Severe and Lethal Neonatal Encephalopathy of Genetic Cause – Diagnostic Approach

Term or late preterm newborn with severe encephalopathy, with or without epileptic seizures, potentially lethal: distinction from hypoxic-ischaemic encephalopathy, treatable and untreatable inborn errors of metabolism, neonatal-onset developmental and epileptic encephalopathies, X-linked forms in boys and the role of rapid genomic sequencing; in-depth coverage of nonketotic hyperglycinaemia, molybdenum cofactor deficiency and isolated sulfite oxidase deficiency.

Literature and regulatory information updated: 28 October 2026.

0. Initial Reference Table

This article addresses a clinical finding rather than a single disease. The first table summarises the finding; the second brings together the entities covered in depth and the main genetic causes to consider.

1. Introduction

1.1. The clinical problem

Neonatal encephalopathy is a syndrome of neurological dysfunction in the first days of life of the term or near-term newborn, with altered consciousness, abnormal tone and reflexes, respiratory dysfunction, feeding difficulties and, often, epileptic seizures [1]. It affects 1 to 6 per 1,000 term live births in recent estimates, and 1 to 7 per 1,000 in other series; in more than half of term newborns no aetiological factor is identified [10, 11]. HIE is the most frequent cause and the main indication for therapeutic hypothermia, but NE is not synonymous with HIE: the diagnosis of HIE is made within a few hours to decide eligibility for cooling, at a time when the phenotype is incomplete, which favours diagnostic anchoring and underdiagnosis of the causes that mimic it [1]. More than 60% of infants with an HIE phenotype have no identifiable acute sentinel event [45].

The central difficulty for the neonatologist and the geneticist is to separate the genetic and metabolic causes of encephalopathy and seizures from those occurring in the context of a hypoxic-ischaemic insult, bearing in mind that there may be seizures without encephalopathy and encephalopathy without seizures [2]. Saudubray and García-Cazorla summarise the principles guiding this search: consider IEM in parallel with common conditions; suspect an IEM when symptoms persist without explanation after initial treatment; accept that any neonatal death may be due to an IEM, especially those attributed to sepsis; remember that true sepsis can trigger decompensation of an underlying IEM; review autopsies critically; and, in an emergency, look first for treatable diseases [3]. Because most IEM are recessive, most cases appear sporadic [3].

1.2. Historical milestones

Nonketotic hyperglycinaemia was described by Gerritsen and colleagues in 1965 and was given the qualifier "nonketotic" to distinguish it from the hyperglycinaemias of the organic acidaemias, in particular propionic acidaemia, then called "ketotic hyperglycinaemias" [46]. Molybdenum cofactor deficiency was described by Johnson and colleagues in 1980 in a girl with extreme developmental delay, combined sulfite oxidase and xanthine oxidase deficiency, lens dislocation, urinary xanthine stones, increased excretion of sulfite, thiosulfate, S-sulfocysteine and taurine, and hypouricaemia; complementation studies in fibroblasts later defined groups A (two-thirds, deficient synthesis of precursor Z, now cyclic pyranopterin monophosphate) and B (one-third, conversion of the precursor into molybdopterin) [47]. Fibroblast co-culture has been replaced by molecular analysis, which today defines types A (MOCS1), B (MOCS2) and C (GPHN) [30].

In the field of neonatal epilepsies, the description of familial neonatal seizures, later called "benign", dates back to Rett and Teubel (1964) and Bjerre (1968); in 2017 the ILAE adopted the term "self-limited" instead of "benign" [4, 8]. From 2013, large-scale sequencing studies showed that many severe infantile encephalopathies result from de novo variants in genes such as KCNQ2, STXBP1, SCN2A and SCN8A, with few distinctive clinical features, and that the same ion channel genes can cause either self-limited familial epilepsies or severe encephalopathies [2, 7].

Rapid genomic sequencing in neonatal intensive care units (NICU) has been tested in randomised trials since 2014 (NSIGHT1, NSIGHT2) and in implementation programmes such as Project Baby Bear (California) and Acute Care Genomics (Australia), with diagnostic yields of 19% to 54% and turnaround times of a few days [48–51].

