures [9, 51]. Adults learn early to avoid protein-rich meals, which cause them malaise and vomiting, and may live undiagnosed until a load exceeds their residual detoxification capacity [1].

3.3 HHH syndrome: frequency of manifestations

3.4 HHH syndrome: liver and coagulation

Liver dysfunction is a particular feature of HHH syndrome among the UCDs. In the Quebec series, liver dysfunction was the presenting feature in 6 of 16 patients; Saudubray states that, in the French-Canadian series, it was the initial symptom in more than one-third [51, 53]. It ranges from isolated elevation of transaminases, with or without mild coagulopathy and without overt hyperammonaemia, to hepatitis-like crises and fulminant liver failure with subdural haematoma, gingival bleeding and melaena [1, 9]. The 2015 review classified the liver disease as mild to moderate (transaminases up to 10 times normal) or severe (more than 10 times), and the coagulopathy as mild to moderate (individual factors at 40–70%, INR 1.5–2.0) or severe (factors below 40%, INR above 2.0 or clinical manifestations) [9]. A three-year-old Italian boy, with moderate protein aversion and mild language delay, presented with lethargy during gastroenteritis and rapidly progressive hepatocellular necrosis, with prothrombin time of 48% on the second day and below 5% on the fifth and peak ammonia of 170 µmol/L; with intravenous arginine and protein restriction, ammonia normalised in about 24 hours and liver transplantation was deferred [44]. Biopsy shows vacuolated hepatocytes with intracytoplasmic glycogen, small nuclei, lipid droplets without fibrosis and mitochondria with crystalloid or lamellar inclusions [9, 44]. Liver dysfunction regresses with treatment of the hyperammonaemia and leaves no long-term sequelae in most patients; in one follow-up study, alpha-fetoprotein was raised and there were hepatic ultrasound abnormalities during follow-up [1]. The 2019 European guideline confirms that liver dysfunction and coagulopathy, with deficiency of factors VII, IX and X, are more frequent in HHH than in the other UCDs, and includes HHH among the UCDs that may present with acute or chronic liver failure [36].

3.5 HHH syndrome: nervous system

Progressive neurological dysfunction is the hallmark of the syndrome, regardless of age and mode of onset [9, 51]. Pyramidal signs range from lower-limb hyperreflexia with Babinski sign to overt spastic paraparesis, often accompanied by cerebellar signs (ataxia, poor fine coordination, dysdiadochokinesia, dysarthria, nystagmus, intention tremor) [9]. Other manifestations are muscle weakness, involuntary hand movements, buccofaciolingual dyspraxia and psychiatric disturbances [9]. In a family with three siblings, the eldest developed gait disturbances at 14 years and at 21 had hyperreflexia, clonus, bilateral Babinski and an IQ of 67; his 18-year-old sister could not run or jump and had an IQ of 60; his 13-year-old brother had brisk reflexes and an IQ of 51 [44]. Seizures, mainly myoclonic but also tonic-clonic, began between 10 months and 18 years in Nyhan's series [44]. According to the 2019 European guideline, spastic paraparesis is an almost constant finding, develops more slowly than in arginase 1 deficiency, is preceded by hyperreflexia and other pyramidal signs and often appears without a history of hyperammonaemia [36].

Neurological deterioration may continue despite good metabolic control. Four patients followed for 11 to 38 years had memory loss, low IQ, tremor, spasticity, bladder incontinence, dystonia, epilepsy and subcortical, cerebral and cerebellar atrophy sparing the basal ganglia; three progressed despite control of ammonia [61]. In contrast, the Rome team describes several patients treated with citrulline and mild protein restriction for more than 20 years without neurological progression, and there are adults with limited manifestations throughout life [1, 51]. The Urea Cycle Disorders Consortium (UCDC) assessed four preschool children with normal developmental quotients in two and below 71 in the other two, and an adult whose full-scale IQ fell from 100 at 21 years to 84 at 26 [1].

Neuroimaging may be normal, especially in patients without coma, or show mild cerebral and cerebellar atrophy, spinal cord atrophy, white matter changes, a thin corpus callosum, cystic lesions, basal ganglia calcifications, diffuse oedema, subdural haematoma and stroke-like lesions [1, 9, 51]. Motor evoked potentials are abnormal in the lower limbs, without involvement of the upper limbs, somatosensory potentials are normal, and electroneuromyography may show altered conduction velocities and signs of anterior horn degeneration [9].

3.6 HHH syndrome: other organs and pregnancy

The eye is usually normal, unlike in gyrate atrophy: papilloedema during a crisis and, rarely, retinal depigmentation, photophobia, night blindness, tapetoretinal degeneration, cataract and an abnormal electroretinogram (retinal changes in 2 of more than 70 patients) have been described [9, 44, 51]. Dysmorphic features, cerebral dysplasia and microcephaly are rarely described [9]. Lysine may limit growth: in Nyhan's patient, linear growth almost stopped until plasma lysine was corrected [44]. Pregnancy and the postpartum period are periods of risk of decompensation, described in section 7 [1, 62].

3.7 Cystinuria: stones and kidney

Cystinuria carries a lifelong risk of stones [34]. Among AR patients, 100% have cystinuria and hyperexcretion of lysine, arginine and ornithine and 94% have nephrolithiasis; 20–25% have chronic kidney disease [10]. The first stone usually appears in the first two decades: the median was 13 years in a European cohort and 16 years in a French study, with a range from childhood to late adulthood and, exceptionally, at birth [4, 10]. The mean age at first presentation in the United States is 12–13 years [47]. Diagnosis often comes after the first stone: according to the French HAS protocol, the median age at diagnosis is around 15–20 years, ranges from the prenatal period to over 60 years, and 60% of diagnoses are made before the age of 40 [32]. Types A and B have similar age at diagnosis and course; one study found greater severity in males, but others did not confirm it [4, 10].

The presentation is that of stone disease: loin pain radiating to the groin and testes, nausea and vomiting, micro- or macroscopic haematuria, spontaneous passage of stones and recurrent urinary tract infections [10]. Stones often appear in clusters separated by long asymptomatic periods; in severe cases hundreds may form per year, and some patients never form stones [10, 34, 47]. Cystine stones are yellow-brown, with a granular surface and radial structure; under alkalinisation they acquire a smooth surface through peripheral incorporation of calcium phosphate [4]. About 10% of patients also form calcium, oxalate, phosphate or struvite stones, owing to coexisting hypercalciuria or hypocitraturia [10]. Expression varies within the same family [10].

Cystinuria damages the kidney more than other stone diseases. Most adults have an estimated glomerular filtration rate (eGFR) below 90 mL/min/1.73 m2, usually between 60 and 89; in a French cohort of 314 adults with a mean age of 38.7 years, 26.8% had an eGFR below 60 and 12% proteinuria, and in another study the proportion below 60 was 17.8% [4]. The risk relates to age and to parenchymal damage (nephrectomies, congenital anomalies, hypoplasia, repeated urological procedures since childhood, staghorn calculi and intratubular obstruction by crystals) [4, 10]. End-stage kidney disease is relatively rare, and cystinuria does not recur after kidney transplantation [4]; in the French cohort published in 2015, 1.1% of patients had chronic kidney disease requiring dialysis, and the French protocol explains the absence of recurrence by normal expression of the b0,+ system in the proximal tubules of the graft [32]. Arterial hypertension affects 29–51% of adults and is associated with male sex, age and chronic kidney disease [4, 10]. Patients with cystinuria have lower bone mineral density than age- and sex-matched controls, independently of chronic kidney disease, according to the only available study [10]. Health-related quality of life is lower than in the general population in patients with active stone disease and improves with stone-free time [4].

3.8 Cystinuria: heterozygotes and transient forms

SLC7A9 heterozygotes have hyperexcretion of cystine and dibasic amino acids in 86–90% (about 14% have a normal profile) and nephrolithiasis in 2–18% [10]. The European recommendation describes excretion in B0 heterozygotes of between 40 and 400 mg/day, compared with less than 100 mg/day in A0 heterozygotes, who usually do not form stones [4]. As a ratio to creatinine, the French protocol describes in A heterozygotes a classically normal value (with published exceptions) and in B heterozygotes a mean of 50 µmol/mmol, with a range of 10 to 100 µmol/mmol [32]. The stone risk of heterozygotes appears to depend on additional factors, such as dehydration [10]. In newborn screening programmes, infants with transient cystinuria have been described, probably owing to immaturity of tubular transport in the first years of life [10, 34].

3.9 Syndromic forms due to 2p21 deletion

In hypotonia-cystinuria syndrome, generalised hypotonia is present at birth, with sucking difficulties, faltering growth, growth hormone deficiency and mild facial dysmorphism (dolichocephaly, ptosis, tented upper lip), followed, at the end of childhood, by hyperphagia and rapid weight gain; motor delay is mild to moderate, the voice is nasal, puberty is delayed in several patients (with hypergonadotropic hypogonadism in some) and nephrolithiasis appears in the first decade [5, 33]. Adults with PREPL deficiency have ptosis, nasal dysarthria, facial and proximal weakness and autonomic signs, with an IQ between 64 and 112 [6]. The atypical form adds mild to moderate intellectual disability and, in some patients, mitochondrial complex IV deficiency [5, 10]. The 2p21 deletion syndrome described by Parvari in seven Bedouin patients combined cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, lactic acidosis and dysmorphism (frontal bossing, almond-shaped eyes, long eyelashes, depressed nasal bridge, large posteriorly rotated ears), with cystine stones from 9 months of age [5, 10].

3.10 Isolated PREPL deficiency: natural history

Isolated PREPL deficiency, without cystinuria, has the same neuromuscular core as hypotonia-cystinuria syndrome and adds to it the difficulty of lacking the urinary marker that leads to diagnosis. Data come from small series: the 2014 index patient, the ten patients characterised in 2018 (five with isolated deficiency, three with hypotonia-cystinuria and two with the atypical form), the three patients with missense variants from 2024 and the retrospective cohort of five Chinese children published in 2026; the total published is around twenty, so no frequency of manifestation should be read as a population estimate [6, 8, 30, 31].

Presentation is neonatal and constant. Severe hypotonia, feeding difficulties with poor sucking and nasal dysarthria were present in all patients in the 2018 series, with a high-arched palate and narrow bitemporal diameter in all; ptosis and facial weakness were present in most, as was the fluctuating character of the weakness, and xerostomia with thick saliva on the tongue is a characteristic sign [6, 7]. Many infants need a nasogastric tube and some a gastrostomy, sometimes for years; ventilatory support is necessary in some cases, and obstructive sleep apnoea treated with positive pressure up to three years of age has been described in one patient [6, 29, 30]. In the 2026 cohort, the median age at diagnosis was five months, ranging from one to twelve months, and ptosis was atypical: only one of the five children had it, unilateral, mild and transient, without evident fluctuation, which shows that absence of ptosis does not rule out the diagnosis [31].

Hypotonia improves spontaneously, but incompletely. Independent walking is delayed, with a median of 23 months in patients with isolated deficiency or hypotonia-cystinuria, and motor delay was described as significant in all children in the 2026 cohort [6, 31]. In adulthood, the residual signs, besides ptosis, are facial weakness, nasal dysarthria and neck flexor weakness; proximal weakness of arm abduction and hip flexion after three years of age occurred in two of five patients with isolated deficiency or hypotonia-cystinuria [6]. Fatigue at the end of the day and with prolonged exertion persists in patients who have normal strength on routine examination [30].

The endocrine component is part of the picture and not a comorbidity. Growth hormone deficiency was detected in four of the five patients with isolated deficiency in the 2018 series, with onset between 20 months and 11 years; hypergonadotropic hypogonadism with delayed puberty occurred in most and, because it is absent in isolated cystinuria and present in both forms with loss of PREPL, was attributed to PREPL deficiency [6, 7]. In the 2026 cohort, two children had a predominant fall in insulin-like growth factor 1 (IGF-1) at 2.3 and 1.6 years [31]. Weight follows a two-phase pattern: faltering growth and thinness in early childhood, followed by rapid weight gain due to hyperphagia from the end of the first decade, with a tendency to obesity, but the pattern is not obligatory, and there are patients followed up to seven years of age with low intake and normal body mass index [6, 30, 38].

The cognitive profile is broad and does not allow prognosis by genotype. In patients with isolated deficiency or hypotonia-cystinuria, the intelligence quotient ranged from 64 to 112, with a median of 70, and it is not known how it evolves over time; there are patients with normal intelligence and appropriate social development, and patients with mild intellectual disability [6, 7]. In the 2026 cohort, one of the five children had language and cognitive delay [31]. Autonomic signs are prevalent and explain part of the picture: besides xerostomia, delayed gastric emptying and the tendency to obesity itself are described, and ptosis, which single-fibre electromyography did not track in patients who still had it, is probably of orthosympathetic and not myasthenic origin [6].

The syndromic forms due to contiguous deletion add manifestations depending on the genes removed, and variability between siblings with the same deletion is real. Atif Towheed and colleagues described two siblings of Dutch ancestry with the same homozygous 2p21 deletion (exons 2 to 10 of SLC3A1 and exons 2 to 14 of PREPL) and a common initial presentation of congenital hypotonia, lactic acidosis and faltering growth, with partial cytochrome c oxidase deficiency on muscle biopsy (40.6% of control in one, 29% in the other): the boy developed severe stone disease, with percutaneous nephrolithotomy of 19 pure cystine stones at seven years, and his sister had late-onset cystinuria, without stones, dominated by neurobehavioural disturbances with autism spectrum disorder, attention deficit, obsessive-compulsive disorder and chronic anxiety [63]. In both, the six-minute walk test, repeated every six months for seven years, improved with age but remained about 150 metres below predicted, without an abnormal fatigue index; the boy's aerobic capacity was more than 50% below average, with peak oxygen consumption of 20.1 and 22.0 mL/kg/min at 15 and 16 years [63]. Mitochondrial dysfunction, described as characteristic of the atypical form and of 2p21 deletion syndrome, may therefore appear in classic hypotonia-cystinuria syndrome [5, 63].

Two neighbouring entities complete the spectrum. The homozygous deletion that removes PREPL and the initial exons of CAMKMT, without involving SLC3A1, gives the so-called 2p21 microdeletion syndrome without cystinuria: Deborah Bartholdi and colleagues described two siblings of unrelated Swiss parents with reduced fetal movements, severe hypotonia, faltering growth and mild to moderate psychomotor delay, dolichocephaly, a hypotonic face with open mouth, cleft palate, mild ptosis, almond-shaped eyes, frontal bossing, midface hypoplasia and small hands and feet, with hypoplasia of the labia majora in the girl and hypospadias in the boy; growth hormone assessment in the boy was normal [7, 58]. Orphanet describes this entity with slightly raised serum lactate and without cystinuria, and classifies it as a partial autosomal monosomy with a prevalence below 1 in 1,000,000 [23]. The second is the association with unexpected extra-neuromuscular manifestations: in a 14-year-old Chinese girl with a homozygous splice-site variant, in addition to severe neonatal hypotonia, growth retardation and cognitive difficulties, absent ovaries, uterine hypoplasia, microcephaly and a short neck were found, which suggests a role for PREPL in gonadal and uterine development that other series have not confirmed [64].

4. Clinical Recognition and Warning Signs

4.1 When to think of HHH syndrome

HHH syndrome should enter the differential diagnosis of any hyperammonaemia, at any age, including in a woman with hyperammonaemia during pregnancy or postpartum [1, 9]. The newborn with hyperammonaemia is usually treated first for sepsis, and the adult with unexplained coma may be thought to have poisoning or encephalopathy of another cause; in the Swedish case described in section 10, the diagnosis was suspected only when the family, after an internet search, suggested a UCD [1, 55].

Ammonia of 80 µmol/L or more (about twice the control value) in a patient with known HHH requires immediate treatment, and clinical manifestations do not always track the rise in ammonia, so the decision considers both [1].

The 2019 European guideline strongly recommends considering a UCD at any age in the situations in the last row of the table, with catabolism or protein load as triggers (recommendation no. 1, moderate quality), measuring ammonia as an emergency test (no. 2, high quality) and, if it is raised, collecting amino acids and acylcarnitines and starting treatment while awaiting results, which should be available within 24 hours (no. 3) [36]. The most frequent misdiagnosis in early onset is neonatal sepsis (no. 4) [36]. The sample should be venous, because capillary samples give falsely high values, and tourniquet use, crying, agitation, seizures and slow processing raise ammonia; rapid bedside methods are used with caution and only in hospital, because some devices do not read above about 280 µmol/L [36]. If the patient dies without a diagnosis, anticoagulated blood is kept for DNA extraction together with frozen aliquots of plasma, serum, urine and cerebrospinal fluid, and if possible fibroblasts or frozen liver (no. 5) [36].

4.2 Newborn screening for HHH syndrome

Newborn screening by tandem mass spectrometry has low sensitivity for HHH. Ornithine rises only after the first days of life, after sampling; in northern Saskatchewan, ornithine measured in the blood spot was normal in newborns homozygous for p.Phe188del, in carriers and in controls, and screening was possible only by genotyping [27]. Erythrocyte arginase may also produce ornithine while the spot dries [35]. OTC, CPS1 and NAGS deficiencies and HHH give low citrulline, and only some programmes detect and report low citrulline reliably [24]. In the review of 111 patients, only one was identified by newborn screening; the United States and Canada include HHH in some panels [9]. The sensitivity of newborn screening for HHH is thus unknown and probably low [35]. The 2019 review maintains this position: it recommends considering newborn screening only for ASS and ASL deficiencies and considers the data insufficient to recommend screening for HHH (recommendation no. 7, moderate quality) [36].

4.3 When to think of cystinuria

The European recommendation, the American consensus and GeneReviews agree on the warning signs: early-onset stone disease, large, multiple or recurrent stones, a staghorn calculus in a child, recurrent urinary tract infections, a family history of stones, and any stone whose composition has never been analysed or in which cystine has never been sought [4, 10, 47]. Cystinuria is the most frequent monogenic cause of kidney stones and accounts for 1–2% of stones in adults and 6–8% (up to 10%) in children, and about 20% of metabolic causes of stone disease [10, 34, 37]. Siblings of a patient should be tested, because they may be asymptomatic [4, 37]. The French protocol lists the circumstances of discovery: renal colic (the most frequent), a stone discovered incidentally on ultrasound, computed tomography or, more rarely, plain abdominal radiography, haematuria, urinary tract infection and family screening; suspicion is raised by the paediatrician, general practitioner, urologist, nephrologist or the sonographer of the prenatal diagnosis centre [32].

A hyperechogenic colon on routine obstetric ultrasound before 36 weeks has a positive predictive value of 89% for cystinuria; the proposed explanation is fetal swallowing of cystine-rich amniotic fluid, which overloads intestinal reabsorption capacity and precipitates in the colon [4]. The diagnosis is confirmed after birth, preferably by genetic testing, because urinary amino acid excretion is difficult to interpret in the first months owing to tubular immaturity [4]. The French protocol adds that prenatal suspicion requires no further investigation during pregnancy, that an information consultation with a paediatric nephrologist at the multidisciplinary prenatal diagnosis centre may be offered and that the postnatal course of these cases is still poorly described; after birth, the child is referred to paediatric nephrology for confirmation and initiation of treatment [32].

Cystinuria with neonatal hypotonia, feeding difficulties, developmental delay or a picture resembling Prader-Willi syndrome suggests a contiguous deletion at 2p21 or 2p16 [4, 34].