1.3. Nomenclature and reclassifications

Glycine encephalopathy. The term collectively designates the neurological disorders caused by disturbed glycine metabolism and transport [23]. "Classic" NKH is due to biallelic variants in GLDC or AMT and is divided, according to final outcome, into severe (no developmental progress and intractable epilepsy) and attenuated (variable progress, treatable or absent epilepsy); the attenuated form is subdivided into good, intermediate and poor [23, 52]. "Variant NKH" corresponds to defects in lipoate synthesis or transport and in iron-sulfur cluster biogenesis, which reduce the activity of the glycine cleavage system (GCS) and other mitochondrial dehydrogenases [52]. The term "atypical NKH" mixed attenuated and variant forms and should no longer be used [23, 52]. The designation "transient NKH" described non-genetic elevations of CSF glycine, now regarded as phenocopies [52].

Molybdenum cofactor deficiency. The subtypes are named by gene, which has therapeutic consequences: type A (MOCS1), type B (MOCS2), type C (GPHN); MOCS3 deficiency has no letter and has only been described in a mild form [30]. Orphanet groups MoCD and ISOD under "sulfite oxidase deficiency encephalopathy" (ORPHA:833) [53].

Neonatal epilepsy syndromes. The 2022 ILAE classification divides the syndromes with onset in the newborn and infant into self-limited epilepsies (self-limited neonatal epilepsy, SeLNE; self-limited familial neonatal-infantile epilepsy, SeLFNIE; self-limited infantile epilepsy, SeLIE), DEE and aetiology-specific syndromes, most of which are also DEE (for example, KCNQ2-DEE, pyridoxine-dependent DEE, PLP-dependent DEE, CDKL5-DEE) [4]. EIDEE, with onset exclusively before 3 months, replaces Ohtahara syndrome and early myoclonic encephalopathy, because the separation between the two was no longer useful for clinical decision-making or prognosis and the two share aetiologies [4]. The term "early infantile epileptic encephalopathy" (EIEE), used in sources before 2022, largely corresponds to EIDEE [4, 7].

1.4. Definitions of the entities in scope

2. Molecular Genetics and Pathophysiology

2.1. Aetiological classes and common mechanisms

The genetic causes of severe neonatal encephalopathy fall into five classes: IEM with neonatal presentation; DEE due to variants in ion channel, synaptic and other neuronal pathway genes; severe X-linked forms in boys; brain malformations and chromosomal anomalies; and genetic mimics of HIE, that is, diseases that reduce tolerance to the stress of labour or worsen the response to a hypoxic-ischaemic insult, with common pathways in energy metabolism and in organs with high metabolic demand [2, 45]. Among IEM, the clinical classification distinguishes small-molecule diseases (intoxication through accumulation or product deficiency), complex-molecule diseases (accumulation, deficiency, cellular trafficking and processing, including congenital disorders of glycosylation, CDG) and energy metabolism diseases (membrane transporters, mitochondria, cytoplasm) [3]. Early infantile monogenic epilepsies with a known biochemical mechanism have also been grouped into three mechanisms that guide diagnosis and treatment: NMDA (N-methyl-D-aspartate) receptor-opathies, phasic GABAergic-opathies and tonic GABAergic-opathies [3].

In intoxication disorders (organic acidaemias, urea cycle disorders, maple syrup urine disease), the newborn is born well, because the placenta clears the toxic metabolites, and deteriorates when the protein load and postnatal catabolism lead to accumulation of ammonia or organic acids [2, 3]. In energy metabolism disorders, the presentation is less suggestive, there is rarely a symptom-free interval, and severe hypotonia, hyperlactataemia and cardiac or hepatic involvement predominate [3]. In DEE, the disturbance of neuronal excitability often begins in utero and the EEG may be abnormal from the first hours [4, 46].

2.2. Nonketotic hyperglycinaemia

Glycine cleavage system. The GCS is a complex of four proteins of the inner mitochondrial membrane: the P protein (GLDC), which contains PLP, decarboxylates glycine and transfers the aminomethyl group to the lipoate of the H protein; the H protein (GCSH), which carries the lipoyl arm; the T protein (AMT), which releases ammonia and transfers the methyl group to tetrahydrofolate, forming 5,10-methylenetetrahydrofolate; and the L protein (DLD), which reoxidises the lipoate [52]. The GCS is the main pathway of glycine degradation and the main source of one-carbon units; it is expressed in the liver, the brain (mainly in astrocytes of the cerebrum and cerebellum, little in the brainstem and not in the spinal cord) and the placental syncytiotrophoblast [46, 52]. Excluding the GCS, endogenous glycine synthesis from serine far exceeds intake, which limits the role of dietary restriction [52].

Neurological mechanism. Glycine accumulates mainly in the brain, and its elevation outside the brain through exogenous loading does not cause symptoms [52]. Glycine is the inhibitory neurotransmitter of the strychnine-sensitive glycinergic receptors in the brainstem and spinal cord, which explains neonatal hypotonia and apnoea, and it is an excitatory allosteric co-agonist of the NMDA receptor, allowing glutamate to excite the neuron at much lower concentrations, which explains the intractable seizures [46, 52]. D-serine, synthesised from glycine, is reduced in the cortex of these children [52]. In newborns who died with classic NKH, myelin spongiosis is seen in the corticospinal tract, optic radiations and brainstem, with separation of the myelin lamellae on electron microscopy, which explains the restricted diffusion on MRI [52]. In mice with deficient GCS there is a deficit of methylated folates in the brain, and restoring one-carbon metabolism improves symptoms and survival; in children with classic NKH, CSF methylfolate is not reduced, so the finding remains preclinical [52].

Genes, proportions and variant spectrum. Classic NKH is AR; GLDC accounts for about 80% of cases and AMT for about 20% [23, 52]. In the series of Coughlin and colleagues (578 families), 410 unique variants were identified, 80% of patients had GLDC variants, missense variants accounted for 52% of GLDC alleles (mostly private), intragenic GLDC copy number variations (CNV) were found in 140 patients (biallelic in 39), and sequencing with CNV analysis identified biallelic variants in 98% of patients [24]. About 20% of pathogenic GLDC alleles are large deletions generated by non-allelic homologous recombination between Alu repeats, on several haplotypes and in several ethnic groups; deletion of exons 1–2 is the most common [23, 46]. GLDC has a full-length processed pseudogene with 97.5% homology, which requires intronic primers to avoid amplifying the pseudogene [23]. In AMT, recurrent missense variants predominate, no intragenic CNV have been described, and some variants in the 5′ untranslated region (around positions −55 to −66) are pathogenic and escape analyses restricted to the coding region [23, 52]. GeneReviews (2019) mentioned for GCSH only two people with biallelic variants, in a conference abstract, without established pathogenicity [23]. OMIM has reclassified as of uncertain significance the first GCSH rearrangement proposed as a cause of NKH and now defines, for this gene (16q23.2), multiple mitochondrial dysfunctions syndrome 7 (MMDS7, AR), with a spectrum from lethal neonatal glycine encephalopathy to an attenuated phenotype with developmental delay, behavioural abnormalities, limited epilepsy and movement disorder [22, 54]. Majethia and colleagues (2021) described three patients from two Indian families homozygous for an initiation codon variant (c.1A>G), with raised plasma and CSF glycine, a lactate peak in the putamen and basal ganglia lesions; Arribas-Carreira and colleagues (2023) described five patients with biallelic variants (missense, nonsense, splicing, exon 2 deletion and duplication of exons 4–5), onset between day 1 and 3 months, burst-suppression in the neonatal forms and death at 7 and 18 days in two of them [22]. The variants variably reduce the two functions of the H protein, glycine exchange and lipoylation, and the phenotype correlates in part with the function most affected [22]. De novo variants have been documented in about 1% of patients [23, 52].