4.4 When to think of PREPL deficiency

Isolated PREPL deficiency presents as severe neonatal hypotonia with feeding difficulties, without cystinuria to guide investigation, which is why diagnosis comes late: in the 2018 series it occurred years after the clinical presentation, and in the 2026 paediatric cohort the median was five months, with one case at twelve [6, 31]. The combination to recognise is neonatal hypotonia with poor sucking, weak cry and dysarthria or nasal voice, high-arched palate and narrow bitemporal diameter, with or without ptosis, accompanied by spontaneous improvement in tone over the first year that does not fully normalise [6, 7]. Xerostomia with thick saliva, delayed gastric emptying and fatigue at the end of the day strengthen the suspicion [6].

The most frequent practical trigger is an investigation for Prader-Willi syndrome that proves negative. In the five patients in the 2018 series and in almost all published cases, Prader-Willi syndrome was the first hypothesis and was excluded in the neonatal period: neonatal hypotonia with severe feeding difficulties, spontaneous improvement, a tendency to obesity in childhood, xerostomia, neuromuscular junction abnormalities and growth hormone deficiency are common to both diseases [6, 7]. Two signs separate them at the bedside: palpebral ptosis, which is present in PREPL deficiency and absent in Prader-Willi syndrome, and severe behavioural disturbances, which are present in Prader-Willi syndrome and not in PREPL deficiency [6]. A methylation or deletion analysis negative for Prader-Willi syndrome in a hypotonic infant with these features should lead to testing of PREPL and not to closure of the investigation [6, 38].

A second trigger is endocrinological. A child with growth retardation and proven growth hormone deficiency, a history of neonatal hypotonia and feeding difficulties, and a negative somatotropic axis gene panel, justifies testing of PREPL; in the patient described in Barcelona, sequencing of the IGF1, IGF1R, IGF2, GH1, GHR, GHRHR, GHSR, NPR2, STAT5B, IGFALS and CCDC8 genes and methylation analysis of the Silver-Russell syndrome region were normal before the exome identified the PREPL variant [38]. The third trigger is neurophysiological: a "seronegative myasthenia" of congenital onset, with a decrement on low-frequency repetitive nerve stimulation or a response to edrophonium, without acetylcholine receptor or anti-MuSK antibodies, requires consideration of the congenital myasthenic syndromes, among them the PREPL form; congenital myotonic dystrophy and spinal muscular atrophy are routinely excluded in these patients [6, 8, 25].

5. Diagnosis: Criteria, Tests and Algorithms

5.1 HHH syndrome: criteria

The biochemical diagnosis rests on the triad of episodic or postprandial hyperammonaemia, persistent hyperornithinaemia and homocitrullinuria, considered pathognomonic [1, 51]. The triad may be incomplete: patients with early protein restriction may never have had hyperammonaemia, patients assessed for school difficulties may have only isolated hyperornithinaemia, and a low-protein diet reduces or eliminates urinary homocitrulline [1]. In the newborn the triad may be absent or masked by the amino acid profile of liver dysfunction and prematurity [1]. The molecular diagnosis is established with biallelic pathogenic or likely pathogenic variants in SLC25A15; biallelic variants of uncertain significance (VUS), or one pathogenic variant with a VUS, neither establish nor exclude the diagnosis [1].

5.2 HHH syndrome: biomarkers

Other urinary findings include 3-aminopiperidin-2-one, the cyclic lactam of ornithine, and increased polyamines [44]. The allopurinol test is not specific: ASS and ARG1 deficiencies, lysinuric protein intolerance and HHH may give a positive result [35, 36]. Among the UCDs, the 2019 guideline identifies HHH in the metabolite profile by raised urinary homocitrulline, often accompanied by increased urinary orotic acid, and points out that the metabolite pattern is more informative than isolated values [36]. Measurement of carbon-14-labelled ornithine incorporation in fibroblasts confirms the defect (null alleles reduce transport by 75–80%), but has been replaced by genetics [9, 35]. The 2019 guideline accepts confirmation by incorporation of labelled ornithine into protein of cultured fibroblasts (or liver tissue) or by identification of the variants, and strongly recommends genetic testing in all UCDs to confirm the diagnosis and enable counselling, carrier testing and prenatal diagnosis (recommendation no. 5, moderate quality); when sequencing is negative, the guideline points to comparative genomic hybridisation array, RNA analysis and MLPA (multiplex ligation-dependent probe amplification) as complementary methods [36].

5.3 HHH syndrome: molecular tests and algorithm

When the biochemistry is characteristic, the first-line test is SLC25A15 sequencing, followed by deletion and duplication analysis if only one variant or none is found; targeted testing for p.Phe188del and p.Arg179Ter may be considered according to ancestry [1]. When the picture is non-specific, a hyperammonaemia or UCD panel with deletion and duplication analysis, or exome or genome sequencing, avoids having to choose the gene, and an exonic array can complement a negative exome [1]. The report should state the gene, the transcript with version (NM_014252.4), the HGVS nomenclature, zygosity, classification according to ACMG/AMP criteria and, in apparent homozygosity, the possibility of an undetected deletion; parental segregation confirms phase. Sequencing detects 99.3% of variants, but the pseudogenes and homology with SLC25A2 require confirmation of unexpected findings [1].

5.4 Cystinuria: criteria

The clinical diagnosis is established by identification of cystine stones and increased urinary cystine excretion [10]. The European recommendation accepts three routes: stone analysis by infrared spectroscopy or X-ray diffraction (wet chemical analysis is outdated), observation of hexagonal cystine crystals in the sediment of the first morning urine or a random sample (present in at least two-thirds of untreated patients), and measurement of cystine and dibasic amino acids in 24-hour urine or as a ratio to creatinine in a spot sample [4]. The American consensus considers diagnostic the stone composition, hexagonal crystals, or a positive cyanide-nitroprusside test (cystine above 75 mg/L) followed by quantification; the European recommendation considers the nitroprusside test outdated because of low reproducibility and sensitivity and because it uses hazardous reagents [4, 47]. For spectroscopic analysis, urine can be studied directly by attenuated total reflectance infrared spectroscopy [10].

The French HAS protocol (2019) accepts the same three tests, any of which is sufficient to confirm the diagnosis, and considers that molecular testing is not necessary for confirmation [32]. It gives performance figures for crystalluria: in undiagnosed and untreated patients, cystine crystals are found in about 83% of samples and in about 88% of first morning urines, which are the most concentrated; storing urine at +4 °C for 48 hours raises this proportion to more than 95% [32]. The presence of cystine crystals indicates a concentration seen only in cystinuria, but the test requires trained technicians and is carried out with qualitative and quantitative analysis in a few specialised laboratories [32]. The protocol's collection rules are practical: use the first morning urine, or fresh urine collected away from meals, and never 24-hour urine, which is unstable; collect the entire void in a clean 500 mL to 1 L screw-top container; and examine it within the next two hours, without the temperature falling below 20 °C for a prolonged period (ideally it would be kept at 37 °C) [32]. The crystals are more or less regular hexagonal plates with a strong tendency to twinning, that is, to nucleate and grow on one another with the same spatial orientation; the stones, formed by stacking of these twinned crystals, are rough, slightly translucent, honey-coloured to yellowish-brown, with a cut surface of coarse radial organisation [32].

Regarding the stone, the French protocol recommends analysing the first stone of any patient, at any age, whether passed spontaneously or removed surgically [32]. In France, infrared spectrophotometry has become the reference technique and is performed by about thirty laboratories; Raman spectroscopy and X-ray diffraction are alternatives, and the infrared spectrum of cystine is specific [32]. More than 85% of cystine stones are pure or consist mainly of cystine, and the presence of other components (calcium oxalate, calcium phosphate, uric acid, struvite) indicates associated metabolic abnormalities that should be investigated and treated separately [32]. For this reason, the protocol requests, after diagnosis, a complete metabolic stone evaluation (calciuria, uricosuria, oxaluria and citraturia), since hypercalciuria, hyperuricosuria and hypocitraturia are frequent in these patients [32].

Values depend on the method and population; ion-exchange chromatography is the reference method and liquid chromatography–tandem mass spectrometry (LC-MS/MS) is acceptable [4]. The sample must be alkalinised after voiding so that cystine does not precipitate [34]. With thiols, colorimetric methods and high-performance liquid chromatography do not distinguish well between free cystine and the drug–cysteine complex, which makes values unreliable for adjusting treatment [37, 47]. Chemical analysis of the stone alone may mislead, because mixed stones are frequent and some contain no cystine [34]. Plasma amino acids are normal or slightly low [34].

The thresholds in the sources do not coincide. The European recommendation classifies as biallelic a ratio above 150 µmol/mmol and excretion above 400 mg/day, whereas the French protocol uses 100 µmol/mmol (210 mg/g) and, in adults, 500 mg/24 h; for the healthy adult, the European recommendation gives less than 30 mg/day and the French protocol less than 100 mg/24 h in the text and less than 50 mg/day in the annex on solubility [4, 32]. This dispersion reflects the dependence of values on the method and laboratory, and intermediate excretion should be interpreted in the light of genotype and clinical picture. There are two further sources of interpretation error: some laboratories express the result as cysteine ("hemicystine"), and the value must then be divided by two to obtain cystine; and the cystine/creatinine ratio of the newborn may be physiologically raised, which requires specialist advice before reaching a conclusion [32]. The French protocol gives indicative values by age, which depend on the laboratory:

Source: annex 5 of the French PNDS [32]. Values are expressed as hemicystine (cysteine equivalents); to compare them with thresholds in cystine they are divided by two.

5.5 Cystinuria: imaging

Cystine stones are visible on ultrasound, but are less radio-opaque than calcium stones and may not appear on plain radiography [34, 47]. Non-contrast computed tomography of the kidneys, ureters and bladder has the highest sensitivity and specificity and retains them with low-dose and ultra-low-dose techniques; Hounsfield units (usually below 800) help little and may confuse cystine with uric acid [4, 10]. In children, ultrasound is the first-line test, and in recurrent stone formers ultrasound is preferred to limit cumulative radiation [4].

5.6 Cystinuria: genetic testing

Genetic testing is not mandatory for diagnosis, but is useful in atypical presentations, uncertain inheritance, family counselling and diagnosis after a fetal hyperechogenic colon; the European recommendation suggests offering it to confirm and classify the disease, whereas the American consensus and the EAU assign it no role in routine management [4, 37, 47]. The French protocol lies between the two: in adults and older children it recognises no defined place for it, because the result is not essential for diagnosis, but it allows it to be offered to patients with intermediate cystine elevation, in the investigation of recurrent stone disease, to distinguish a heterozygote from a patient with variants on both alleles, and in research protocols; in the presence of a hyperechogenic colon in the third trimester or a suspicion in a very young infant, it considers it the only means of early confirmation, because cystine measurement is difficult to interpret with immature tubular transporters [32]. In France, the decision to request the genotype rests with the nephrologist [32]. The preferred method is a next-generation sequencing (NGS) panel that includes copy-number variation analysis, or sequencing with MLPA or quantitative PCR, because large deletions and duplications are frequent [4, 10]. If stone analysis is lacking, a nephrolithiasis panel with MLPA is preferable; if there are extrarenal manifestations, the exome or genome, and chromosomal microarray for contiguous deletions [10, 34]. Stone panels in patients without suspicion may reveal variants in SLC3A1 and SLC7A9 whose causality must be consistent with the clinical picture and biochemistry [4].

The limits of the test are real. In 5–7% of patients with a typical phenotype no explanation is found, and in about 5% only one heterozygous variant is found [10, 34]. The detection rate in a paediatric series was 54% for SLC3A1 and 25% for SLC7A9, and 5–10% of adults have no variant identified [28]. A patient with the phenotype and a single variant may have an undetected second variant (intronic, regulatory or structural) or a variant in another gene [10]. Apparent homozygosity may result from an undetected deletion on the other allele or from uniparental isodisomy [10]. The proportion of variants detectable by each method (80–85% by sequencing, 15–20% by deletion analysis) is not equivalent to the clinical sensitivity of the test.

5.7 PREPL deficiency: tests and algorithm

The diagnosis of PREPL deficiency is molecular, and the first step is urine amino acid analysis, which confirms nothing but divides the path: cystinuria indicates that SLC3A1 is involved and points to a contiguous deletion, and its absence in an infant with congenital hypotonia, ptosis, feeding difficulties and proximal weakness points to isolated deficiency [6, 7]. Confirmation requires a method sensitive to copy-number variation, because the most frequent mechanism is a deletion: exome or genome sequencing, or a neuromuscular panel including PREPL with deletion and duplication analysis, and chromosomal microarray when a contiguous deletion is suspected [6, 10]. Sequencing alone is not enough, and apparent homozygosity should prompt a search for a deletion on the other allele or for uniparental disomy, with parental testing [14, 38].

Two technical limitations were documented by the authors of the 2018 series: in two of eight families, technical and interpretation problems with the microarray delayed detection of relatively large deletions, and the resulting recommendation is to ensure good coverage of the PREPL region and not to classify deletions in this region as benign from the outset [6]. The 31-kilobase deletion restricted to the gene in the Barcelona patient appeared only on a microarray with targeted exonic coverage [38].

The functional blood assay developed in 2018 is the available non-molecular confirmatory test. It measures, by immunoblotting in stimulated lymphocytes, the presence of the PREPL protein and its reactivity with an activity-based probe, biotinylated fluorophosphonate (FP-biotin), reliably distinguishes patients from controls with 200 micrograms of protein and gives a normal result in Prader-Willi syndrome and other neuromuscular diagnoses, which makes it useful precisely in the scenario in which doubt arises [6, 7]. In the two heterozygotes tested, reactivity was half that of the control, but there are no age-specific normal values [6]. The limit of the assay was demonstrated in 2024: missense variants from patients with congenital myasthenic syndrome 22 retained hydrolytic activity and reacted normally with the probe, and of the 18 ClinVar missense variants tested only four altered this binding, so a normal assay does not exclude the diagnosis in a child with a compatible phenotype [30].

The neurophysiological study is supportive and has its own pitfalls. Low-frequency repetitive nerve stimulation shows a decrement of the compound muscle action potential in some patients, but in others reveals it only after prolonged 20-hertz stimulation, and the 2014 patient had no decrement at 2 hertz when not taking pyridostigmine, a finding also observed in myasthenic infants; as in other presynaptic forms, an increment rather than a decrement may occur on high-frequency stimulation [8, 25, 29]. In several recent series no electrodiagnostic studies were performed, and the diagnosis was molecular from the outset [30]. Muscle biopsy with PREPL immunostaining, absent in fibres and endplates, and microelectrode study of neuromuscular transmission are reserved for specialised centres [7, 8]. Creatine kinase is normal, as is brain magnetic resonance imaging, which helps to exclude myopathies and central causes of hypotonia [8, 38]. The report should state the gene, the transcript with version (NM_001171613.2 as the MANE Select transcript, with conversion to NM_006036.4 when comparing with the literature), the variant in HGVS, zygosity with phase confirmed in the parents and the class according to the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) [12, 30].

5.8 Prenatal and preimplantation diagnosis

For both diseases, prenatal diagnosis (chorionic villi or amniocytes) and preimplantation genetic testing are possible when the familial variants have been identified [1, 10, 35]. In HHH, genotype does not predict severity, owing to intrafamilial variability [1]; before the molecular era, ornithine incorporation in amniocytes was used [9]. The 2019 European guideline strongly recommends molecular analysis as the preferred prenatal method in all UCDs (recommendation no. 6, high quality) and, for HHH, indicates as first choice analysis of the variants in DNA from chorionic villi or amniocytes, with the functional assay in villi or cultured amniocytes as an alternative; prenatal testing requires a confirmed diagnosis in the index case [36]. In cystinuria, ultrasound showing a hyperechogenic colon without a family history justifies molecular testing [10]. The French protocol specifies that this test is done after birth: there is no indication for amniocentesis to confirm suspected fetal cystinuria, and, when the appearance of a hyperechogenic colon is difficult to distinguish from a hyperechogenic small bowel, the couple should be referred to a multidisciplinary prenatal diagnosis centre, to exclude aneuploidies, cystic fibrosis and congenital infections [32].

6. Differential Diagnosis

6.1 HHH syndrome

The differential of HHH syndrome is organised around each element of the triad. In the presence of hyperammonaemia, the first filter is acid–base balance and blood glucose: UCDs give isolated hyperammonaemia with respiratory alkalosis and raised glutamine, organic acidaemias metabolic acidosis and ketonuria with normal or low glutamine, β-oxidation defects hypoketotic hypoglycaemia, liver dysfunction and raised CK, pyruvate carboxylase deficiency and carbonic anhydrase VA deficiency lactic acidosis and hypoglycaemia, and hyperinsulinism-hyperammonaemia severe hypoglycaemia [1, 9]. In the presence of hyperornithinaemia, only ornithine aminotransferase deficiency gives chronic elevation of ornithine, but without hyperammonaemia outside the neonatal period, without homocitrullinuria and with gyrate atrophy of the choroid and retina [1, 51]. In the presence of homocitrullinuria, a dietary origin (formulas and heated milk), lysinuric protein intolerance, OTC deficiency and hyperlysinaemia must be excluded [1, 9, 51]. In the presence of spastic paraparesis, arginase 1 deficiency is the only other UCD with prominent pyramidal involvement, usually earlier, and hereditary spastic paraplegias and cerebral palsy are the non-metabolic differentials [1, 9]. Urinary excretion of Krebs cycle intermediates and lactate may suggest primary mitochondrial disease [1].

6.2 Cystinuria

Cystinuria is distinguished from other stone diseases by stone composition and urinary biochemistry, but there are three pitfalls. The first is mixed composition: about 10% of patients also form calcium stones, and excessive alkalinisation may induce calcium phosphate stones, so a new stone should not be presumed to be cystine [10, 47]. The second is radiology: cystine and uric acid have overlapping densities on tomography [10]. The third is homonymy: cystinosis is a lysosomal disease with intracellular cystine accumulation and Fanconi syndrome, and homocystinuria is a defect of methionine metabolism, both unrelated to the b0,+ transporter. In the fetal period, the differential is different: the hyperechogenic colon of cystinuria has to be separated from hyperechogenic small bowel, which is associated with aneuploidies, cystic fibrosis and congenital infections [32].

6.3 PREPL deficiency and neonatal hypotonia

The differential of PREPL deficiency is not that of stone disease, but that of neonatal hypotonia with feeding difficulties, and spans three groups: acquired and common causes (congenital infection, heart disease, hypothyroidism, drug toxicity, hypoxic-ischaemic encephalopathy), Prader-Willi syndrome and the other congenital myasthenic syndromes [6, 29, 38]. The similarity with Prader-Willi syndrome has a molecular correlate, with dysfunction of the hypothalamic–pituitary axis and deficient mitochondrial bioenergetics in both conditions, but the hypothesis that it resulted from underexpression of PREPL was ruled out by the normal functional assay in these patients [6, 60]. Among the congenital myasthenic syndromes, the presynaptic forms due to defective acetylcholine synthesis or recycling (CHAT, SLC5A7, SLC18A3) share neonatal hypotonia, ptosis and feeding difficulties, but have episodic apnoea as the dominant sign and a more consistent and lasting response to acetylcholinesterase inhibitors [25, 29, 57].

The specific pitfall of this family of entities is the wrong attribution of a deletion. An infant in whom microarray shows a 2p21 deletion should be assessed for the myasthenic and endocrine manifestations of PREPL loss, and not only for the cystinuria that prompted the request; conversely, a deletion that does not involve SLC3A1 does not cause cystinuria and should not be dismissed for that reason [23, 58]. Partial cytochrome c oxidase deficiency on muscle biopsy, which points to mitochondrial disease, has been described in siblings whose cause was a classic SLC3A1 and PREPL deletion, so a mitochondrial finding neither excludes the contiguous deletion nor requires a search for a second diagnosis [63].

The starting point common to both diseases is amino acid analysis: in HHH syndrome plasma ornithine is raised and homocitrulline appears in the urine; in cystinuria ornithine is raised only in the urine, together with cystine, lysine and arginine, and the plasma is normal; in lysinuric protein intolerance the dibasic amino acids are low in plasma and high in urine [1, 34].

7. Treatment and Risk Management Strategies

7.1 HHH syndrome: sources of the recommendations

HHH syndrome is treated according to the principles of the UCDs. The 2012 European guideline (Häberle and colleagues, E-IMD group) has a specific statement (no. 50, grade D): low-protein diet, L-citrulline and, perhaps, creatine; the impact of these measures on neurological outcome remains to be demonstrated [35]. The first revision of that guideline, published in 2019 by a group of 16 European and Israeli authors, is the current European reference and grades all recommendations using the GRADE methodology (Grading of Recommendations, Assessment, Development and Evaluation), with a vote by the 11–12 panel members on the quality of evidence [36]. For HHH, recommendation no. 38 proposes a low-protein diet with L-citrulline or L-arginine supplementation and declares the effect of these measures on pyramidal dysfunction uncertain (moderate quality: 5/12 votes moderate, 5/12 low, 2/12 high) [36]. The text of the revision adds that a low-protein diet with citrulline prevents hyperammonaemia and liver disease, but apparently not spastic paraparesis, and that creatine may be given to patients with low plasma creatine, owing to the secondary deficiency described in HHH [36]. Compared with 2012, the revision now accepts arginine as an alternative to citrulline in maintenance, includes intravenous arginine in the HHH crisis, widens the citrulline dose range and no longer excludes HHH from the list of UCDs already treated by liver transplantation (sections 7.2 to 7.4) [35, 36]. The 2020 GeneReviews and the 2025 UCD overview complete the HHH-specific guidance [1, 24]. There are no clinical trials in HHH; doses result from consensus and extrapolation from the other UCDs, and the 2019 guideline records that there are no studies comparing the efficacy of citrulline and arginine in HHH [35, 36, 68].

7.2 HHH syndrome: treatment of the hyperammonaemic crisis

The crisis is treated as in the other UCDs, but with one particular feature: the patient with HHH responds more quickly to intravenous glucose and withdrawal of protein than a patient with OTC or ASS deficiency, and ammonia often normalises without scavenger drugs [1]. Treatment should be carried out with a metabolic diseases team, and the steps are as follows [1, 35]:

1. Assessment: ammonia, full biochemistry, arterial blood gases, full blood count with differential, urinalysis, plasma and urine amino acids, urinary orotic acid and organic acids, viral studies and search for the trigger (infection, gastrointestinal bleeding, fasting, surgery, drugs). Ammonia of 80 µmol/L or more is treated immediately [1].
2. Stop all protein and oral intake until stable (as a rule 24 hours, without exceeding 24–48 hours without protein or essential amino acids) [1, 9, 35]. Glycerol phenylbutyrate (Ravicti®) should be stopped during the crisis, because it depends on pancreatic lipases and acts only with food [1].
3. Anabolism: 10% dextrose with quarter-strength saline and 20 mEq/L of potassium chloride at twice maintenance, with a glucose infusion rate of 10–15 mg/kg/min and target blood glucose of 100–150 mg/dL; increase glucose concentration (12.5–15%) to avoid fluid overload, especially with cerebral oedema; do not reduce the infusion for hyperglycaemia above 150 mg/dL, but add insulin for blood glucose of about 150–160 mg/dL; intravenous lipids 2–3 g/kg/day; monitor ammonia, glucose and electrolytes every 2 hours or with each neurological change [1].
4. If there is no clinical improvement: intravenous arginine and nitrogen scavenger drugs, as a loading dose over 90–120 minutes followed by the same dose over 24 hours (following table) [1, 24, 35].
5. Dialysis: if ammonia rises or neurological status worsens despite treatment, with arginine and scavengers continued during dialysis (threshold table) [1].
6. Reintroduction: 24–36 hours after admission, start oral intake with essential amino acids, carnitine, vitamins and lipids, avoiding non-essential amino acids (glutamine, proline, glycine); protein is reintroduced when ammonia falls below 100 µmol/L, up to the safe level over 2–4 days [1, 35].

The 2019 European guideline organises the crisis by ammonia level in a symptomatic patient and applies the same scheme to the undiagnosed patient and to the patient with a known UCD; the first measures are to stop protein, start intravenous glucose with sodium and potassium, start the first-line drugs, collect plasma and urine for diagnosis without delaying treatment and transfer to a specialised centre [36]. Recommendation no. 8 (strong; moderate quality) requires immediate reversal of endogenous protein catabolism and promotion of ammonia detoxification, and no. 12 (strong; moderate) requires establishing anabolism with high-dose glucose, with insulin if necessary, and adding lipids as soon as fatty acid oxidation disorders have been excluded, without exceeding 24 (to 48) hours of protein-free nutrition [36].

There are two differences from the GeneReviews protocol: the European guideline gives lipids of 1–2 g/kg/day, compared with 2–3 g/kg/day in GeneReviews, and grades the glucose rate by age (6–10 mg/kg/min) instead of the 10–15 mg/kg/min of GeneReviews [1, 36]. If enteral feeding is not possible, intravenous amino acids start at 0.5 g/kg/day and increase by 0.5 g/kg/day, according to ammonia, up to the safe level for age; energy should be around 120% of requirements for age, and protein is reintroduced over 2–4 days, adding essential amino acids if ammonia rises [36]. After a scavenger bolus, ondansetron (0.15 mg/kg) may be given to prevent vomiting; repeated boluses or very high doses of benzoate or phenylacetate saturate the conjugation systems and increase the risk of accumulation and toxicity [36]. In the patient not yet diagnosed, the guideline adds carglumic acid, carnitine, hydroxocobalamin and biotin to the scavengers, and recommendation no. 21 (high quality) keeps carglumic acid as an emergency drug in hyperammonaemia of unknown cause [36].

The loading solution mixes arginine, benzoate and phenylacetate in 25 mL/kg of 10% dextrose and runs over 90 minutes; all preparations should be checked two to three times, because of the risk of toxicity with high doses and of persistent neurotoxicity with low doses [1]. In the United States, the 10%/10% combination of sodium phenylacetate and sodium benzoate (Ammonul®, approved on 17/02/2005, and generics) is adjunctive treatment of acute hyperammonaemia with encephalopathy in urea cycle enzyme deficiencies, diluted in at least 25 mL/kg of 10% dextrose and given through a central line; on the label, the dose for 0–20 kg is 2.5 mL/kg (250 mg/kg of each component) and, above 20 kg, 55 mL/m2 (5.5 g/m2 of each component), with 10% arginine 2 mL/kg (200 mg/kg) in CPS and OTC deficiencies, as a 90–120-minute loading dose repeated as maintenance over 24 hours; the label advises against repeating the loading dose, because phenylacetate persists in plasma, and defines no other maximum dose [69]. The 2019 European guideline accepts this equimolar solution when available (loading 250 mg/kg over 90–120 minutes and then 250 mg/kg as a continuous 24-hour infusion) and records that in the European Union it is obtainable only on a named-patient basis [36]. In the European Union there is no centralised authorisation for sodium benzoate: the application for Prohippur® was withdrawn in 2018, and benzoate has had orphan designation for ornithine transporter deficiency since 2016 [70, 71].

Continuous venovenous haemodiafiltration is the preferred method in newborns and children, especially with haemodynamic instability; in adults, haemodialysis or continuous haemofiltration are first line; peritoneal dialysis is much less effective and exchange transfusion should not be used [1, 35]. In adults, susceptibility to intracranial hypertension justifies an earlier approach [35]; the 2019 revision recommends considering extracorporeal detoxification as first-line treatment in adult decompensation (recommendation no. 10, low quality), considering it above 200 µmol/L according to comorbidities and drug tolerance, because haemodialysis or continuous venovenous haemofiltration is available in most intensive care units and can start before transfer [36]. The same revision strongly recommends preparing extracorporeal detoxification in parallel with medical treatment in any patient with severe neurological symptoms and starting it as soon as possible, unless there is sufficient improvement (no. 9, moderate quality), and recommends haemodialysis or continuous venovenous haemodiafiltration as the method of choice in the newborn, peritoneal dialysis only as a bridge and, strongly, that exchange transfusion should not be used (no. 11, high quality) [36]. After haemodialysis is stopped, hyperammonaemia may relapse, and effective detoxification does not guarantee a good cognitive prognosis [36]; the Swedish case in section 10 illustrates the late cerebral oedema that can appear even after ammonia has normalised [55].

7.3 HHH syndrome: maintenance treatment

Chronic treatment combines diet, supplements and, when necessary, nitrogen scavengers, with the aims of keeping ammonia, glutamine, arginine, ornithine and the essential amino acids (lysine, isoleucine, leucine and valine) within normal limits [1]. Plasma ornithine rarely normalises; keeping it as low as possible may help to prevent some of the neurological complications, but without formal demonstration [1, 9]. In the review of 111 patients, all were on a low-protein diet, 22% on protein restriction alone, 30% on arginine, 22% on citrulline, 10% on ornithine and 36% on nitrogen scavengers (21% benzoate, 12% phenylbutyrate, 3% both) [9].

Diet. The low-protein diet, adapted to age, should control ammonia and allow growth; natural protein is titrated individually against ammonia, starting from the FAO/WHO/UNU safe levels, and the ratio of natural protein to essential amino acid mixture is around 60:40 [1, 35]. From 12 years of age, the total protein requirement is about 0.7–1.0 g/kg/day [1]. Shih observed that 1.5 g/kg/day prevented crises and that 2 g/kg/day caused symptomatic hyperammonaemia in the first patient [44]. The supplements used include essential amino acid mixtures (Cyclinex®-1 and -2, Vitaflo essential amino acid mix), protein-free calorie supplements and branched-chain amino acid mixtures when these are low owing to the action of the scavengers [1]. Non-prescribed protein supplements, such as those used in sport, should be avoided [1].

The 2019 European guideline strongly recommends that the diet be managed by a dietitian specialised in metabolic diseases, starting from the safe levels of protein intake and the energy requirements of FAO/WHO/UNU 2007 (recommendation no. 13, moderate quality), and acknowledges that the low-protein diet is not based on controlled studies, but on physiology, patients' self-selection of low-protein foods and clinical experience [36]. Excessive restriction compromises growth and well-being and may cause metabolic instability, so optimal intake is titrated in each patient; less active patients may need less protein or energy than the table indicates [36].

Daily protein is divided over three to four meals, with a late-evening snack that shortens the overnight fast, and the last meal before sleep should contain about 25% of the day's energy, natural protein, essential amino acids and citrulline or arginine [36]. The diet should combine protein of low and high biological value, because some low-protein foods, such as cereals, are low in lysine [36]. When protein tolerance is below the safe level, the 2019 guideline proposes that essential amino acids provide up to 20–30% of total protein, a lower value than the 60:40 ratio of natural protein to essential amino acid mixture in the 2012 guideline and GeneReviews; the mixture should be rich in branched-chain amino acids and low in tryptophan, phenylalanine and tyrosine, precursors of serotonin and dopamine, and is taken with meals (recommendation no. 14, moderate quality, which includes patients treated with phenylbutyrate, because of branched-chain amino acid depletion) [1, 36]. The low-protein diet exposes patients to deficiencies of iron, zinc, copper, calcium and cobalamin and, when very restricted, of essential fatty acids, which can be offset with fortified formulas or walnut, rapeseed or sunflower oils [36]. A nasogastric tube or gastrostomy should be considered early when there is inability to suck or swallow, food refusal, unpleasant taste of supplements, vomiting or reflux, or for the emergency regimen (recommendation no. 16, low quality); the guideline also suggests written treatment sheets for parents, pharmacists and carers (no. 17) and recommends training of parents and patients for lifelong dietary management (no. 15) [36].

Citrulline and arginine. L-citrulline is preferred to arginine, because it gives better metabolic control, avoids secondary creatine deficiency and, by accepting an aspartate in the ASS reaction, eliminates two amino groups per cycle; in a European registry, citrulline gave higher plasma arginine than arginine or no supplement in patients with OTC deficiency, CPS1 deficiency or HHH [1, 9]. GeneReviews gives 0.17 g/kg/day or 3.8 g/m2/day [1]. The 2012 guideline gives, for HHH, L-citrulline or L-arginine at doses of 100–200 mg/kg/day below 20 kg and 2.5–6 g/m2/day above, with a maximum of 6 g/day, divided into three to four doses with meals, with a fasting plasma arginine target of 70–120 µmol/L [35]. The 2019 revision, which is the reference dose, keeps L-arginine (hydrochloride or free base) at 100–200 mg/kg/day below 20 kg and 2.5–6 g/m2/day above, with a maximum of 6 g/day, and widens L-citrulline to 100–250 mg/kg/day, maximum 6 g/day; when citrulline is given, arginine is generally not needed, and doses are divided over three to four meals throughout the day [36]. Recommendation no. 20 (strong; moderate quality) requires L-arginine or L-citrulline supplementation in all UCDs except arginase 1 deficiency, accepts that it may not be needed in mild phenotypes and recommends measuring plasma arginine in all patients, with a fasting target of 70–120 µmol/L [36]. To convert doses, 100 mg corresponds to 0.571 mmol of citrulline, 0.475 mmol of arginine hydrochloride and 0.574 mmol of arginine base [36]. Ornithine supplementation, tried in the past with contradictory results, is not recommended [9].

Nitrogen scavengers. Sodium phenylbutyrate is given in three doses: 450–600 mg/kg/day up to 25 kg or 9.9–13.0 g/m2/day above (FDA and EMA label doses), when glycerol phenylbutyrate is not available [1, 35]. The 2012 guideline proposes lower doses (up to 250 mg/kg/day below 20 kg, 5 g/m2/day above, maximum 12 g/day) and considers it second choice for maintenance, to be combined with benzoate when benzoate alone is insufficient; sodium benzoate is used up to 250 mg/kg/day, maximum 12 g/day [35]. The 2019 revision keeps these doses for HHH (benzoate up to 250 mg/kg/day; sodium or glycerol phenylbutyrate up to 250 mg/kg/day below 20 kg and 5 g/m2/day above; maximum of 12 g/day each), keeps phenylbutyrate as second choice combined with benzoate and cites the higher doses from the FDA studies (450–600 mg/kg/day below 20 kg; 9.9–13.0 g/m2/day above) as necessary in some patients by expert opinion; these doses coincide with those on the labels and are above the guideline reference dose [36]. Recommendation no. 18 (moderate quality) defines scavengers as a pillar of treatment with individualised dosing, and the guideline records the divergence between continents: in the United States most patients receive only sodium or glycerol phenylbutyrate, whereas many European centres use benzoate as first line because of long experience, fewer adverse effects and price [36]. Glycerol phenylbutyrate has practically the same phenylbutyrate content (5% more) as sodium phenylbutyrate, contains no sodium, is a tasteless liquid and is given in three equal doses; benzoate and sodium phenylbutyrate are given in four doses with meals and plenty of fluids, because undiluted granules, tablets or liquids may cause mucositis or gastritis [36]. The adverse effects recorded by the guideline are benzoate toxicity above 2 mmol/L in plasma, amenorrhoea or menstrual dysfunction in about 25% of women treated with sodium phenylbutyrate, reduced appetite, dysgeusia and body odour, branched-chain amino acid depletion with low albumin, hypokalaemia due to renal potassium loss and metabolic acidosis with high doses [36]. Urinary hippurate helps to assess adherence to benzoate and urinary phenylacetylglutamine to adjust the dose of glycerol phenylbutyrate [36]. Plasma levels of benzoate and phenylbutyrate and their metabolites (phenylacetate, phenylacetylglutamine) help to adjust the dose and assess adherence [1, 35]. Glycerol phenylbutyrate (Ravicti®) is the only formulation whose European indication names HHH (section 7.5); the dose for patients not previously on phenylbutyrate is 4.5–11.2 mL/m2/day (5–12.4 g/m2/day), starting at 4.5 mL/m2/day when there is residual activity, taken with food [71, 72]. The paediatric patient described in section 10 was maintained on glycerol phenylbutyrate and citrulline [56].

Other supplements. Lysine is given when plasma lysine is low, because this is associated with growth and developmental delay [1, 44]. Creatine is recommended when plasma creatine is low, owing to inhibition of AGAT by ornithine [1, 9, 35]. Carnitine may be needed because of dietary deficiency and conjugation with scavenger metabolites [1]; the 2019 guideline requires monitoring of plasma carnitine in all patients with UCDs and treatment of severe deficiency [36]. The same guideline considers the practical consequences of low creatine in HHH uncertain and makes no recommendation on oral neomycin or metronidazole to reduce ammonia-producing intestinal flora, for lack of controlled studies [36].

7.4 HHH syndrome: liver and transplantation

Three patients with fulminant liver failure and coagulopathy stabilised rapidly with protein restriction and arginine or citrulline, which indicates that liver failure in HHH responds to metabolic treatment [1]. Liver transplantation is not indicated when metabolic control is achieved with diet, citrulline and scavengers: the transporter is expressed in the brain, kidney and astrocytes, and transplantation corrects hyperammonaemia but not the tissue changes that contribute to the neuropathology [1]. Two transplants have been published, in a 6-year-old child with persistent biochemical abnormalities, protein intolerance, delay and abnormal posture, and in a 7-year-old child with poor control after hyperammonaemic coma at 4 weeks (ammonia of 2300 µmol/L) [1]. The 2012 guideline recorded transplantation in all UCDs except NAGS and HHH [35]; the 2019 revision records it in all UCDs except NAGS deficiency, with no specific recommendation for HHH [36]. The general 2019 recommendations are to consider transplantation in severe UCDs without sufficient response to standard treatment and with poor quality of life, without severe neurological damage and preferably in metabolic stability (no. 24, moderate quality), and to consider it strongly in severe and progressive liver disease or in recurrent decompensations requiring admission despite medical treatment (no. 25, high quality) [36]. In paediatric programmes, survival after transplantation is around 95% at 1 year and 90% at 5 years; in a series of 186 children with UCDs, 5-year survival was 88% when transplanted before 2 years of age and 99% from 2 years onwards, and the guideline advises against transplantation before 3 months of age or below 5 kg [36]. Transplantation does not reverse established neurological damage, and after it de novo arginine synthesis remains absent in all UCDs except arginase 1 deficiency, usually without clinical impact [36]. In HHH, these data from cohorts with other UCDs add to the GeneReviews reservation about extrahepatic expression of the transporter [1, 36].

7.5 HHH syndrome: regulatory status

7.6 HHH syndrome: agents and situations to avoid

Excess dietary protein, non-prescribed protein supplements, prolonged fasting during illness or to lose weight, oral and intravenous corticosteroids, valproic acid (which induces and worsens hyperammonaemia in UCDs) and unnecessary exposure to communicable diseases should be avoided [1]. Triggers described in adults include surgery, infection, fasting, pregnancy, internal gastrointestinal bleeding and drugs [1, 55]. The emergency card and the action plan for infections and surgery derive from these rules, which apply to all UCDs. The 2019 guideline adds carbonic anhydrase inhibitors, such as topiramate, to the list of triggering drugs, because the bicarbonate produced by carbonic anhydrase VA in hepatic mitochondria is a substrate of CPS1 [36].