The following table presents the GLDC and AMT variants with population or prognostic relevance, normalised in VariantValidator to the MANE Select transcripts (Matched Annotation from NCBI and EMBL-EBI) (GLDC NM_000170.3; AMT NM_000481.4), with the historical nomenclature in parentheses [23, 52, 55]. The conversion confirmed that the Finnish variant c.2284G>A encodes p.(Gly762Arg), and not p.Gly761Arg as it appears in part of the literature and in the OMIM entry, and that c.1166C>T corresponds to p.(Ala389Val), resolving the inconsistency of the GeneReviews table, which also assigns it p.Ala398Val; OMIM records Ala389Val [23, 55, 56]. In OMIM, the Finnish variant Ser564Ile accounts for 14 of 20 GLDC alleles in Finnish patients, Gly761Arg (historical numbering) for 8% of Finnish alleles, Met1Thr is homozygous in 8 Arab patients from Jerusalem, Ala802Val retains 32% activity and is associated with persistent hyperglycinaemia with transient or absent symptoms, and a silent exonic variant that alters splicing (c.2607) causes an atypical form in 9 members of a large Bedouin family; in AMT, His42Arg was found in homozygosity in the 14 affected members of a consanguineous Israeli Arab family and can give ambiguous enzyme activity in chorionic villi [20, 21, 56, 57].

GeneReviews also lists as common in GLDC the variants c.1382G>A p.(Arg461Gln), c.2368C>T p.(Arg790Trp) and c.2714T>C p.(Val905Gly), with residual activity, c.2311G>A p.(Gly771Arg), and the variants without activity c.1009C>T p.(Arg337Ter), c.1270C>T p.(Arg424Ter), c.499G>T p.(Glu167Ter) and c.2919+1G>A; in AMT, c.2T>C, c.217C>T p.(Arg73Cys), c.471+2T>C, c.878-1G>A, c.887G>A p.(Arg296His) and c.921G>A, a synonymous variant that creates a new splice site [23]. These additional variants were transcribed from GeneReviews on transcript NM_000170.2 or NM_000481.2 and were not projected individually; for both genes, the c. numbering of the old transcript coincided with that of MANE for all variants checked [55].

Genotype-phenotype correlation. There are no clinical differences between GLDC and AMT [23]. Two variants without residual activity (exonic CNV, frameshift, nonsense, canonical splicing) always predict severe NKH; at least one variant with residual activity is required for the attenuated form; two variants with residual activity are associated with the best neurocognitive outcome [52, 58]. Residual activities of only about 1% in expression studies may suffice for an attenuated outcome [23]. The exceptions described in GLDC are c.2203-2A>G and c.2999delG p.(Cys1000LeufsTer31), a very distal frameshift that retains activity [23]. In T protein defects, overall GCS activity is about 10% according to Van Hove and Van Coster, and 20 to 25% according to Nyhan's Atlas and GeneReviews, through cycling via the P protein [23, 46, 52].

Variant NKH. Lipoate is a cofactor of the GCS H protein, pyruvate dehydrogenase (PDH), 2-ketoglutarate dehydrogenase, branched-chain keto acid dehydrogenase and 2-ketoadipate dehydrogenase [52]. LIAS, LIPT2 and GCSH deficiencies reduce the GCS; LIPT1 deficiency affects the E2 subunits without raising glycine; defects of iron-sulfur cluster biogenesis (BOLA3, NFU1, ISCA2, GLRX5, IBA57) also affect complexes I and II of the respiratory chain [52]. About 5% of patients with deficient GCS activity have no variants in GLDC or AMT and belong to this group, to PLP defects or to glycine transport defects [23, 52].

Glycine transport. Deficiency of the astrocytic glycine transporter GlyT1 (SLC6A9), due to biallelic variants, causes a glycine encephalopathy with raised CSF glycine and normal plasma glycine [23, 34]. Pyridox(am)ine 5′-phosphate oxidase (PNPO) deficiency reduces the PLP available for all PLP-dependent reactions, including the GCS P protein [52].

2.3. Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are part of the differential diagnosis.