The 2019 European guideline calls for a written plan for parents, nursery and school, with instructions on when to contact the metabolic team or the local hospital and a written prescription for emergency treatment, and recommends training visits to the nursery and school by a specialist nurse or a metabolic dietitian [36]. During intercurrent illness, the protein-free emergency regimen starts at home and, without rapid improvement, the metabolic unit is contacted urgently; the glucose polymer solution is used at 10% up to 6 months (40 kcal/100 mL; 150 mL/kg/day, maximum 1200 mL), 10–15% from 7 to 12 months (120 mL/kg/day, maximum 1200 mL), 15% at 1 year, 20% from 2 to 9 years and 25% above 10 years (100 kcal/100 mL), in doses every 2 or 3 hours, day and night, or by continuous tube feeding; above 10 kg the volume is calculated as 100 mL/kg for the first 10 kg, 50 mL/kg for the next 10 kg and 25 mL/kg thereafter, up to 2500 mL/day [36]. Vaccinations follow the national schedule (recommendation no. 22, strong, moderate quality), because they have not been identified as a relevant cause of decompensation, and the guideline suggests an antipyretic above 38 °C (low quality) [36]. Elective surgery is performed in a centre experienced in metabolic diseases (no. 23, expert opinion), with the patient well, ammonia and amino acids normal and no intercurrent illness; on the day before, drugs are switched to the intravenous route with 10% glucose and electrolytes, the patient is first on the operating list, and return to oral intake and withdrawal of glucose are done only in metabolic stability; midazolam, ketamine, fentanyl and isoflurane, with infiltration of the surgical field with ropivacaine, have been described as safe [36].

7.7 HHH syndrome: pregnancy, delivery and lactation

Pregnancy and the postpartum period are periods of risk owing to catabolism, nausea and vomiting, the patient's natural aversion to protein and uterine involution. Ho and colleagues described four women (one with two pregnancies of her own and three from the literature), with complications in three: mild hyperammonaemia at the end of the first trimester, a decompensation with ammonia of 295 µmol/L before the second trimester treated with phenylacetate and benzoate and arginine, absence seizures in the second trimester, hyperammonaemia ten hours after caesarean section treated with oral benzoate and intravenous arginine, and ammonia of 43–126 µmol/L during labour [1, 62]. There were two normal pregnancies, one of them in an asymptomatic woman maintained on citrulline and diet, and a woman treated with arginine and lactulose had a child with an IQ of 130 at five years [1, 15, 44].

The published recommendations are as follows [1, 62]: continue the protein restriction, citrulline and scavengers that the patient was taking before pregnancy, after assessment of benefit and risk; monitor plasma amino acids and ammonia weekly or every two weeks, especially in the first and third trimesters, and closely immediately after delivery; adjust protein (the patients described took 0.8–1.3 g/kg/day, against a reference intake of 1.1 to 1.2–1.52 g/kg/day); and prepare a protocol for delivery and the postpartum period. GeneReviews recommends caesarean section to optimise metabolic control; this indication comes from a case series and not from comparison with vaginal delivery [1]. The 2012 guideline has a table of protein and energy requirements in pregnancy and lactation [35]; the 2019 revision updates it with the FAO/WHO/UNU 2007 values: additional protein of 1, 10 and 31 g/day in the first, second and third trimesters, and of 19 g/day in the first 6 months of lactation and 13 g/day thereafter; additional energy of 90, 287 and 466 kcal/day by trimester, and of 669 and 460 kcal/day in the same periods of lactation [36]. The revision asks that protein malnutrition be avoided and that monitoring be close during delivery and the first 5 days after delivery, a period in which hyperammonaemia has to be recognised [36]. Recommendation no. 19 (low quality) considers it generally necessary to continue nitrogen scavengers in pregnancy and suggests sodium benzoate, for biochemical reasons and by group opinion, as a safer choice than phenylbutyrate, for which there are only isolated reports of successful pregnancies; evidence is insufficient to assess fetal outcome [36]. This preference for benzoate diverges from the guidance of GeneReviews and of Ho and colleagues, who continue the scavenger the patient was already taking after assessment of benefit and risk [1, 36, 62].

The fetal outcomes described in five children of three women treated mainly with diet or lactulose were: three healthy newborns, one child with intrauterine growth restriction and normal development at two years, and one with transient respiratory distress [1]. In a woman with HHH treated with sodium phenylbutyrate (5.5 g four times daily) during her first pregnancy, the child had typical growth and development at five years; in the second pregnancy (5 g four times daily, plus phenylacetate and benzoate during crisis), the child was born preterm and growth-restricted and had language delay and autism at two years, without it being possible to attribute the cause to decompensation, ornithine or the drugs [1]. There are no controlled studies of the fetal effects of benzoate, phenylacetate or phenylbutyrate; benzoate caused malformations in zebrafish larvae and phenylbutyrate is a histone deacetylase inhibitor, with teratogenic potential in mice. Ho and colleagues prefer sodium benzoate when a scavenger is needed in pregnancy, but do not advise changing maintenance medication during gestation [1]. The offspring of women and men with HHH were apparently normal [51].

7.8 HHH syndrome: developments and research

GeneReviews mentions no clinical trials in HHH [1]. On ClinicalTrials.gov, up to 31/10/2026, there are no specific trials; the only ongoing trial that includes the ornithine transporter among its conditions is a phase 1/2, randomised, placebo-controlled study of KRRO-121 (Korro Bio), an RNA editing therapy that produces a stabilised variant of glutamine synthetase to increase ammonia clearance, in healthy volunteers and in adults with UCDs, recruiting since 28/09/2026 (NCT07773246), without results [74]. A phase 2 trial of the microbiome modulator KB195 in UCDs was terminated in 2021 for business reasons (NCT03933410) [75]. A 2026 Japanese series, with seven adults with HHH in a national survey, described two 52-year-old adults homozygous for nonsense variants, both with intellectual disability, and noted that in HHH intellectual disability appeared regardless of age at onset and severity of hyperammonaemia [54].

7.9 Cystinuria: guidelines and general strategy

Treatment of cystinuria is based on four documents that agree on the stepwise strategy but diverge on targets and doses. The ERKNet and eUROGEN European clinical practice recommendation (Servais and colleagues, 2021) resulted from a systematic review (115 articles selected from 1754) and a consensus conference in Paris, in January 2019, with 20 statements that could not be graded because of the poverty of the evidence [4]. The American consensus promoted by the International Cystinuria Foundation (Eisner and colleagues, 2020) assessed 25 articles out of 2335 using the GRADE system and classifies the recommendations as standard with grade C evidence or expert opinion [47]. The European Association of Urology (EAU) guidelines on urolithiasis, 2026 edition, have three strong recommendations for cystine stones, with level of evidence 3 [37]. The guideline of the Brazilian Society of Nephrology (2025) covers cystinuria within nephrolithiasis in general [76]. The French National Diagnosis and Care Protocol (PNDS) of the Haute Autorité de Santé (HAS), of November 2019, was prepared by the MARHEA, Néphrogones and SORARE rare kidney disease reference centres of the ORKiD network, following the 2012 HAS method; it is contemporary with the European conference and shares some authors, but is more operational, with doses in grams and in tablets, collection rules, dietary tables and its own surveillance schedule, and does not grade its recommendations [32]. In this article, the European recommendation is the reference and the others serve for comparison.

The aim is to keep urinary cystine concentration below the solubility limit in all portions of urine over 24 hours: below 250 mg/L (1 mmol/L), with urinary pH of 7.5–8.0, which allows crystals to dissolve; to prevent and dissolve stones, supersaturation should remain below 0.6 mmol/L in 24-hour urine, to leave a margin for the nocturnal rise [4, 10]. The strategy is stepwise: hydration, diet and alkalinisation for all, and thiols in refractory cases [4, 47]. The French protocol summarises the aims in four mechanisms: lowering cystine concentration (hyperdiuresis), increasing its solubility (alkalinisation and, only as second line, thiols), reducing its production (less methionine) and reducing its excretion (less sodium); it insists that treatment is lifelong and that the basis is alkaline hyperdiuresis [32].

7.10 Cystinuria: hydration and diet

Fluid intake should ensure a urine output that keeps cystine below 250 mg/L; in most adults at least 3 L of urine per day are needed (to excrete 1–1.5 g of cystine), with urinary specific gravity of 1.005 or less, and in children more than 2 L/1.73 m2 [4]. The American consensus defines minimum outputs by age: more than 750 mL/24 h in infants, more than 1000 mL up to 5 years, more than 1500 mL up to 10 years, more than 2000 mL in older children and adolescents and more than 3000 mL in older adolescents and adults, which corresponds to 2.5–4 L of fluids per day in adults; a retrospective study associated 3.1 L/day of urine with fewer recurrences than 2.4 L/day [47]. Fluids are distributed throughout the day and night: drink at bedtime and on waking, and at each nocturnal awakening; the D-penicillamine label advises about half a litre at bedtime and another during the night, and Saudubray gives 500 mL at bedtime and, if possible, 500 mL during the night [4, 10, 34, 77]. In refractory children, nocturnal water by nasogastric tube or gastrostomy may be considered [4]. Drinks high in sugar or sodium should be avoided [10].

The French protocol sets in adults a urine output above 3 L per day distributed over 24 hours, with about half a litre at bedtime, at each nocturnal awakening and after each void [32]. Because the target is urine volume and not volume ingested, intake must compensate for insensible losses (about 700 mL/day in adults and 400 mL/m2 in children, at 20 °C) and increase with vomiting, diarrhoea, heavy sweating, physical exertion, heat, long journeys and occupations exposed to extreme temperatures [32]. The practical indicator is urinary specific gravity, which should remain at 1.005 or less in all voids and especially in the morning urine (a specific gravity of 1.005 corresponds to about 3 L/day well distributed); the patient monitors it with reagent strips at different times of day or learns to recognise it by the colour of the urine [32]. The intensity of treatment depends on the magnitude of cystine loss, so it is useful to know, at the outset, the order of magnitude of 24-hour cystinuria, despite its daily variation [32]. In children, the French protocol asks for more than 2–3 L/m2/day (2 L/m2/day in the dietary summary table of the same document), compared with more than 2 L/1.73 m2 in the European recommendation; if the target is not reached and there is active stone disease, nocturnal hydration by nasogastric tube may be proposed and, depending on the course, gastrostomy discussed [4, 32]. In a child diagnosed in the prenatal period and still without stones, the protocol recommends getting the child used to drinking at night from an early age, leaving a bottle in the cot, and does not indicate a nasogastric tube until there has been a stone episode [32].

Sodium restriction reduces cystine excretion, which increases linearly with natriuresis, although no study has shown that it prevents stones [4, 47]. The European recommendation gives 1–1.5 mEq/kg/day (in adults, 100 mEq/day or 6 g of sodium chloride), the American consensus less than 2500 mg of sodium per day and the EAU less than 2 g of sodium (5 g of sodium chloride) [4, 37, 47]. The French protocol considers the effect of sodium restriction little studied and modest and advises not exceeding 6 g of salt (100 mmol of sodium), which corresponds to a 24-hour natriuresis below 100 mmol; its dietary summary table accepts 6–8 g/day and a natriuresis below 120 mmol, which shows that the target is approximate [32]. The same document proposes estimating salt intake from 24-hour urine: salt (g/day) ≈ urinary sodium (mmol/24 h) ÷ 17 [32].

Reducing methionine, the precursor of cystine, decreases cystine production, without going below physiological requirements (1200–1400 mg/day in adults); in adults, protein should remain below 1 g/kg of ideal body weight per day (GeneReviews gives 0.8–1 g/kg/day of protein, preferring plant protein) [4, 10]. Restriction of animal protein also reduces the acid load and facilitates alkalinisation [4, 47]. In children protein is not restricted, so as not to compromise growth, but methionine-rich foods are reduced [4, 47]. The Brazilian guideline recommends a diet rich in vegetables and fruit, of the DASH pattern [76]. The EAU notes that adherence to a low-methionine diet is difficult and that sodium restriction is easier and more effective [37].

The French protocol recommends, in adults, limiting animal protein so that total protein intake is 0.8–1 g/kg/day, and adjusting the amount of methionine-rich foods, without eliminating them, because methionine is an essential amino acid (requirements of about 1200–1400 mg/day in the text; recommended intake of 1100–1500 mg/day according to weight, in the dietary annex); it acknowledges that the effect of dietary protein on cystine excretion has been little studied [32]. Protein intake is estimated from 24-hour urinary urea: protein (g/day) ≈ urea (mmol/24 h) ÷ 5, or urea (g/24 h) × 3.5, with a target urinary urea below 5.5 mmol/kg of ideal body weight per day in adults [32]. The sample menus in the annex give an idea of the order of magnitude: in a 60 kg woman, 48–50 g of protein per day (0.8 g/kg) correspond to 1100–1200 mg of methionine; in an 80 kg man, 62–64 g of protein correspond to 1400–1500 mg [32]. In children, the diet is low in salt and normoproteic, without excess; the protocol proposes, simply, a single daily portion of animal protein, preferably at lunch, and dietetic follow-up every 3–6 months, especially in infants, to avoid deficiencies [32]. The same text states that plant proteins do not count because they are not a source of methionine; its own table nevertheless assigns 60–70 mg/100 g to cooked pulses, which makes this rule a practical simplification and not a real absence of methionine [32].

Source: European recommendation, adapted from Bouzidi and Daudon [4]. Dietary and hair supplements rich in methionine should also be avoided [10].

The dietary annex of the French protocol adds standard portions and low-content foods, useful for building the diet [32]:

The values of the French protocol and those of the European recommendation differ for some foods (for example, 450 versus 390 mg/100 g for eggs and 900 versus 930 mg/100 g for parmesan), a difference explained by the food composition tables used [4, 32].

Diet also influences urinary pH, and the French protocol uses the PRAL index (potential renal acid load), which estimates the acid load of 100 g of a food from its acidic and basic minerals and protein [32]. Food intake should have a negative PRAL over the day: vegetables, potatoes or fruit at every meal, 2–3 pieces of fresh or dried fruit per day, lemon juice diluted in water and bicarbonate-rich waters; acidifying foods (meat, PRAL about +15; poultry and fish, about +12; eggs, +7; cheeses, +10 to +27; cereals and their products) are consumed in the necessary amounts and spread over the day, with a single daily portion of cheese and only occasional cola drinks [32]. Among alkalinising foods, the annex lists vegetables (−2 to −10), herbs and spices (−15 to −20), potatoes (−5), fresh fruit (−2 to −7) and dried fruit (−10 to −27) and bicarbonate waters (from −3 to −10); milk, yoghurt, oils and sugar are neutral [32].

7.11 Cystinuria: alkalinisation

Alkalinisation is a pillar of treatment for all patients, because cystine solubility depends on pH: a pH above 7.5 triples solubility relative to physiological pH [4, 10]. Potassium citrate is the preferred alkalinising agent, because it does not increase natriuresis; potassium bicarbonate is an alternative, and sodium bicarbonate or sodium citrate are reserved for severe renal impairment or potassium intolerance, bearing in mind that sodium increases cystinuria; acetazolamide is poorly tolerated and not superior [4, 47]. The last dose should be taken at bedtime, and pH should be measured in fresh urine from each void, with strips, dipsticks or electronic devices [4, 10].

Acalka® is contraindicated in renal impairment (glomerular filtration below 0.7 mL/kg/min), hyperkalaemia, active urinary tract infections, urinary obstruction, active peptic ulcer, delayed gastric emptying and with potassium-sparing diuretics, among other situations; the SmPC recommends monitoring electrolytes, creatinine and full blood count every four months [78]. Alkalinisation should be reinforced whenever the patient undergoes a urological procedure [4].

The French protocol expresses potassium citrate doses in grams of salt and not in milliequivalents. Assuming tripotassium citrate monohydrate (324 g/mol, 3 mEq of potassium per mmol), 4 g/day corresponds to about 37 mEq/day, the 2 g steps to about 18 mEq and the maximum of 16 g/day to about 148 mEq/day; the paediatric dose of 150 mg/kg/day corresponds to about 1.4 mEq/kg/day. The French starting dose is thus below the 60 mEq/day with which the European recommendation starts, and the ceiling described exceeds the 100 mEq/day that the Acalka® SmPC says to avoid; the reference for dosing is the guideline, and titration is by urinary pH in either scheme [4, 32, 78]. Potassium citrate and bicarbonate, in pharmacy-prepared capsules or sachets, are preferred to sodium bicarbonate because they alkalinise without a sodium load; sodium bicarbonate, in 1 g capsules (4–8 g/day) or in bicarbonate waters, remains acceptable, because the benefit of alkalinisation outweighs the small increase in cystinuria caused by sodium [32]. The protocol gives the composition of three French waters: Vichy Célestins® (2989 mg/L of bicarbonate and 1172 mg/L of sodium), Vichy Saint-Yorre® (4368 mg/L of bicarbonate and 1708 mg/L of sodium) and Salvetat® (500 mg/L of bicarbonate and 5 mg/L of sodium), the last practically sodium-free but with a much smaller alkalinising effect [32].

Gastrointestinal tolerance of alkalinising agents is often poor (abdominal discomfort and pain, epigastric pain, nausea, vomiting, diarrhoea, aphthae and gastric ulcers); diluting potassium citrate improves it and, with it, adherence, sodium bicarbonate is better tolerated than potassium salts, and the various alkalinising agents can be combined [32]. Doses are spread over 24 hours, not forgetting the bedtime dose, and an alkaline water can be drunk on a nocturnal awakening; pH is measured with indicator paper, reagent strips or, at greater cost, an electronic meter [32]. The theoretical risk of alkalinisation is calcium phosphate precipitation, monitored by crystalluria and systematic analysis of each stone, although the proportion of patients who form pure or very calcium phosphate-rich stones is small [32]. From an eGFR below 45 mL/min/1.73 m2, or with a renin–angiotensin system inhibitor, potassium citrate requires potassium monitoring; in stage 4–5 chronic kidney disease, treatment of cystinuria is no longer useful, because urinary cystine output becomes negligible [32]. In an infant diagnosed before birth, alkalinisation is debatable, because urinary pH is often alkaline in the first months and citrate is unpalatable and poorly tolerated; the French protocol recommends starting it after the first stone episode or in the presence of positive crystalluria [32].

7.12 Cystinuria: thiols

Thiols (tiopronin, also called 2-mercaptopropionylglycine, and D-penicillamine) break the disulphide bond of cystine and form with cysteine a mixed disulphide up to 50 times more soluble; they reduce free cystine but not total excretion [4, 10, 47]. They are indicated when stones recur despite good adherence to the preceding measures, or after three months of ineffective conservative treatment [4, 10]. The French protocol reserves them for second line and asks that, before starting them in the face of a recurrence, dietary intake and adherence to general measures be reassessed [32]. The EAU recommends tiopronin when cystine exceeds 3 mmol/day (720 mg/day) or when other measures are insufficient [37]. Efficacy is based on non-randomised retrospective studies, and most patients do not reach the cystine target [10, 47]. The two drugs appear to have similar efficacy; the largest comparative study, cited by the American consensus, found fewer adverse effects with tiopronin, and the 2014 American Urological Association guideline recommended it as first line, but a more recent French study, cited by the European recommendation, found a similar incidence of adverse effects [4, 47].

The European recommendation's tiopronin doses in adults (800–1500 mg/day) are higher than those of GeneReviews (600–800 mg/day), although the latter states that it follows the same recommendation; D-penicillamine label doses (up to 4 g/day in adults in the USA and 2.1 g/day in adults in Portugal) exceed the 1.2 g/day maximum of the European recommendation [4, 10, 77, 82]. In adults, the French protocol also exceeds the European D-penicillamine ceiling, with 1.8 g/day, and allows up to 2 g/day of tiopronin, above the 1500 mg/day of the European recommendation; the reference dose is that of the European recommendation, and the French and label maxima serve for comparison [4, 32]. Tiopronin doses above 1 g/day did not further reduce free cystine in one study [10]. The dose is adjusted to keep cystine below 250 mg/L (FDA tiopronin label) or, where the assay exists, free cystine below 100 µmol/mmol creatinine (European recommendation), or to obtain a positive "capacity" above 50 mg/L in the US commercial assay (American consensus) [4, 47, 81]. The D-penicillamine label indicates limiting cystine to 100–200 mg/day in patients without stones and below 100 mg/day in patients with stones or pain [77].

Adverse effects and monitoring. Both thiols may cause taste disturbance (through chelation of trace elements), mucocutaneous lesions, rash, arthritis, immune-mediated diseases, proteinuria up to nephrotic syndrome due to immune-complex membranous glomerulopathy, vitamin B6 deficiency, neutropenia, thrombocytopenia or aplastic anaemia, gastrointestinal intolerance and hepatotoxicity [4, 10, 81]. D-penicillamine has been associated with rapidly progressive glomerulonephritis; tiopronin rarely with minimal change glomerulopathy [10]. In the uncontrolled trial underpinning the tiopronin label (66 patients aged 9 to 68 years), 34 of 49 patients had stopped D-penicillamine because of toxicity and 22 of these continued on tiopronin; among the 17 without previous treatment, 6% stopped tiopronin because of serious reactions; the most frequent effects were nausea (25% and 12% in the two groups), diarrhoea (18% and 6%), oral ulcers (12% and 18%), rash (14% and 12%), fatigue (14% and 0%) and proteinuria (10% and 6%) [81]. Pyridoxine is recommended, 50 mg/day according to GeneReviews, the American consensus and the Brazilian guideline (25 mg/day according to the Kelatine® SmPC) [10, 47, 76, 82]; the French protocol, less categorical, states only that reduced vitamin B6 activity due to D-penicillamine may require supplementation [32]. The same protocol adds pulmonary, immunological and allergic reactions and dysgeusia to the list of adverse effects, recalls that thiols chelate copper and zinc and may cause deficiency of these trace elements, without recommending measurement of serum copper or zinc during treatment, and requires contraception in women of childbearing potential, because of the teratogenic effect [32].

Monitoring includes regular full blood count and proteinuria according to the European recommendation [4]; the American consensus recommends full biochemistry and urine protein/creatinine ratio at baseline, 3–6 months after starting and then every 6–12 months, annual vitamin B6 with penicillamine, and withdrawal in the presence of significant proteinuria [47]. The tiopronin label asks for urinary cystine one month after starting and then every three months and proteinuria before starting and every 3–6 months, with withdrawal if proteinuria appears and possible reintroduction at a lower dose after resolution; the Thiola EC® label no longer requires full blood count or liver function tests [47, 81]. The Kelatine® SmPC asks for monitoring every two weeks in the first six months and then every two months (urine, leucocytes with differential, haemoglobin, platelets), withdrawal with leucocytes below 3.5 × 10^9/L or platelets below 100 × 10^9/L, liver function every six months and annual renal radiography in cystinuria, and contraindicates combination with gold salts and antimalarials [82]. The French protocol asks for a tighter initial schedule than the European and American documents: full blood count and protein/creatinine ratio on a spot urine sample every week with D-penicillamine and every 15 days with tiopronin in the first months, then twice a month with D-penicillamine and monthly with tiopronin until the end of the first year, and at longer intervals thereafter, to which it adds liver function tests [32]. Thiols accumulate in renal impairment: the European recommendation advises reducing or stopping with an eGFR of 60–89 mL/min/1.73 m2 and always stopping below that [4].

Captopril. Captopril, an angiotensin-converting enzyme inhibitor with a thiol group, has no reproducible efficacy in preventing stones in adults and is not recommended routinely (the French protocol no longer recommends it) [32]; at doses of 0.3–1.5 mg/kg/day it may be used to treat hypertension in patients with cystinuria or in children who cannot take tiopronin capsules [34, 47].

7.13 Cystinuria: regulatory status

In France, according to the 2019 protocol, the only marketed thiol was D-penicillamine (Trolovol®, 300 mg tablets), with paediatric authorisation above 6 years of age; tiopronin was no longer marketed and the medicines agency (ANSM) temporarily authorised its importation as Thiola® and Captimer®, the latter dispensed only by hospital pharmacies once the Thiola® stock was exhausted [32]. Potassium citrate is available there as an extemporaneous preparation (powder to dilute or capsules, sometimes refused reimbursement), as a non-reimbursed food supplement (Lithosolv®, 1 g tablets), as Urocit-K® through a nominative temporary authorisation for use, and in combinations with sodium citrate (Foncitril®, the least recommended form, and Alcaphor®, with trometamol); in pregnancy the pharmacy preparation is preferred to supplements, because they contain unassessed excipients [32]. Cystinuria is recognised in France as a long-term condition (ALD 30, hereditary metabolic diseases), which guarantees coverage of care [32].

7.14 Cystinuria: colic and surgery

For colic, the European recommendation gives a non-steroidal anti-inflammatory drug as first choice, in the absence of contraindications, and urgent decompression by ureteric catheter or nephrostomy in infected or obstructed kidneys; there is no specific evidence for medical expulsive therapy in cystinuria [4]. In stone disease in general, spontaneous passage occurs in 89% of stones below 5 mm, 49% of those of 5–7 mm and 29% of those larger than 7 mm (52% in the proximal ureter and 83% in the distal ureter), and patients with cystinuria are sometimes able to pass larger stones [4].

The two European sources diverge mainly on lithotripsy: the European recommendation only allows it to be offered, whereas the French protocol places it as first line in children, citing the pliability of the urinary tract, which lets large fragments pass, and the experience of paediatric centres that consider it more effective than in adults, with the caveat that the literature on the subject is scarce [4, 32]. The French protocol stresses that the surgical indications are those of common stone disease, but that cystine stones recur and grow faster, which exposes patients to multiple interventions over their lifetime; complete extraction is a priority, because any residual fragment acts as a matrix for a new stone, and in children repeated interventions may compromise renal function, so the least invasive ones are preferred, stones are sought while they are small and X-ray exposure is limited [32]. In renal colic, non-steroidal anti-inflammatory drugs are the most widely used analgesics, with caution when renal function deteriorates [32]. All surgery should be accompanied by preventive treatment [34]. Recurrent urinary tract infections are treated according to the antibiogram; chronic kidney disease and hypertension are treated according to the general rules, because hypertension contributes to renal progression [10].

7.15 Cystinuria: pregnancy

Absence of stones should be checked before pregnancy, preferably with the patient stone-free, because pregnancy increases stone risk through anatomical factors and hypercalciuria [4, 10]. Thiols are contraindicated in pregnancy according to the European recommendation, GeneReviews and the US D-penicillamine label; the Portuguese Kelatine® SmPC allows it to be continued "in some difficult cases of cystinuria", with a fetal risk of connective tissue changes at high doses [4, 10, 77, 82]. The tiopronin label identifies no risk in case reports or in animal studies up to twice the human dose of 2 g/day, but advises against breastfeeding [81]. Alkalinisation is considered safe and should be continued if necessary [10]. The French protocol requires D-penicillamine and tiopronin to be stopped in pregnancy, asks for urine output to be optimised, because of physiological hypercalciuria and the anatomical changes of gestation, and recommends continuing potassium citrate: published data and the experience of the French reference centre on teratogenic agents (CRAT) show no increase in malformation risk with first-trimester exposure, and abstention exposes to an increased risk of stones, with an increased risk of preterm delivery, and of loss of renal function [32]. The French position therefore diverges from the Kelatine® SmPC, which allows D-penicillamine to be continued in difficult cases, and coincides with the European recommendation [4, 32, 82].

7.16 Cystinuria: trials, developments and research

The evidence for emerging therapies is, for now, preclinical or of very low quality. Alpha-lipoic acid reduced stone formation in mice and in a few human reports, and the completed randomised trial had not yet been published in a peer-reviewed journal in searches up to 31/10/2026 [10, 85]. An observational series of ten patients treated off-label with dapagliflozin described fewer stone events over a median of 13.5 months, but the small four-week trial showed no effect on urinary cystine and the long-term trial was abandoned [10, 86]; the 2026 narrative review considers sodium-glucose cotransporter 2 inhibitors off-label and without a basis for recommendation outside research [87]. Tolvaptan improved cystine capacity in a pilot study, but cost and hepatic monitoring limit it to highly selected cases [10, 87]. Bucillamine, a dithiol, had a phase 2 trial without published results [87]. L-cystine crystallisation inhibitors (L-cystine diamides, such as L-cystine bis(N-methylpiperazide), with European orphan designation since 2018) distort hexagonal crystals in vitro and reduce stones in animal models, without validated clinical use [4, 10, 70]. L-ergothioneine has had European orphan designation for cystinuria since 2021 [70]. Gene therapy with adeno-associated viral vectors and nanoparticles, and genome editing, are at the preclinical stage [10]. None of these interventions is authorised for cystinuria. In 2019, the French protocol mentioned an ongoing study of a new long-acting alkalinising agent in cystinuria (code B13); the registered phase 2/3 trials of ADV7103, a prolonged-release alkalinising agent, were later withdrawn by business decision [32, 84].

7.17 PREPL deficiency: targeted treatment

There is no gene-targeted therapy or authorised drug for PREPL deficiency, and there are no consensus guidelines specific to the congenital myasthenic syndromes; treatment has two mechanism-directed axes, acetylcholinesterase inhibition for the neuromuscular transmission defect and hormone replacement for growth hormone deficiency, plus treatment of cystinuria when SLC3A1 is co-deleted [6, 25]. All pyridostigmine doses are extrapolated from the label, whose only indication is myasthenia gravis, because no source publishes a dosage specific to this condition; the extrapolation is declared here and should be declared to the patient [88, 89].

Pyridostigmine (Mestinon® and generics) is a reversible acetylcholinesterase inhibitor that prolongs the presence of acetylcholine in the synaptic cleft and partly compensates for the reduction in quantal content. The evidence comes from small series and is qualified. In the 2014 prospective assessment, the infant girl with isolated deficiency responded markedly to edrophonium and pyridostigmine at 11 weeks of life, and of the three patients with hypotonia-cystinuria syndrome treated, aged one, two and twelve years, only the infant improved, with a marked change on the Alberta Infant Motor Scale within one day; the two older patients, assessed with the six-minute walk test, hand-held myometry, maximal inspiratory and expiratory pressures and video recording of ptosis and speech, did not respond [8]. In the two infants who responded, withdrawal of the drug after nine and two months of treatment did not cause worsening, which the authors interpreted as spontaneous improvement of the myasthenia with age; the index patient stopped pyridostigmine at 12 months without deterioration, while still having weakness [8, 29]. GeneReviews translates this body of evidence into the statement that acetylcholinesterase inhibitors are "possibly beneficial in infancy" in this form, with a frequency of less than 1% of congenital myasthenic syndromes [25]. The 2026 retrospective cohort is more favourable: the five children received pyridostigmine from a median age of six months (range three to fourteen months) and four of the five had significant improvement in motor development [31]. The practical consequence is to carry out an early therapeutic trial, with outcomes defined before starting, and to formally reassess the response rather than continue the drug by inertia; the decision to stop should be tested, because improvement may be age-related and not pharmacological [8, 25].

Growth hormone deficiency is the second targeted therapy and the one with the most consistent result. The 2018 review states that growth hormone therapy restores normal growth in these patients, and individual reports confirm it with figures: in the Barcelona patient, height was 72 cm at 20 months (−3.8 standard deviations), with IGF-1 of 27 ng/mL, binding protein 3 of 1.78 mg/L, peak growth hormone of 3.22 ng/mL on the glucagon test and bone age of nine months at a chronological age of fourteen months; with somatropin started at two years and eight months, growth velocity rose from 7 to 11 cm per year and at seven years and five months height was 116.5 cm (−1.8 standard deviations) [6, 38]. In the 2018 series, age at onset of the deficiency ranged from 20 months to 11 years, which requires growth to be monitored throughout childhood and not only in the first years; a patient with hypotonia-cystinuria started somatropin at 11 years, when growth velocity fell, and received oestrogens at 14 years for delayed puberty with hypergonadotropic hypogonadism [6]. In one of the siblings with a classic deletion, somatropin started at 12 years, with height below the 5th percentile, brought him to the 17th percentile five years later [63]. The decision to treat follows the usual criteria for growth hormone deficiency, with a stimulation test and exclusion of coeliac disease and hypothyroidism, and there is no dose specific to this condition: the label dose is used, with the extrapolation declared [38, 90]. IGF-1 is a useful early marker, because in the 2026 cohort its fall preceded the endocrine diagnosis, but it may also normalise without treatment, as in one patient in whom the slight fall corrected spontaneously, so an isolated value does not indicate treatment [30, 31]. The mechanism of the deficiency remains unknown; the hypothesis is a role of PREPL in the secretion or processing of peptide hormones, and the good response to exogenous administration shows that signalling downstream of the receptor is intact [38, 60].

The adjuvant drugs used in other congenital myasthenic syndromes may be considered, with the specific rules for each subtype and always off-label. Salbutamol syrup was used with benefit in combination with pyridostigmine in a child with missense variants, who at four years of age ran, walked and got up from the floor normally, with fatigue only after intense exertion, and the 2026 review of the presynaptic forms notes that patients in this subgroup may benefit from drugs that improve neuromuscular transmission, such as pyridostigmine and salbutamol [30, 57]. Amifampridine (3,4-diaminopyridine) requires caution in young children, after the deaths of two children with fast-channel syndrome who started it, and there are no data supporting it in this form [25]. The finding that coenzyme Q10 corrects mitochondrial dysfunction in PREPL-deficient cell lines is preclinical and does not justify clinical use [60].

The rest of treatment is supportive and multidisciplinary, and it is what determines the outcome in the first years: nasogastric tube feeding and, when necessary, gastrostomy, which in one patient was removed only at six and a half years; physiotherapy, occupational therapy and speech therapy; ventilatory support and sleep study, with continuous or bilevel positive airway pressure when there is obstructive apnoea; and monitoring of the risk of respiratory decompensation with fever and intercurrent infection, with a written plan and training of parents in resuscitation [6, 25, 30]. In the contiguous deletion forms involving SLC3A1, complete treatment of cystinuria described in sections 7.9 to 7.14 is added, under nephrological supervision [4, 25]. None of these entities has a drug with marketing authorisation in the United States or the European Union for the indication, and the two studies registered on ClinicalTrials.gov are stalled.

The primary outcome of the sulfamethoxazole trial was the change in ptosis on the myasthenia gravis composite scale and photographic indices of ptosis, lip length and lip contraction, comparing the third week of treatment with baseline and with the value one week after withdrawal; exclusion criteria included hypersensitivity to sulphonamides and reduced renal function [91]. Without published results, sulfamethoxazole has no place in clinical practice for this condition, and the absence of publication is equivalent neither to absence of effect nor to proof of safety [91].

8. Reproductive Options and Family Counselling

8.1 HHH syndrome

HHH syndrome has autosomal recessive inheritance [1]. The parents of a patient are obligate heterozygotes, and molecular testing of the parents confirms phase and allows a reliable risk calculation; no de novo variant has been described, although these occur at low frequency in recessive diseases [1]. Each sibling of a patient has, at conception, a 25% chance of being affected, 50% of being an asymptomatic carrier and 25% of inheriting neither variant; the children of a patient are obligate carriers, and each uncle or aunt has a 50% chance of being a carrier [1]. Carriers have no symptoms or biochemical abnormalities, so only molecular testing identifies them [1, 51].

Counselling has to explain that genotype does not predict the course: siblings with the same variants may have very different phenotypes, from almost asymptomatic to severely affected [1, 53]. At-risk siblings should be tested; if there was no prenatal diagnosis, the at-risk newborn has targeted testing for the familial variants while kept on a diet with protein limited to 1.2 g/kg/day or less (breastfeeding and protein-free formula), with monitoring of ammonia, plasma amino acids and urinary orotic acid [1]. Once the familial variants are known, prenatal diagnosis and preimplantation genetic testing are possible [1, 35]. The ideal time for these discussions is before pregnancy, and genetic counselling should be offered to young adults who are affected, carriers or at risk of being carriers [1]. In founder populations with a very high carrier frequency, such as the communities of northern Saskatchewan (1 in 19), newborn screening by genotyping is an option that has already been applied [27]. In cases without a molecular diagnosis, storage of the proband's DNA should be considered [1].

8.2 Cystinuria

Counselling in cystinuria depends on the genotype, because three inheritance patterns coexist [3, 10].

Siblings of a patient should be screened with a 24-hour urine or an amino acid/creatinine ratio on a spot sample; if cystine is raised, targeted molecular testing for the familial variants confirms it, and asymptomatic cystinuria above 80 mg/24 h justifies renal ultrasound [4, 10, 47]. The French protocol gives an age rule for screening siblings: a normal 24-hour urinary cystine, or cystine/creatinine ratio, after 4 years of age excludes the disease, whereas a raised value before 4 years requires specialist advice, because it does not always reflect cystinuria [32]. In type B cystinuria, all children of a patient are SLC7A9 heterozygotes; the family enquiry should inform heterozygotes (parents, children and more distant relatives) of a theoretically increased stone risk, without firm epidemiological proof, and offer them a consultation to measure cystine excretion, assess the risk and institute preventive measures; because genetic testing is not systematic in France, screening of descendants is by cystine/creatinine ratio [32]. Consanguinity increases the probability of a homozygous child [32].

Disclosure of the diagnosis, according to the French protocol, takes place after confirmation (markedly raised urinary cystine, cystine stone by spectrophotometry or positive crystalluria), in a dedicated consultation, in a quiet place, by a doctor experienced in the disease, in the presence of a relative if the adult so wishes, or of both parents and the child in a paediatric diagnosis; information is given progressively and in several stages, on transmission, mechanisms, dietary, medical and surgical treatment and possible complications (obstruction, increased risk of hypertension and chronic kidney disease, a very low but non-zero risk of end-stage renal failure) [32]. In the same consultation the care network is organised (urologist, nephrologist, dietitian, geneticist, social worker, psychologist, school doctor and nurse) and the contacts of patient associations are provided; any patient diagnosed from stone analysis in urology should have regular nephrological follow-up [32]. Carrier testing should be considered in the reproductive partners of patients or carriers, especially in populations with founder variants (Ashkenazi and Libyan Jews, Mennonites) [10]. The European recommendation points out that, in a recessive disease, the risk of transmission to affected children is very low, unless the other parent carries a pathogenic variant in the same gene [4]. Molecular prenatal diagnosis and preimplantation testing are possible when the variants have been identified; a fetal hyperechogenic colon is a suggestive ultrasound sign [4, 10]. The decision to use these techniques in a treatable disease with mainly renal morbidity is personal and should be discussed non-directively [10].

In the 2p21 deletion syndromes, inheritance is autosomal recessive; the parents are heterozygous for the deletion, and the recurrence risk is 25% [5, 13].