Cofactor biosynthesis. Molybdenum cofactor (MoCo) is synthesised from GTP in four steps: cyclic pyranopterin monophosphate (cPMP, formerly "precursor Z"), molybdopterin (MPT), adenylated MPT and MoCo [30]. The protein encoded by MOCS1, which through alternative splicing gives rise to two proteins, converts GTP into cPMP (type A); MOCS2 encodes the two subunits of molybdopterin synthase, which converts cPMP into MPT (type B); MOCS3 is the sulfurase that reactivates molybdopterin synthase; gephyrin (GPHN) adenylates MPT and inserts molybdenum (type C) [30, 59]. Gephyrin also has a role in the formation of inhibitory synapses [34, 59].

Mechanism of injury. Four human enzymes depend on MoCo: sulfite oxidase, mitochondrial amidoxime reducing component, xanthine oxidase and aldehyde oxidase; loss of sulfite oxidase is necessary and sufficient for the neurological phenotype [30]. Sulfite oxidase catalyses the last step of cysteine and methionine catabolism, oxidising sulfite to sulfate and transferring the electrons to cytochrome c [30, 32]. Without it, sulfite accumulates in blood, urine and CSF, is converted into thiosulfate and reacts with cystine to form S-sulfocysteine [59]. Sulfite depletes intracellular ATP and impairs mitochondrial respiration in neuronal lines; S-sulfocysteine is stereochemically similar to glutamate and activates NMDA receptors, which is consistent with the MRI picture of energy failure and excitotoxic injury [30]. Sulfite breaks the cystine cross-links of the lens suspensory ligament, causing ectopia; secondary PLP deficiency, with elevation of urinary α-aminoadipic semialdehyde (α-AASA), may contribute to the seizures and causes confusion with pyridoxine-dependent epilepsy [30, 59]. Loss of xanthine oxidase in MoCD (and not in ISOD) raises xanthine and hypoxanthine and lowers uric acid [59].

Genes and proportions. Among published patients with MoCD, MOCS1 accounts for about 49%, MOCS2 for about 45%, GPHN for about 4% and MOCS3 for 2% [30]. The mechanism is loss of function, with no known genotype-phenotype correlation and no described allelic disorders [30]. Mild phenotypes are associated with certain MOCS1 and MOCS2 variants, including a MOCS2 allele prevalent in the Roma community, in which the variant rs776441627 had an allele frequency of 3.6% in a control group [59, 60]. In GPHN, homozygosity caused severe MoCD in two patients, but a heterozygous variant appears to have caused epileptic encephalopathy in an adult, and other heterozygous variants may predispose to epilepsy or hyperekplexia [34, 59]. OMIM classifies the GPHN variant proposed in hyperekplexia as of uncertain significance and records heterozygous deletions of exons 3–5 in 6 of 8,775 patients with autism, schizophrenia or epilepsy, versus 3 of 27,019 controls [61].

The recurrent European MOCS1 variants described in Nyhan's Atlas with historical protein nomenclature were searched for in ClinVar on transcript NM_001358530.2: p.R73W corresponds to c.217C>T p.(Arg73Trp), with conflicting classifications; p.R319Q to c.956G>A p.(Arg319Gln), likely pathogenic; p.G126D to c.377G>A p.(Gly126Asp), with conflicting classifications; p.R67W to c.199C>T p.(Arg67Trp), of uncertain significance in ClinVar [47, 62]. The variants MOCS1 c.1523_1524del and MOCS2 c.726_727del (written "c.726delAA" in the source), the latter responsible for 11 of 28 MOCS2 alleles in a European series, are kept in the Atlas notation, because they were not found in ClinVar with that spelling [47, 62]. OMIM confirms, in historical cDNA numbering, the MOCS1 deletion 1523_1524delAG in homozygosity in two Turkish patients and one Italian patient (frameshift at amino acid 90 of MOCS1B) and indicates that, in the series of 24 European and Israeli patients of Reiss and colleagues (variant detection in 70% of chromosomes), p.Arg319Gln was the most common variant, with 14% of alleles, found only in English patients [63]. MOCS1 (6p21.2) is bicistronic: exons 1–9 encode MOCS1A and exon 10 MOCS1B; in an Afghan patient with a mild form, the deletion c.1338delG left a functional MOCS1B fragment, which explains the residual activity [26, 63].