8.3 PREPL deficiency

Isolated PREPL deficiency is autosomal recessive: the parents are obligate carriers, the recurrence risk is 25% in each pregnancy, 50% of unaffected siblings are carriers and the proband's children are obligate carriers, unless the partner carries a variant in the same gene [6, 7]. Loss of a single copy is not pathogenic: ClinGen assigned PREPL the haploinsufficiency score reserved for genes associated with recessive phenotypes and no evidence of triplosensitivity, which allows reassurance of parents who carry a 2p21 deletion, a frequent situation in these families [30].

Three situations alter the calculation and should be checked before giving figures. The first is compound heterozygosity for different mechanisms: the typical pattern is a point variant on one allele and a deletion on the other, so testing both parents is mandatory to confirm phase and to guide cascade testing, which has to include both methods [8, 38]. The second is uniparental disomy: in the infant girl with complete maternal uniparental disomy of chromosome 2, the mother was heterozygous and the father was not a carrier, and the recurrence risk for the couple is not that of a classic recessive disease, because the second allele did not come from the father; this scenario can be identified only by testing the trio, and apparent homozygosity with a non-carrier parent should raise suspicion of it [14]. The third is intrafamilial heterogeneity of expression: siblings with the same deletion had divergent courses, one dominated by stone disease and the other by neurobehavioural disturbances, so counselling cannot promise an affected sibling the course of the other [63].

Prenatal diagnosis and preimplantation genetic testing are possible when the familial variants have been identified, with the caveat that prenatal detection of a deletion requires a copy-number-sensitive method and that of a point variant a targeted analysis; counselling should set out the real spectrum of severity, which ranges from normal intelligence with residual fatigue to mild intellectual disability with prolonged dependence on assisted feeding, and the fact that the treatable manifestations are precisely the most limiting in the first years [6, 30]. In asymptomatic at-risk siblings, early assessment is recommended, as in the other congenital myasthenic syndromes, to start treatment and preventive measures before a respiratory decompensation [25]. In the contiguous deletion forms, the cystinuria counselling described in section 8.2 is added, and in the forms involving CAMKMT or PPM1B the neurological prognosis and mortality are worse, which changes the content of the conversation [6, 58].

9. Special Considerations: Population Diversity and Epidemiology

9.1 HHH syndrome

HHH syndrome is panethnic, but published cases are concentrated in Canada, Italy and Japan, which together account for more than half: 25 Canadian patients (23%), 18 Italian (17%) and 14 Japanese (13%) in a review of 111 [9]. There is no direct population incidence; the study by Summar and colleagues, based on two North American registries and the European E-IMD registry, estimated that HHH represents 1% of UCDs in the United States and 3% in Europe and has a frequency below 1:2,000,000 live births in the United States, the overall incidence of UCDs being at least 1:35,000–1:51,946 [1, 24, 26]. The 2015 review gives a proportion of 1–3.8% of UCDs [9]. Orphanet classifies the prevalence as below 1/1,000,000 in Europe and at birth in the United States, and records 111 published cases [2]. These figures measure diagnosed and reported cases; the UCD overview notes that undiagnosed defects may make the true frequency much higher, which is plausible in HHH given the frequency of mild presentations and late diagnoses [9, 24].

Founder populations are the main epidemiological data. In Quebec, the p.Phe188del variant accounts for most cases (15 of 16 homozygous patients) [53]. In northern Saskatchewan, an isolated population of French-Canadian and Aboriginal ancestry (94% Aboriginal, according to Nyhan's Atlas), genotyping of all newborns in an at-risk area for seven years estimated a heterozygote frequency of about 1 in 19 and an incidence of 1:1550 live births (about one affected child every 12 years), probably the highest in the world [27, 44]. The p.Arg179Ter variant is frequent in Japanese and Middle Eastern patients; in a Japanese national survey of adults with UCDs, seven with HHH were identified [1, 54]. The sources consulted contain no data from Portugal or Brazil.

9.2 Cystinuria

The average prevalence of cystinuria is about 1:7000 births, with wide variation: 1:2500 in Jews of Libyan origin in Israel and 1:100,000 in Sweden [3, 10, 34]. The American consensus uses the same estimate of 1:7000 live births in the United States [47]. Orphanet records birth prevalence estimates of 50/100,000 in the United Kingdom, 25/100,000 in Australia, 8/100,000 in Canada, 5.9/100,000 in the United States and 5.5/100,000 in Japan, a point prevalence of 5/100,000 in Europe (EMA data from 2018) and 14/100,000 worldwide [19]. These estimates have different origins (publications, expert opinions and EMA data) and are not directly comparable [19]; many patients are not diagnosed because they do not form stones or form them rarely [34].

In 2026, a population genetics study calculated the expected prevalence from the frequencies of pathogenic variants in the 1000 Genomes project: allele frequency of 0.64% for SLC3A1 (1 carrier in 79) and 0.40% for SLC7A9 (1 in 126), with 1 carrier in 48 and 1 affected in 17,612 (95% confidence interval 1:39,505 to 1:11,332) when the two genes are combined; this value was 1.74 times higher than that calculated in 2016 because of the variants discovered in the meantime, but remains below the clinical prevalence of 1:7000, which suggests genes, non-coding variants or modifiers yet to be identified [28].

Cystinuria accounts for 1–2% of stones in adults and 6–8% in children [10, 37]; in two large French studies, 1% of all stones and 4–5% of paediatric stones [4]. The French HAS protocol (2019) adopts the prevalence of 1:7000 with wide geographical variation and gives less than 1% of adult stones and 3–4% of paediatric stones; in the French cohort of 112 patients, type A predominated (74 of 99 genotyped, compared with 22 with type B), and large rearrangements accounted for 18% of mutated alleles [32]. The known founder variants are NM_000341.4(SLC3A1):c.808C>T p.(Arg270Ter) in Ashkenazi Jews, NM_014270.5(SLC7A9):c.508G>A p.(Val170Met) in Libyan Jews and NM_000341.4(SLC3A1):c.1136+2T>C and NM_014270.5(SLC7A9):c.1166C>T p.(Thr389Met) in Mennonite communities (Weaverland and Groffdale) [10, 49]. The sources consulted contain no epidemiological data from Portugal or Brazil; in Brazil, tiopronin is not available and D-penicillamine (Cuprimine®) is the thiol used [76].

9.3 2p21 deletion syndromes

Orphanet records 22 patients with hypotonia-cystinuria syndrome, two with the atypical form and seven with 2p21 microdeletion syndrome [13, 20, 21]. Jaeken's first patients were from Flemish and French families, and 2p21 deletion syndrome was described in a Bedouin family [5]. The 2p21 microdeletion without cystinuria, which spares SLC3A1, was described in a Swiss family and Orphanet classifies its prevalence as below 1 in 1,000,000 [23, 58]. The 2021 review cited by Towheed counted 26 cases of hypotonia-cystinuria syndrome resulting from eight different deletions, and hypotonia-cystinuria syndrome had been described in 19 families up to 2018 [6, 63].

Isolated PREPL deficiency is rarer and more dispersed. It represents less than 1% of congenital myasthenic syndromes, a proportion within a rare group and not a population rate, and Orphanet does not give it its own entry: it classifies it in the subtype of congenital myasthenic syndromes due to defects of acetylcholine synthesis or recycling, without a prevalence estimate [22, 25]. The published patients come from Belgium, Sweden, the Netherlands, Italy, Spain, the United States, South Korea and China: the 2018 series brought together ten patients from Belgian, Swedish, Dutch, Italian and Spanish centres; the three patients with missense variants were identified in the United States and South Korea; the 2026 cohort brought together five Chinese children; and there are cases described in Russia and China with distinct molecular mechanisms [6, 14, 30, 31, 59, 64]. In 2026 a recurrent frameshift variant, NM_006036.4:c.1282_1285del p.(Phe428ArgfsTer18) in the article's numbering, = MANE NM_001171613.2:c.1015_1018del p.(Phe339ArgfsTer18) [12], was detected in two unrelated Chinese patients, which suggests mutational recurrence rather than a founder effect [31]. The sources consulted contain no data from Portugal or Brazil on this entity, and the authors of the 2018 series acknowledge that most patients remain undiagnosed because the condition is not recognised [6].

9.4 Special populations and transition

In childhood, cystinuria requires hydration and alkalinisation to be adjusted to body surface area, the protein intake necessary for growth to be maintained, ultrasound to be preferred for surveillance and miniaturised surgical techniques to be used [4, 47]. In HHH syndrome, protein requirements and surveillance intervals change with age, and adolescence brings the risk of abandoning the diet and of sports protein supplements [1]. Pregnancy is a risk situation in both diseases (sections 7.7 and 7.15). Transition to adult care should preserve the metabolic diseases team in HHH and nephrological and urological follow-up in cystinuria. The French protocol asks, for the child with cystinuria, for a transition process prepared well in advance to a multidisciplinary adult team trained in the disease and coordinated by a referring doctor, usually the nephrologist, and for the involvement of the school doctor, with an individualised reception plan (PAI) that adapts meals and access to water at school [32]. Therapeutic education is central, because the main difficulty of treatment is long-term adherence to alkaline hyperdiuresis, which is very demanding at the start: the patient learns to measure urinary pH and specific gravity and to record them in a notebook to adjust doses, and the programme, guided by a shared educational diagnosis, also includes workshops on stress and pain management, acceptance of the disease and occupational integration, together with national patient days; patients with a single functioning kidney should carry a card stating this [32]. In PREPL deficiency, transition must ensure continuity of neurology, because the residual signs (ptosis, nasal dysarthria, facial and neck flexor weakness, fatigue) persist into adulthood, and of endocrinology, for the decision to continue or stop somatropin at the end of growth and for the hypergonadotropic hypogonadism, which requires hormone replacement in adolescence [6, 38].

9.5 European networks and patient associations

MetabERN, the European Reference Network for hereditary metabolic disorders, includes the amino acid and organic acid disorders subnetwork, within which the UCDs fall [39]. ERKNet, the European Reference Network for rare kidney diseases, has a metabolic disease and stone group and maintains the European cystinuria registry EUROCYS; the 2021 recommendation was prepared jointly with eUROGEN, the European Reference Network for rare and complex urogenital diseases [4, 10, 40]. For HHH, GeneReviews lists the National Urea Cycle Disorders Foundation (nucdf.org) and its international registry, the UCD Foundation (Connecting Families), the Urea Cycle Disorders Consortium of the North American rare diseases network, Metabolic Support UK and the European registry for intoxication-type metabolic diseases [1]. For cystinuria, the International Cystinuria Foundation (cystinuria.org), which promoted the American consensus, and the Rare Kidney Stone Consortium, with a registry and biobank at the Mayo Clinic [10, 47]. In France, cystinuria is followed within the ORKiD rare kidney disease health network, with three reference centres (MARHEA at Hôpital Necker-Enfants Malades, in Paris; Néphrogones at Hôpital Femme Mère Enfant of CHU de Lyon, in Bron; SORARE at CHU de Toulouse) and a network of competence centres; the national protocol also lists about sixty public and private laboratories that study crystalluria, distributed across all regions, and the Maladies Rares Info Services telephone service [32]. The French patient associations are AIRG-France (Association pour l'Information et la Recherche sur les maladies Rénales Génétiques, airg-france.fr and cystinurie.com), which publishes a book on the disease and organises information days, LUNNE (Lithiases UriNaires Network, a mixed association of patients and professionals dedicated to preventing recurrence and to therapeutic education tools) and France Rein [32]. For PREPL deficiency, the relevant reference network is EURO-NMD, the European Reference Network for rare neuromuscular diseases, which maintains a registry with data specific to congenital myasthenic syndromes, with associated endocrine follow-up and, in the contiguous deletion forms, nephrological follow-up; Orphanet lists for the 2p21 microdeletion without cystinuria 189 reference centres, 11 networks of centres, 106 patient associations and 26 registries [23, 41].

10. Illustrative Clinical Case

10.1 Late-diagnosed HHH syndrome in an adult

Silfverberg and colleagues described, in 2018, a 48-year-old Caucasian man in Sweden, admitted to the emergency department with headache, low back pain, a sensation of pressure in the ears and vomiting during the night, with progressive alteration of consciousness [55]. He had a history of depression and reading and writing difficulties, recurrent seizures with vomiting and loss of consciousness between one and eight years of age, interpreted as febrile seizures, a peptic ulcer ten years earlier and periods in which he had excluded protein from his diet on his own initiative. In the emergency department, the Glasgow coma scale fell from 11 to 8 in 15 minutes; he was afebrile, agitated, with slight miosis, saccadic eye movements and bilateral Babinski sign. Routine tests, including liver function tests and toxicology screening, and brain computed tomography were normal; arterial pH was 7.5. Twelve hours later he had haematemesis, was intubated and transferred to intensive care, received antibiotics, antivirals and betamethasone, and magnetic resonance imaging was normal [55].

At 24 hours, his sister suggested a UCD to the team, after an internet search with her brother's symptoms. Ammonia was 213 µmol/L (reference 11–32), and continuous haemodiafiltration was started [55]. At 48 hours, upper gastrointestinal bleeding from a peptic ulcer required several endoscopic haemostatic procedures and 25 units of packed red cells, 20 of plasma and 3 of platelets, with an estimated loss of 10 litres of blood; ammonia rose to 497 µmol/L and partial seizures appeared. He received sodium benzoate as a loading dose of 250 mg/kg over 120 minutes and maintenance of 250 mg/kg/day, with carnitine and arginine, intravenous glucose and lipids (115% of requirements) and no protein. On the third day, plasma ornithine was 420 µmol/L, urinary homocitrulline 23 mmol/mol creatinine, glutamine 1321 µmol/L and urinary orotic acid 156 mmol/mol creatinine, with normal citrulline, arginine and lysine, and ammonia normalised by the end of that day [55].

On the fourth day, with ammonia already normal, he became deeply unconscious, with stereotyped extension of the limbs; tomography showed generalised cerebral oedema, with intracranial hypertension and seizures treated with sedation and levetiracetam. Magnetic resonance imaging on the fifth day showed symmetrical cytotoxic oedema of the cortex and subcortical white matter, most marked in the insula, cingulate gyrus and hippocampus, a pattern considered typical of hyperammonaemic encephalopathy; that on day 31, regression of the oedema with frontotemporal gliosis and haemorrhagic necrosis; and that at seven months, marked atrophy, gliosis and enlarged ventricles [55]. The images are in figure 2 of the article (https://pmc.ncbi.nlm.nih.gov/articles/PMC6151189/figure/Fig2/), and the course of ammonia and haemoglobin in figure 1 (https://pmc.ncbi.nlm.nih.gov/articles/PMC6151189/figure/Fig1/). SLC25A15 sequencing identified c.337G>A p.(Gly113Ser) and c.712C>T p.(Gln238Ter) in trans, confirmed by segregation (the mother and daughter carried the nonsense variant and the sister the missense variant) [12, 55].

The patient remained in hospital for ten months, with aphasia, perceptual deficits, progressive spastic paraparesis and dystonia. He was discharged on sodium benzoate, citrulline, arginine, essential amino acids and protein of 0.7 (0.6–0.8) g/kg/day; at 15 months he ate unaided and moved his wheelchair with his legs, and ammonia remained normal or slightly raised during intercurrent infections [55]. The case illustrates the typical late presentation (self-restriction of protein and unrecognised symptoms in childhood), a particularly intense catabolic and protein trigger (the gastrointestinal bleeding), the 24-hour delay until ammonia was measured and the cerebral oedema that may develop after ammonia has normalised. The use of corticosteroids at the start, before diagnosis, is contrary to the recommendation to avoid them in UCDs [1].

10.2 HHH syndrome with acute liver failure in a child

Mosher and colleagues described, in 2026, a previously healthy 26-month-old girl with lethargy, food refusal and vomiting [56]. The initial transaminases (AST 125 U/L, ALT 255 U/L) were attributed to a common coronavirus and Epstein-Barr virus infection; after rehydration, she worsened, with AST of 4323 U/L, ALT of 3830 U/L, INR of 3.1 and ammonia of 219 µmol/L, and was transferred to intensive care with a diagnosis of paediatric acute liver failure. Liver biopsy showed scattered hepatocyte necrosis without significant inflammation, glycogenated nuclei without zonal distribution and irregular perisinusoidal fibrosis (figure 1, https://pmc.ncbi.nlm.nih.gov/articles/PMC13403024/figure/jpr370206-fig-0001/). With intravenous glucose, rifaximin and lactulose, ammonia fell to 65 µmol/L on the third day, with a peak of 182 on the fourth, without the need for dialysis (figure 2, https://pmc.ncbi.nlm.nih.gov/articles/PMC13403024/figure/jpr370206-fig-0002/). On the eighth day, plasma ornithine of 495 nmol/mL (10–163), plasma and urinary homocitrulline and orotic and lactic aciduria led to the diagnosis; the family then reported that the child refused meat, eggs, cheese and milk, which made her vomit [56]. She was started on intravenous lipids, a low-protein diet, citrulline and glycerol phenylbutyrate, with clinical and laboratory improvement and discharge. Sequencing showed the SLC25A15 variant c.22C>T p.(Gln8Ter) in homozygosity; after discharge, she had mild neurodevelopmental delay and no further crises [12, 56]. The case shows the hepatic presentation of HHH, the usefulness of collecting plasma and urine amino acids at the onset of any paediatric acute liver failure and the value of the dietary history.

10.3 Cystinuria with a staghorn calculus in an adolescent girl

Schaefer and colleagues described, in 2025, an 11-year-old girl who attended the emergency department with right-sided colic and passed a stone two days later; she had had intermittent left loin pain since the age of 7 [92]. Non-contrast tomography showed a 3.5 cm staghorn calculus in the right kidney (figure 1B, https://pmc.ncbi.nlm.nih.gov/articles/PMC12452493/figure/reports-08-00163-f001/), and stone analysis revealed 100% cystine. She started potassium citrate, hydration and sodium restriction, but had recurrences with four endourological procedures, including percutaneous nephrolithotomy for stones larger than 2 cm. Genetic testing found a homozygous pathogenic variant in SLC7A9 (type B cystinuria), and urinary cystine was 658 mg/24 h (807 mg/1.73 m2/24 h) [92]. Tiopronin, recommended, was refused by the insurer; she started D-penicillamine, which the article describes variously as 250 mg once or twice daily and as 33 mg/kg/day in three doses, with pyridoxine. Cystine fell to the target, rose during an interruption of the drug on a trip and normalised again on reintroduction; during treatment she needed one further nephrolithotomy and two ureteric stents, without adverse effects [92]. The case illustrates the diagnostic delay in paediatric cystinuria (stone analysis and genetics came only after several interventions), the surgical burden of the disease and D-penicillamine as an alternative when tiopronin is not accessible, a situation that corresponds to that of Brazil and Portugal [76, 83].

10.4 Isolated PREPL deficiency with growth hormone deficiency

Laura Sayol-Torres and colleagues described, in 2023, a girl followed at Hospital Universitari Vall d'Hebron, in Barcelona, the daughter of healthy, non-consanguineous Caucasian parents, with a healthy younger brother [38]. She was born by caesarean section at 39 weeks for breech presentation, after a monitored and uneventful pregnancy, with a weight of 2855 g (−0.9 standard deviations), length of 47 cm (−1.5 standard deviations) and head circumference of 34.5 cm (+0.2 standard deviations). At birth a broad nasal bridge, micro-retrognathia, mild thenar hypoplasia and bilateral fifth-finger clinodactyly were noted, with neonatal hypotonia and poor responsiveness to stimuli. She had neonatal hypoglycaemia due to feeding difficulties and required a nasogastric tube during the first month. Infection, heart disease and toxic causes were excluded; metabolic investigation, electrocardiogram and brain magnetic resonance imaging were normal, there was no cystinuria, creatine kinase and thyroid hormones were normal and electromyography at four months was normal. Microarray and multiplex ligation-dependent probe amplification analysis for Prader-Willi syndrome showed no abnormalities [38].