SUOX. SUOX (12q13.2) encodes a 466-amino acid molybdo-haemoprotein of the mitochondrial intermembrane space, with an N-terminal haem domain, a central molybdenum domain and a C-terminal dimerisation domain; the variants alter the active site, dimer stability or MoCo binding [32]. The MANE Select transcript is NM_001032386.2; the c. numbering coincides with that of transcript NM_000456.2 used in GeneReviews [55]. The variant described in a patient with paternal uniparental disomy of chromosome 12 is NM_001032386.2:c.1200C>G p.(Tyr400Ter); GeneReviews writes "c.1400C>G", which does not correspond to the reference sequence [32, 55]. Other selected variants are c.650G>A p.(Arg217Gln), c.794C>A p.(Ala265Asp), c.1589G>A p.(Gly530Asp) and c.520delG [32, 55].

2.4. Neonatal-onset developmental and epileptic encephalopathies

Genetic architecture. More than 100 genes cause neonatal-onset DEE; the most frequent are ARX (X-linked), SCN2A, SCN8A, STXBP1 and KCNQ2 (AD, de novo) [8]. Pathogenic variants are identified in more than half of patients with EIDEE, and neuroimaging, metabolic and genetic studies allow a precise aetiological classification in about 80% [4]. Channelopathies (SCN1A, SCN2A, SCN8A, KCNQ2, KCNT1) are a common cause of rare monogenic epilepsies, and the same genes can cause self-limited familial forms [7]. Other mechanisms include defects of the synaptic vesicle cycle (STXBP1, SPTAN1, TBC1D24, SIK1) and of vesicular trafficking (CNPY3, ATP6AP2, HCFC1) [3]. EIDEE with burst-suppression can result from biallelic variants in SLC25A22 (mitochondrial glutamate carrier) and early myoclonic forms from variants in ERBB4, PIGA, SETBP1, SIK1 or SLC25A22 [7]. Mitochondrial glutamate carrier deficiency (SLC25A22, AR, missense variants), with about 15 patients described, presents with epileptic spasms and migrating focal seizures of neonatal onset with burst-suppression, microcephaly, hypotonia, abnormal electroretinogram and severe psychomotor delay; MRI in infancy shows cerebellar hypoplasia and abnormalities of the corpus callosum, temporoparietal gyration and myelination of the temporal poles; diagnosis is mainly genetic (glutamate oxidation in fibroblasts is a possible functional test), there may be hyperprolinaemia and fibroblasts with lipid vacuoles, and there is no specific treatment [34]. In epilepsy of infancy with migrating focal seizures (EIMFS), KCNT1 accounts for 30 to 50% of cases, with SCN2A, SCN1A, SLC12A5, BRAT1 and TBC1D24 among more than 25 genes [4, 7].

KCNQ2. The Kv7.2 and Kv7.3 channels (KCNQ2, KCNQ3) generate the neuronal M current [8]. Haploinsufficiency (loss of function) causes the self-limited end of the spectrum (SeLNE); de novo missense variants in hotspots with a dominant-negative effect, with more marked loss of function, cause KCNQ2-DEE; and de novo gain-of-function variants cause a neonatal encephalopathy with non-epileptic myoclonus, progressive hypomyelination and volume loss [4, 8]. The direction of the functional change is therapeutically relevant, because channel openers only have a rationale in loss-of-function forms [8].

SCN2A, SCN8A, STXBP1, CDKL5, KCNT1. SCN2A accounts for 1 to 2% of all epileptic encephalopathies (3% of 400 patients with early-onset seizures in one series; estimated frequency of 1 in 78,608 births in Denmark) [39, 40]; gain-of-function variants cause early-onset DEE, with seizures from birth, and often respond to sodium channel blockers, often at high doses [4, 40]. STXBP1 takes part in the synaptic vesicle cycle (docking and release) [3]; the variants are mostly de novo and loss-of-function [9]. OMIM records a family with a homozygous variant (p.Leu446Phe) and AR inheritance, an asymptomatic father with somatic mosaicism (5.3 to 16.9% of alleles depending on the tissue) who transmitted the variant, and 9q34.11 deletions including STXBP1 and SPTAN1, found by array in 2 of 28 patients (7.1%) with cryptogenic early epileptic encephalopathy; in a review of 271 patients, the most common recurrent variants were p.Arg406His, p.Arg406Cys and p.Arg292His, and variants at Arg406 were more often associated with burst-suppression and spastic tetraplegia [38]. In SCN2A, gain-of-function variants are associated with onset before 3 months and response to sodium channel blockers, and loss-of-function variants with later onset and poor response, with exceptions; the same gene causes benign familial neonatal-infantile seizures (BFIS3, AD, clusters of seizures in the first year without sequelae) and episodic ataxia type 9, often preceded by neonatal or infantile seizures that remit [39, 64, 65]. In ARX (Xp21.3), DEE1 is X-linked recessive, with tonic seizures or spasms and burst-suppression that evolve into West syndrome in about 75%, in an allelic spectrum ranging from X-linked lissencephaly with ambiguous genitalia to non-syndromic intellectual disability; female carriers may be affected [66]. The semiological correlation described by the ILAE is as follows: sequential seizures with a tonic component or tonic seizures in KCNQ2; sequential seizures with tonic and autonomic features in SCN2A; focal seizures in SCN8A; asymmetric tonic or sequential seizures in STXBP1; sequential hyperkinetic-tonic-spasm seizures in CDKL5; focal tonic seizures with autonomic symptoms in KCNT1; and predominantly myoclonic seizures in UBA5 [4].

2.5. Severe X-linked forms in boys

In boys with severe neonatal encephalopathy, the sources recommend considering variants in ARX, IKBKG (NEMO) and MECP2 [2]. MECP2 encodes a methylated CpG dinucleotide-binding protein that maintains transcriptional repression; the spectrum includes girls with Rett syndrome, boys with severe neonatal encephalopathy and boys with intellectual disability and progressive spasticity [67]. Boys hemizygous for a MECP2 loss-of-function variant more often have a neonatal-onset encephalopathy with a relentless course, abnormal tone, involuntary movements, severe seizures, respiratory abnormalities and death often before 2 years of age [44]. The Xq28 duplication including MECP2 is a disorder of increased dosage, with early hypotonia, feeding difficulties, recurrent respiratory infections in about 75%, seizures in about 50% and complete penetrance in boys, and is not detected by single-gene sequencing [44, 67]. X-linked methylmalonic acidaemia with homocystinuria of the cblX type (HCFC1) causes neonatal seizures in boys with raised glycine [23].

2.6. Allelic disorders, modifiers and dual pathology

No other phenotypes associated with biallelic GLDC or AMT variants have been described; two studies suggested an association of heterozygous variants with neural tube defects, not observed in the parents of children with NKH [23]. Dextromethorphan metabolism depends on CYP2D6 and, to a lesser extent, on CYP3A4 and UGT1A1, which introduces pharmacogenomic variability in the response [52]. A newborn with a metabolic or genetic disease may tolerate labour poorly, so HIE and a genetic disease can coexist; genetic diagnoses have been found in newborns with a sentinel event, abnormal blood gases, low Apgar score and hypoxic injury on MRI [2, 68, 69].

3. Clinical Spectrum and Manifestations by System

3.1. Clinical repertoire of the newborn and patterns by class

The newborn has a limited repertoire of responses to severe illness (respiratory distress, hypotonia, poor suck, vomiting, diarrhoea, dehydration, lethargy, seizures), which are easily attributed to sepsis or another common cause; the death of an affected sibling may have been wrongly attributed to sepsis, heart failure or intraventricular haemorrhage [3]. Neonatal seizures can be difficult to recognise and may manifest as apnoeas, repetitive sucking movements, hiccups, subtle jerks or fasciculations; in newborns on anticonvulsants, sedated, paralysed and ventilated, neurological assessment is difficult [2].