At five months global hypotonia with axial predominance persisted, with apparent improvement over time; she stood alone at one year and began walking at 18 months, with motor rehabilitation in a specialised centre. At 20 months, height was 72 cm (−3.8 standard deviations), IGF-1 was low (27 ng/mL), as was binding protein 3 (1.78 mg/L); the glucagon test showed no response, with the maximum growth hormone peak at baseline (3.22 ng/mL) and 0.48 ng/mL at 60 minutes, and bone age was nine months at a chronological age of fourteen months. Coeliac disease and hypothyroidism were excluded, and hypothalamic–pituitary magnetic resonance imaging was normal. With a diagnosis of growth hormone deficiency, replacement was started at two years and eight months, with growth velocity rising from 7 to 11 cm per year [38].

The genetic diagnosis required three steps. A panel with the IGF2, IGF1R, IGF1, NPR2, GH1, GHR, GHRHR, IGFALS, STAT5B, CCDC8 and GHSR genes was normal, as was methylation analysis of the Silver-Russell syndrome region. The exome identified an apparently homozygous variant in PREPL, c.1528C>T p.(Arg510Ter) in long-transcript numbering, corresponding to NM_001171613.2:c.1261C>T p.(Arg421Ter) in MANE nomenclature, of paternal origin; a microarray with targeted exonic coverage of PREPL revealed a 0.031-megabase deletion at 2p21 restricted to the gene, of maternal origin [12, 38]. At seven years and five months, on continued somatropin (figure 1 of the article, https://pmc.ncbi.nlm.nih.gov/articles/PMC10234053/figure/f1/), she weighed 18 kg (−1.75 standard deviations), measured 116.5 cm (−1.8 standard deviations), was prepubertal, had left ptosis associated with fatigue, nasal voice, high-arched palate, epicanthus, retrognathism, ligamentous laxity and clinodactyly, with normal axial tone and reflexes and normal social and school development; body mass index was 13.2 kg/m², at the 8th percentile, without the obesity the literature describes at the end of the first decade [38].

The case shows that a negative result for Prader-Willi syndrome in a hypotonic infant does not close the investigation, that detection of the deletion depended on the exonic coverage of the microarray and that apparent homozygosity required testing of the parents. The approach in the report, in which genetic testing came only after an extensive endocrine panel, is not presented as routine: in an infant with neonatal hypotonia, feeding difficulties and ptosis, testing of PREPL with deletion analysis belongs in the first line [6, 38].

11. Prognosis and Long-Term Follow-Up

11.1 HHH syndrome: prognosis

Survival is high: 94% (109/116) in the GeneReviews series, and 7 deaths among 106 patients with known vital status (6.6%; median age 14 years, 1 month to 57 years) in the 2015 review; OMIM gives survival of about 95% after diagnosis and treatment [1, 9, 15]. Mortality is higher with neonatal onset, 15% (2/13) [1]. With treatment, lifespan is almost normal, patients become metabolically stable and rarely have further hyperammonaemia, and the hepatic signs regress [9, 51]. Neurological outcome is the determinant of prognosis: it ranges from mild involvement to severely disabling disease, and chronic therapy prevents hyperammonaemia and liver disease but does not appear to modify spastic paraparesis, which may progress despite normal ammonia for decades [9, 35, 61]. Neurodevelopmental outcome correlates, although not always, with the severity and duration of crises, and early diagnosis improves prognosis at all ages of onset [1]. The experience of the Rome team with patients stable for more than 20 years on citrulline and mild protein restriction shows that progression is not inevitable [51].

11.2 HHH syndrome: initial assessment and surveillance

After diagnosis, the following are recommended: anthropometry, neurological examination with a validated ataxia scale (SARA, ICARS or BARS) and assessment of spasticity, referral to physical medicine and rehabilitation, electroencephalogram if seizures are suspected, baseline magnetic resonance imaging and spectroscopy in patients with significant neurological deficit or after coma, developmental and language assessment, nutritional and swallowing assessment, screening for sleep disturbances, attention deficit hyperactivity disorder and anxiety after 12 months, liver biochemistry and ultrasound, and genetic counselling, social support and a metabolic dietitian [1].

The 2019 European guideline strongly recommends regular clinical, biochemical and nutritional surveillance by a multidisciplinary metabolic team, with an individual schedule (recommendation no. 26, expert opinion), and sets targets of ammonia below 80 µmol/L (with an upper limit of normal of 50 µmol/L), glutamine below 1000 µmol/L, arginine in the high-normal range and essential and branched-chain amino acids within normal limits (no. 27, high quality) [36]. It also recommends brain magnetic resonance imaging with spectroscopy even without neurological or cognitive deficit, with a case-by-case schedule (no. 28), assessment of IQ, development and neuropsychological strengths and weaknesses in all patients (no. 29) and psychological monitoring and support for patients and families (no. 30, high quality) [36]. The guideline cites liver ultrasound as especially indicated in HHH, considers blood urea and aminoaciduria of little value for surveillance and urinary orotic acid of doubtful value, and recalls the theoretical risk of osteoporosis with a low-protein diet, without studies in UCDs [36]. The GeneReviews intervals in the surveillance table (monthly in the first year, every 2 or 3 months up to 2 years and every 3 or 4 months thereafter) are compatible with the guideline's minimum frequency [1, 36].

11.3 Cystinuria: prognosis

Cystinuria is a lifelong disease, with recurrent stones, repeated urological interventions and progressive loss of renal function. About 20–25% of patients have an eGFR below 60 mL/min/1.73 m2 (26.8% in a French cohort of 314 adults, 17.8% in another series), hypertension affects 29–51% of adults, but end-stage kidney disease is relatively rare and cystinuria does not recur after kidney transplantation [4, 10]. Patients with active stone disease have lower quality of life than the general population, which improves with stone-free time; many consider medical treatment ineffective or poorly tolerated [4]. Adherence to treatment, including alkalinisation, has been associated with fewer urological interventions [4], and close follow-up favours this adherence [47]. The lower bone mineral density described in one study justifies assessing bone health in adults [10]. In the 2015 French cohort, 1.1% of patients were on dialysis, and the French protocol describes the risk of end-stage renal failure as very low but not zero [32].

11.4 Cystinuria: initial assessment and surveillance

The initial assessment includes 24-hour urine (volume, pH, cystine, sodium, citrate, urate, calcium and creatinine), renal and bladder ultrasound or non-contrast tomography to establish the stone burden, serum creatinine, albuminuria, blood pressure and genetic counselling [10, 47]. The European recommendation does not set the frequency: it varies from every three months to annually depending on stone activity, and is annual in patients with fewer than one event per year [4]. The French protocol proposes an explicit schedule: visits every 2–3 months at diagnosis or in severe disease (every 3 months, in the summary for the attending doctor) and every 6 months when targets are met and stone activity is low [32]. At each visit, colic, passage of stones or gravel, macroscopic haematuria and urinary tract infections, blood pressure, adherence to diet and drugs and their distribution over the 24-hour cycle, achievement of the pH and specific gravity targets recorded in the patient's notebook (measured, at the start, about four times a day until the alkalinising dose is right and, thereafter, at varied times, with emphasis on waking) and tolerance of treatment are assessed [32].

In biological surveillance, the French protocol gives the main role to crystalluria and not to urinary cystine [32]. The cystine crystal volume per cubic millimetre of urine (VCys), calculated from the number of crystals per mm3 and their mean size, measured in at least 20 consecutive crystals, ideally in the first morning urine (the most concentrated and most acidic), separates patients: kept always below 3000 µm3/mm3, it is associated with a very low risk of recurrence, and above this threshold recurrence exceeded 90% [32]. The test is not easily accessible in routine practice, but the protocol considers it the only indicator associated with the risk of recurrence, considers that cystine concentration alone does not predict it and asks that any positive crystalluria lead to intensification of treatment [32]. For this reason, after diagnosis, the French protocol does not recommend repeating 24-hour cystine, unlike the European recommendation, GeneReviews and the American consensus, which adjust treatment by cystine concentration or cystine capacity; 24-hour urine is still requested regularly to measure urine output, confirm the quality of the collection by creatinine and estimate salt and protein intake from sodium and urea [4, 10, 32, 47]. The 24-hour collection starts after discarding the first urine of the first day and ends with the first urine of the following day, at the same time, including all nocturnal voids [32].

11.5 PREPL deficiency: prognosis and surveillance

The prognosis of isolated PREPL deficiency is more favourable than that of Prader-Willi syndrome, with which it is confused, and it is this contrast that makes the differential diagnosis decisive [6]. Severe neonatal hypotonia improves spontaneously, but incompletely: independent walking comes at a median of about 23 months, and in adulthood ptosis, facial weakness, nasal dysarthria and neck flexor weakness persist, with proximal weakness in two of five patients over three years of age; intelligence ranges from mild deficit to normal, with a median quotient of 70 and a range of 64 to 112, and neither the long-term cognitive course nor final height on somatropin is known [6, 38]. Assisted feeding may be prolonged, with removal of the gastrostomy at six and a half years in one patient, and exercise capacity remains reduced even in patients who walk: in the two siblings with a contiguous deletion followed for seven years, the six-minute walk distance improved with age but remained about 150 metres below predicted [6, 63]. The deletion forms that include CAMKMT or PPM1B have poorer development and higher mortality, with deaths of undiagnosed hypotonic siblings and of children with 2p21 deletion syndrome [6, 58].

In the 2p21 deletion syndromes, follow-up combines surveillance of cystinuria with that of growth, growth hormone, feeding and development [6, 33]. The mitochondrial dysfunction described in some of these patients, with partial cytochrome c oxidase deficiency and raised lactate, justifies measuring lactate when there is acidosis or exercise intolerance, although there is no validated specific treatment [58, 63].

12. Summary and Final Considerations

HHH syndrome and cystinuria are two amino acid transporter disorders with very different consequences. In HHH syndrome, loss of the mitochondrial ornithine transporter ORNT1, encoded by SLC25A15, deprives ornithine transcarbamylase of its substrate and ornithine aminotransferase of its degradation pathway, and produces the triad of episodic or postprandial hyperammonaemia, hyperornithinaemia and homocitrullinuria [1, 51]. The disease is rare (about 120 published patients), panethnic, with founder populations in Quebec and northern Saskatchewan, and with frequent variants in Japan and the Middle East [1, 9, 27]. It presents at any age, more often in childhood than in the neonatal period, with crises of encephalopathy, liver dysfunction that may progress to liver failure with coagulopathy, protein aversion and, in the long term, pyramidal signs and intellectual disability in about two-thirds of patients [1]. Diagnosis requires thinking of the disease in any hyperammonaemia or unexplained acute hepatitis and requesting plasma and urine amino acids; newborn screening often fails, because ornithine rises only after the first days of life [27, 35]. Treatment follows the principles of the UCDs, with protein restriction, citrulline, nitrogen scavengers and creatine and lysine supplements when deficient; the crisis responds well to glucose and withdrawal of protein, and survival exceeds 90%, but spastic paraparesis may progress despite good metabolic control, and liver transplantation is not indicated when diet and drugs control ammonia [1, 9, 35].

In cystinuria, failure of the apical b0,+ transporter, formed by the heavy chain rBAT (SLC3A1) and the light chain b0,+AT (SLC7A9), leaves filtered cystine in the urine, where its low solubility produces stones from childhood [4, 10]. It is the most frequent monogenic cause of stone disease, with a prevalence of about 1:7000 and founder populations among Libyan and Ashkenazi Jews and Mennonite communities; the genetic classification (A, B, AB and the notations AA, BB, A0, B0) replaced the old classification into types I to III, and recessive, digenic and dominant inheritance with reduced penetrance coexist [3, 4, 10]. Diagnosis is by stone analysis by infrared spectroscopy or X-ray diffraction, by hexagonal crystals and by urinary cystine; genetic testing is useful, but not mandatory, and should include deletion and duplication analysis [4, 10]. Treatment is stepwise: urine output of at least 3 L per day, sodium and methionine restriction, potassium citrate for a urinary pH of 7.5–8.0 and, in refractory cases, tiopronin or D-penicillamine, with monitoring of proteinuria and full blood count [4, 37, 47]. The sources diverge on pH targets and doses, and tiopronin is neither authorised in Portugal nor available in Brazil, where D-penicillamine is the alternative [76, 83]. The French HAS protocol adds to the European recommendation a surveillance pathway centred on crystalluria and cystine crystal volume, instead of repeating 24-hour cystine, doses of alkalinising agents and thiols in grams and tablets with maxima higher than the European ones, and lithotripsy as first line in children [4, 32]. Chronic kidney disease and hypertension affect a substantial proportion of adults, and quality of life depends on stone-free time [4]. Emerging therapies, such as alpha-lipoic acid, crystallisation inhibitors and gene therapy, still lack sufficient clinical demonstration [10, 85, 87].

The third part of this article concerns the gene adjacent to SLC3A1. Biallelic loss of PREPL, isolated or within a contiguous 2p21 deletion, causes congenital myasthenic syndrome 22, with severe neonatal hypotonia that improves spontaneously but incompletely, feeding difficulties, nasal dysarthria, ptosis, high-arched palate, xerostomia and autonomic signs, growth hormone deficiency with onset between 20 months and 11 years, hypergonadotropic hypogonadism and hyperphagia with obesity at the end of the first decade [6–8]. The mechanism is twofold, a (thio)esterase with a long mitochondrial isoform and a regulator of AP-1-dependent transport in the trans-Golgi network, with reduced quantal content and miniature endplate potential amplitude and normal acetylcholine receptors [8, 30, 50]. Diagnosis is molecular and requires copy-number sensitivity, with urine amino acids separating the contiguous from the isolated form and the functional lymphocyte assay distinguishing the disease from Prader-Willi syndrome, although a normal activity assay does not exclude missense variants [6, 30]. Targeted treatment has two components with unequal evidence: pyridostigmine, extrapolated from the myasthenia gravis label, with a response mainly in infancy and possibly age-related, which requires formal reassessment and controlled withdrawal; and somatropin, which consistently restores growth [8, 31, 38]. The prognosis is better than that of Prader-Willi syndrome, with intelligence ranging from mild deficit to normal, which makes recognition of this entity a priority in the assessment of the hypotonic infant [6, 60].

In both diseases, genetic counselling needs to explain the absence of genotype–phenotype correlation and, in cystinuria, the coexistence of several inheritance patterns and the risk to SLC7A9 heterozygotes; and the contiguous 2p21 deletions, which combine cystinuria and PREPL deficiency, are a reminder that cystinuria with neonatal hypotonia or a picture resembling Prader-Willi syndrome should be investigated by microarray, and that in PREPL deficiency trio testing is necessary because of compound heterozygosity for a point variant and a deletion and the possibility of uniparental disomy [1, 6, 10, 14].

13. References

1. Camacho J, Rioseco-Camacho N. Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993–2026. Published 31/05/2012; updated 13/02/2020; revised 10/04/2025. Available from: https://www.ncbi.nlm.nih.gov/books/NBK97260/
2. Orphanet. Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (ORPHA:415). Available from: https://www.orpha.net/en/disease/detail/415
3. Online Mendelian Inheritance in Man, OMIM®. MIM #220100 — Cystinuria; CSNU. Baltimore: Johns Hopkins University. Available from: https://omim.org/entry/220100
4. Servais A, Thomas K, Dello Strologo L, Sayer JA, Bekri S, Bertholet-Thomas A, et al. Cystinuria: clinical practice recommendation. Kidney Int. 2021;99(1):48-58. doi:10.1016/j.kint.2020.06.035 PMID: 32918941.
5. Online Mendelian Inheritance in Man, OMIM®. MIM #606407 — Hypotonia-cystinuria syndrome. Available from: https://omim.org/entry/606407
6. Régal L, Mårtensson E, Maystadt I, Voermans N, Lederer D, Burlina A, et al. PREPL deficiency: delineation of the phenotype and development of a functional blood assay. Genet Med. 2018;20(1):109-118. doi:10.1038/gim.2017.74 PMID: 28726805.
7. Online Mendelian Inheritance in Man (OMIM). #616224 — Myasthenic syndrome, congenital, 22; CMS22 (PREPL deficiency). Johns Hopkins University, Baltimore. Accessed 01/11/2026. Available from: https://www.omim.org/entry/616224
8. Régal L, Shen XM, Selcen D, Verhille C, Meulemans S, Creemers JW, et al. PREPL deficiency with or without cystinuria causes a novel myasthenic syndrome. Neurology. 2014;82(14):1254-1260. doi:10.1212/WNL.0000000000000295 PMID: 24610330. PMC4001208.
9. Martinelli D, Diodato D, Ponzi E, Monné M, Boenzi S, Bertini E, et al. The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. Orphanet J Rare Dis. 2015;10:29. doi:10.1186/s13023-015-0242-9 PMID: 25874378. PMC4358699.
10. Spasiano A, Halbritter J, Ferraro PM. Cystinuria. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993–2026. Published 20/11/2025. Available from: https://www.ncbi.nlm.nih.gov/books/NBK619248/
11. Online Mendelian Inheritance in Man (OMIM). *609557 — Prolyl endopeptidase-like; PREPL. Johns Hopkins University, Baltimore. Accessed 01/11/2026. Available from: https://www.omim.org/entry/609557
12. Freeman PJ, Hart RK, Gretton LJ, Brookes AJ, Dalgleish R. VariantValidator: Accurate validation, mapping, and formatting of sequence variation descriptions. Hum Mutat. 2018;39(1):61-68. doi:10.1002/humu.23348 PMID: 28967166. Accessed 01/11/2026. Available from: https://variantvalidator.org
13. Orphanet. Hypotonia-cystinuria syndrome (ORPHA:163690). Available from: https://www.orpha.net/en/disease/detail/163690
14. Zhang P, Wu B, Lu Y, Ni Q, Liu R, Zhou W, et al. First maternal uniparental disomy for chromosome 2 with PREPL novel frameshift mutation of congenital myasthenic syndrome 22 in an infant. Mol Genet Genomic Med. 2020;8(3):e1144. doi:10.1002/mgg3.1144 PMID: 31985178. PMC7057094.
15. Online Mendelian Inheritance in Man, OMIM®. MIM #238970 — Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; HHHS. Baltimore: Johns Hopkins University. Available from: https://omim.org/entry/238970
16. Online Mendelian Inheritance in Man, OMIM®. MIM *603861 — Solute carrier family 25 (mitochondrial carrier, ornithine transporter), member 15; SLC25A15. Available from: https://omim.org/entry/603861
17. Online Mendelian Inheritance in Man, OMIM®. MIM *104614 — Solute carrier family 3 (cystine, dibasic, and neutral amino acid transporter), member 1; SLC3A1. Available from: https://omim.org/entry/104614
18. Online Mendelian Inheritance in Man, OMIM®. MIM *604144 — Solute carrier family 7 (cationic amino acid transporter, y+ system), member 9; SLC7A9. Available from: https://omim.org/entry/604144
19. Orphanet. Cystinuria (ORPHA:214). Available from: https://www.orpha.net/en/disease/detail/214
20. Orphanet. Atypical hypotonia-cystinuria syndrome (ORPHA:238523). Available from: https://www.orpha.net/en/disease/detail/238523
21. Orphanet. 2p21 microdeletion syndrome (ORPHA:163693). Available from: https://www.orpha.net/en/disease/detail/163693
22. Orphanet. Congenital myasthenic syndrome due to defective synthesis or recycling of acetylcholine (ORPHA:716893). Accessed 01/11/2026. Available from: https://www.orpha.net/en/disease/detail/716893
23. Orphanet. 2p21 microdeletion syndrome without cystinuria (ORPHA:369881). Accessed 01/11/2026. Available from: https://www.orpha.net/en/disease/detail/369881
24. Simpson KL, MacLeod EL, Kakajiwala A, Gropman AL, Ah Mew N. Urea cycle disorders overview. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993–2026. Published 29/04/2003; updated 03/07/2025. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1217/
25. Abicht A, Müller JS, Lochmüller H. Congenital Myasthenic Syndromes Overview. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993–2026. Published 09/05/2003; updated 23/12/2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1168/
26. Summar ML, Koelker S, Freedenberg D, Le Mons C, Haberle J, Lee HS, et al. The incidence of urea cycle disorders. Mol Genet Metab. 2013;110(1-2):179-180. doi:10.1016/j.ymgme.2013.07.008 PMID: 23972786. PMC4364413.
27. Sokoro AA, Lepage J, Antonishyn N, McDonald R, Rockman-Greenberg C, Irvine J, et al. Diagnosis and high incidence of hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome in northern Saskatchewan. J Inherit Metab Dis. 2010;33 Suppl 3:S275-81. doi:10.1007/s10545-010-9148-9 PMID: 20574716.
28. Wu CW, Chang J, Lovrenert K, Bodner D, Hildebrandt F, Schumacher FR. How variant discovery redefines genetic prevalence: the case of cystine stone disease. Eur J Hum Genet. 2026;34(7):956-963. doi:10.1038/s41431-026-02085-y PMID: 41957517. PMC13341753.
29. Ohno K, Ohkawara B, Shen XM, Selcen D, Engel AG. Clinical and pathologic features of congenital myasthenic syndromes caused by 35 genes — a comprehensive review. Int J Mol Sci. 2023;24(4):3730. doi:10.3390/ijms24043730 PMID: 36835142. PMC9961056.
30. Monnens Y, Theodoropoulou A, Rosier K, Bhalla K, Mahy A, Vanhoutte R, et al. Missense variants in CMS22 patients reveal that PREPL has both enzymatic and nonenzymatic functions. JCI Insight. 2024;9(17):e179276. doi:10.1172/jci.insight.179276 PMID: 39078710. PMC11385081.
31. Wang T, Zhou S, Li W. Clinical manifestations and genetic insights into congenital myasthenic syndrome-22 in pediatric patients. Pediatr Neurol. 2026;176:68-76. doi:10.1016/j.pediatrneurol.2025.12.027 PMID: 41547082.
32. Haute Autorité de Santé. Protocole National de Diagnostic et de Soins (PNDS): La Cystinurie. Centres de référence des maladies rénales rares MARHEA, Néphrogones et SORARE, filière ORKiD; coordinated by Lemoine and Heidet. Saint-Denis La Plaine: HAS; HAS page dated 28 Oct 2019; document dated November 2019. Available from: https://www.has-sante.fr/jcms/p_3115039. Accessed 6 Nov 2026.
33. Jaeken J, Martens K, Francois I, Eyskens F, Lecointre C, Derua R, et al. Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome. Am J Hum Genet. 2006;78(1):38-51. doi:10.1086/498852 PMID: 16385448. PMC1380222.
34. Niinikoski H, Schiff M, Tanner L. Disorders of amino acid transport at the cell membrane. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 25, section 25.1. doi:10.1007/978-3-662-63123-2_25
35. Häberle J, Boddaert N, Burlina A, Chakrapani A, Dixon M, Huemer M, et al. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis. 2012;7:32. doi:10.1186/1750-1172-7-32 PMID: 22642880. PMC3488504.
36. Häberle J, Burlina A, Chakrapani A, Dixon M, Karall D, Lindner M, et al. Suggested guidelines for the diagnosis and management of urea cycle disorders: First revision. J Inherit Metab Dis. 2019;42(6):1192-1230. doi:10.1002/jimd.12100 PMID: 30982989.
37. European Association of Urology. EAU Guidelines on Urolithiasis. Edn. presented at the EAU Annual Congress London 2026. Arnhem: EAU Guidelines Office; 2026. Section 4.9, Cystine stones. Available from: https://uroweb.org/guidelines/urolithiasis/chapter/metabolic-evaluation-and-recurrence-prevention
38. Sayol-Torres L, Valenzuela MI, Tomasini R, Fernández-Alvarez P, Clemente M, Yeste D. Prolyl endopeptidase-like deficiency associated with growth hormone deficiency. J Clin Res Pediatr Endocrinol. 2023;15(2):205-209. doi:10.4274/jcrpe.galenos.2021.2021.0128 PMID: 34693706. PMC10234053.
39. MetabERN — European Reference Network for Hereditary Metabolic Disorders. AOA subnetwork (Aminoacid and organic acids related disorders). Available from: https://metab.ern-net.eu/
40. ERKNet — European Rare Kidney Disease Reference Network. "Metabolic & Stone Disorders" working group; EUROCYS registry. Available from: https://www.erknet.org/
41. ERN EURO-NMD — European Reference Network for rare neuromuscular diseases. Available from: https://ern-euro-nmd.eu
42. Nyhan WL, Hoffmann GF, Al-Aqeel AI, Barshop BA. Lysinuric protein intolerance. In: Atlas of Inherited Metabolic Diseases. 4th ed. Boca Raton: CRC Press; 2020. p. 253–258.
43. Shih VE, Efron ML, Moser HW. Hyperornithinemia, hyperammonemia, and homocitrullinuria. A new disorder of amino acid metabolism associated with myoclonic seizures and mental retardation. Am J Dis Child. 1969;117(1):83-92. doi:10.1001/archpedi.1969.02100030085009 PMID: 5782534.
44. Nyhan WL, Hoffmann GF, Al-Aqeel AI, Barshop BA. Hyperornithinemia, hyperammonemia, homocitrullinuria syndrome. In: Atlas of Inherited Metabolic Diseases. 4th ed. Boca Raton: CRC Press; 2020. p. 247–252.
45. National Center for Biotechnology Information. ClinVar — PREPL and SLC25A15 (gene submissions, accessed 01/11/2026). Bethesda (MD): National Library of Medicine. Available from: https://www.ncbi.nlm.nih.gov/clinvar/?term=PREPL%5Bgene%5D and https://www.ncbi.nlm.nih.gov/clinvar/?term=SLC25A15%5Bgene%5D
46. Camacho JA, Obie C, Biery B, Goodman BK, Hu CA, Almashanu S, et al. Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter. Nat Genet. 1999;22(2):151-158. doi:10.1038/9658 PMID: 10369256.
47. Eisner BH, Goldfarb DS, Baum MA, Langman CB, Curhan GC, Preminger GM, et al. Evaluation and Medical Management of Patients with Cystine Nephrolithiasis: A Consensus Statement. J Endourol. 2020;34(11):1103-1110. doi:10.1089/end.2019.0703 PMID: 32066273. PMC7869875.
48. Calonge MJ, Gasparini P, Chillarón J, Chillón M, Gallucci M, Rousaud F, et al. Cystinuria caused by mutations in rBAT, a gene involved in the transport of cystine. Nat Genet. 1994;6(4):420-425. doi:10.1038/ng0494-420 PMID: 8054986.
49. Feliubadaló L, Font M, Purroy J, Rousaud F, Estivill X, Nunes V, et al. Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT. Nat Genet. 1999;23(1):52-57. doi:10.1038/12652 PMID: 10471498.
50. Rosier K, McDevitt MT, Smet J, Floyd BJ, Verschoore M, Marcaida MJ, et al. Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function. iScience. 2021;24(12):103460. doi:10.1016/j.isci.2021.103460 PMID: 34888501. PMC8634043.
51. Baumgartner MR, Valle D, Dionisi-Vici C. Disorders of ornithine and proline metabolism. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 21, section 21.2. doi:10.1007/978-3-662-63123-2_21
52. Häberle J, Rubio V. Disorders of the urea cycle and related enzymes. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 19.
53. Debray FG, Lambert M, Lemieux B, Soucy JF, Drouin R, Fenyves D, et al. Phenotypic variability among patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome homozygous for the delF188 mutation in SLC25A15. J Med Genet. 2008;45(11):759-764. doi:10.1136/jmg.2008.059097 PMID: 18978333.
54. Kido J, Sugawara K, Yano N, Nakamura K. Genotypic and clinical characteristics of long-term surviving adult patients with urea cycle disorders in Japan. Orphanet J Rare Dis. 2026;21(1):380. doi:10.1186/s13023-026-04524-x PMID: 42947209. PMC13715130.
55. Silfverberg T, Sahlander F, Enlund M, Oscarson M, Hårdstedt M. Late onset hyperornithinemia-hyperammonemia-homocitrullinuria syndrome - how web searching by the family solved unexplained unconsciousness: a case report. J Med Case Rep. 2018;12(1):274. doi:10.1186/s13256-018-1794-9 PMID: 30243302. PMC6151189.
56. Mosher TL, Czepiel KS, Neu MB, Fernandez ACC, Browne LW, Niehaus AD, et al. Biochemical testing and pathology reveal rare cause of pediatric acute liver failure: Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. JPGN Rep. 2026:e70206 (published online on 27/07/2026). doi:10.1002/jpr3.70206 PMID: 42516736. PMC13403024.
57. Maselli RA. Presynaptic congenital myasthenic syndromes. Muscle Nerve. 2026;74(Suppl 1):S41-S51. doi:10.1002/mus.70106 PMID: 42383533. PMC13599454.
58. Bartholdi D, Asadollahi R, Oneda B, Schmitt-Mechelke T, Tonella P, Baumer A, et al. Further delineation of genotype-phenotype correlation in homozygous 2p21 deletion syndromes: first description of patients without cystinuria. Am J Med Genet A. 2013;161A(8):1853-1859. doi:10.1002/ajmg.a.35994 PMID: 23794250.
59. Shchagina O, Bessonova L, Bychkov I, Beskorovainaya T, Poliakov A. A family case of congenital myasthenic syndrome-22 induced by different combinations of molecular causes in siblings. Genes (Basel). 2020;11(7):821. doi:10.3390/genes11070821 PMID: 32707643. PMC7397044.
60. Bhalla K, Rosier K, Monnens Y, Meulemans S, Vervoort E, Thorrez L, et al. Similar metabolic pathways are affected in both congenital myasthenic syndrome-22 and Prader-Willi syndrome. Biochim Biophys Acta Mol Basis Dis. 2024;1870(5):167175. doi:10.1016/j.bbadis.2024.167175 PMID: 38626828.
61. Kim SZ, Song WJ, Nyhan WL, Ficicioglu C, Mandell R, Shih VE. Long-term follow-up of four patients affected by HHH syndrome. Clin Chim Acta. 2012;413(13-14):1151-1155. doi:10.1016/j.cca.2012.03.015 PMID: 22465082.
62. Ho B, MacKenzie J, Walia J, Geraghty M, Smith G, Nedvidek J, et al. Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome in pregnancy: Considerations for management and review of the literature. JIMD Rep. 2019;46(1):28-34. doi:10.1002/jmd2.12025 PMID: 31240152. PMC6498866.
63. Towheed A, Hietanen CL, Kamath VG, Singh LN, Ho A, Engelstad K, et al. Hypotonia-cystinuria 2p21 deletion syndrome: intrafamilial variability of clinical expression. Ann Clin Transl Neurol. 2021;8(11):2199-2204. doi:10.1002/acn3.51464 PMID: 34612606. PMC8607452.
64. Yang Q, Hua R, Qian J, Yi S, Shen F, Zhang Q, et al. PREPL deficiency: a homozygous splice site PREPL mutation in a patient with congenital myasthenic syndrome and absence of ovaries and hypoplasia of uterus. Front Genet. 2020;11:198. doi:10.3389/fgene.2020.00198 PMID: 32218803. PMC7078161.
65. Nyhan WL, Hoffmann GF, Al-Aqeel AI, Barshop BA. Argininemia. In: Atlas of Inherited Metabolic Diseases. 4th ed. Boca Raton: CRC Press; 2020. p. 241–246.
66. Groothoff JW, Metry E, Deesker L, Garrelfs S, Acquaviva C, Almardini R, et al. Clinical practice recommendations for primary hyperoxaluria: an expert consensus statement from ERKNet and OxalEurope. Nat Rev Nephrol. 2023;19(3):194-211. doi:10.1038/s41581-022-00661-1. PMID 36604599
67. Edvardsson VO, Sahota A, Palsson R. Adenine phosphoribosyltransferase deficiency. 2012 Aug 30 [updated 2019 Sep 26]. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews®. Seattle (WA): University of Washington, Seattle; 1993–2026. Available from: https://www.ncbi.nlm.nih.gov/books/NBK100238/
68. Morris AAM, Jones S. Medications used in the treatment of inborn errors of metabolism. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 45.
69. U.S. Food and Drug Administration. Sodium phenylacetate and sodium benzoate injection 10%/10% (Ammonul; NDA020645, approved 17/02/2005) — prescribing information (Zydus generic, label of 30/10/2023). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=0f17c50f-3761-4470-bf3b-63837a287b8a
70. European Medicines Agency. Orphan designations: EU/3/10/738 (glycerol phenylbutyrate, HHH), EU/3/16/1730 (sodium benzoate, ornithine translocase deficiency), EU/3/18/2036 (L-cystine bis(N-methylpiperazide), cystinuria), EU/3/19/2239 (tripotassium citrate monohydrate/potassium hydrogen carbonate, cystinuria), EU/3/21/2445 (L-ergothioneine, cystinuria). Available from: https://www.ema.europa.eu/en/medicines/human/orphan-designations/eu-3-19-2239
71. European Medicines Agency. Ravicti (glycerol phenylbutyrate) — EPAR; authorisation of 26/11/2015; Ammonaps (07/12/1999); Pheburane (30/07/2013). Available from: https://www.ema.europa.eu/en/medicines/human/EPAR/ravicti
72. U.S. Food and Drug Administration. Ravicti (glycerol phenylbutyrate) oral liquid — prescribing information (Horizon; NDA203284; label of 29/10/2025). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=900e7dc0-9afe-4f50-80de-90567bb78519
73. U.S. Food and Drug Administration. Buphenyl (sodium phenylbutyrate) tablets and powder — prescribing information (Horizon; NDA020572/NDA020573; label of 30/10/2024). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=463a36fa-3eb2-4326-8bd0-c8c7a11bca3a
74. ClinicalTrials.gov. NCT07773246 — Phase 1/2 Study of KRRO-121 in Healthy Volunteers and Patients With UCD (recruiting). Available from: https://clinicaltrials.gov/study/NCT07773246
75. ClinicalTrials.gov. NCT03933410 — UNLOCKED: A Phase 2, Open-label Trial With KB195 in Subjects With a Urea Cycle Disorder (terminated on 02/03/2021). Available from: https://clinicaltrials.gov/study/NCT03933410
76. Carvalho M, Matos ACC, Santos DRD, Barreto DV, Barreto FC, Rodrigues FG, et al. Brazilian Guidelines on evaluation and clinical management of Nephrolithiasis: Brazilian Society of Nephrology. J Bras Nefrol. 2025;47(2):e20240189. doi:10.1590/2175-8239-jbn-2024-0189en PMID: 40080792. PMC11913452.
77. U.S. Food and Drug Administration. Depen (penicillamine) titratable tablets — prescribing information (Viatris; NDA019854; label of 21/09/2025). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=38f8ae60-b354-11de-8a39-0800200c9a66
78. INFARMED. Acalka 1080 mg comprimido de libertação modificada (citrato de potássio) — Resumo das Características do Medicamento. Infomed (med_id 8921). Available from: https://extranet.infarmed.pt/INFOMED-fo/download-ficheiro.xhtml?med_id=8921&tipo_doc=rcm
79. INFARMED. Uralyt-U granulado (ácido cítrico + citrato de potássio + citrato de sódio) — Resumo das Características do Medicamento (28/06/2023). Infomed (med_id 8905). Available from: https://extranet.infarmed.pt/INFOMED-fo/download-ficheiro.xhtml?med_id=8905&tipo_doc=rcm
80. U.S. Food and Drug Administration. Urocit-K (potassium citrate) extended-release tablets — prescribing information (Mission Pharmacal; NDA019071; label of 02/12/2021). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=72cdea1b-2240-41db-987d-86d5c6aaa978
81. U.S. Food and Drug Administration. Thiola EC (tiopronin) delayed-release tablets — prescribing information (Mission Pharmacal; NDA211843; label of 07/10/2025); Thiola (NDA019569; approved 11/08/1988; label of 27/08/2026); Venxxiva (ANDA216990; label of 22/12/2025). Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=20298a52-a194-a161-8632-d84b6f26e23c
82. INFARMED. Kelatine 300 mg comprimidos revestidos (D-penicilamina) — Resumo das Características do Medicamento (approved on 01/04/2022). Infomed. Available from: https://extranet.infarmed.pt/INFOMED-fo/
83. INFARMED. Infomed — search for "tiopronina" (no authorised medicines), accessed 31/10/2026. Available from: https://extranet.infarmed.pt/INFOMED-fo/
84. ClinicalTrials.gov. NCT04137978 and NCT04147871 — ADV7103 in cystinuria (withdrawn). Available from: https://clinicaltrials.gov/study/NCT04147871
85. ClinicalTrials.gov. NCT02910531 — The Effect of Lipoic Acid Natural Supplement on Cystine Stone Formation (results posted in the registry; last update 15/04/2026). Available from: https://clinicaltrials.gov/study/NCT02910531
86. ClinicalTrials.gov. NCT04818034 — The Effect of SGLT2 Inhibitors on Cystine Stone Formation: A Preliminary Study (results in the registry); NCT05058859 (withdrawn). Available from: https://clinicaltrials.gov/study/NCT04818034
87. Zambudio Munuera A, Rodriguez Parras P, Galan Llopis JA, Grases Freixedas F, Cano Garcia MDC, Arrabal-Martin M, et al. Cystinuria: Diagnosis, Medical and Surgical Management, and Emerging Therapies. A Narrative Review. Res Rep Urol. 2026;18:629402. doi:10.2147/rru.s629402 PMID: 42542789. PMC13428097.
88. INFARMED. Resumo das Características do Medicamento — Mestinon 60 mg comprimidos revestidos (brometo de piridostigmina). Text approved on 08/07/2020. Available from: https://extranet.infarmed.pt/INFOMED-fo/
89. U.S. Food and Drug Administration. MESTINON (pyridostigmine bromide) tablets, oral solution and TIMESPAN tablets — prescribing information. Bausch Health US. Available from: https://www.accessdata.fda.gov/scripts/cder/daf/
90. INFARMED. Resumo das Características do Medicamento — Genotropin (somatropina), pó e solvente para solução injetável. Available from: https://extranet.infarmed.pt/INFOMED-fo/
91. ClinicalTrials.gov. Sulfamethoxazole for the treatment of primary PREPL deficiency (NCT02640443). Universitair Ziekenhuis Brussel and KU Leuven. Registry accessed 01/11/2026. Available from: https://clinicaltrials.gov/study/NCT02640443
92. Schaefer B, Garba A, Wu X. Outcomes of Tiopronin and D-Penicillamine Therapy in Pediatric Cystinuria: A Clinical Comparison of Two Cases. Reports (MDPI). 2025;8(3):163. doi:10.3390/reports8030163 PMID: 40981121. PMC12452493.