Intoxication. The suggestive picture is that of a term newborn, with normal pregnancy and delivery, who after a symptom-free interval of hours to weeks deteriorates inexorably, without response to symptomatic therapy, with normal chest radiograph, CSF, septic screen and cranial ultrasound [3]. The first sign is usually poor suck, followed by unexplained coma; at an advanced stage autonomic abnormalities appear (respiratory abnormalities, hiccups, apnoeas, bradycardia, hypothermia) [3]. In coma, the movements are characteristic: hypertonic episodes with opisthotonus and slow "boxing" or pedalling movements in maple syrup urine disease; axial hypotonia with limb hypertonia, rapid large-amplitude tremors and myoclonus mistaken for seizures in organic acidaemias [3]. Because most non-metabolic causes of coma are accompanied by hypotonia, a "normal" peripheral tone in a comatose newborn reflects relative hypertonia [3]. Organic acidaemias and urea cycle disorders (UCD) typically present on days 2–3, with the protein load of feeding, and rarely have seizures without encephalopathy or hypoglycaemia [2]. The odour of maple syrup (maple syrup urine disease) or sweaty feet (isovaleric acidaemia, glutaric aciduria type II) guides the diagnosis [3].

Energy deficiency. No symptom-free interval in most, with severe generalised hypotonia, rapidly progressive neurological deterioration, possible dysmorphism or malformations, frequent hyperlactataemia and common cardiac and hepatic involvement; lethargy and coma are rarely the initial signs [3]. Severe peroxisome biogenesis disorders combine dysmorphism and severe neurological dysfunction; severe CDG, hypotonia, seizures, dysmorphism, malformations and visceral involvement; cellular trafficking disorders, congenital microcephaly with brain malformations [3].

Metabolic seizures. These are often a mixture of partial and erratic myoclonus of the face and extremities, or tonic seizures; the EEG frequently shows burst-suppression, but myoclonus may occur without an electroencephalographic correlate [3]. Severe NKH, GlyT1 deficiency encephalopathy and sulfite oxidase deficiency are the most frequent untreatable causes of severe neonatal epilepsy; D-glyceric acidaemia, very rare, shares most of the signs of NKH [3].

Hypotonia and hypertonia. The most severe forms of acute metabolic hypotonia occur in defects of pyruvate oxidation and of the respiratory chain, UCD, NKH, the sulfite oxidase/MoCo spectrum and peroxisomal disorders; severe hypotonia mimicking neuromuscular disease occurs in treatable IEM such as aromatic L-amino acid decarboxylase (AADC) deficiency, with paroxysmal limb movements and oculogyric crises, and in reversible myopathy due to specific mt-tRNAGlu variants [3]. Neonatal hypertonia is very common in sulfite oxidase deficiency and in hyperekplexia [3].

3.2. Nonketotic hyperglycinaemia

Forms of presentation. With neonatal onset (the majority), 85% progress to the severe form and 15% to the attenuated form; with onset between 2 weeks and 3 months, 50% to each form; onset after 3 months always corresponds to an attenuated form [23]. Severity (severe or attenuated) is consistent within families [23].

Neonatal NKH. The newborn is normal at birth; after an interval usually of up to 48 hours (from a few hours to 8 days), lethargy, food refusal and poor suck appear and progress to coma; 24 to 48 hours after the first symptom the newborn becomes flaccid, unresponsive and apnoeic [46]. In 80% the ventilatory drive slows, with prolonged apnoea and death if there is no intubation and ventilation; 20% maintain spontaneous ventilation [23]. The great majority recover spontaneous breathing within the first 3 weeks, even without glycine-lowering treatment; spontaneous weaning from the ventilator occurs between days 10 and 22 [23, 46]. Hiccups are frequent and persistent, often with a history of recurrent fetal hiccups; there are almost continuous myoclonus or tonic-clonic seizures, erratic eye movements, sometimes pinpoint pupils, and burst-suppression on EEG described as early as 30 minutes after birth [46, 52]. Some newborns have associated malformations (clubfoot, cleft lip or palate) [52]. At least 15 to 30% of patients with classic NKH die in the neonatal period, often after withdrawal of intensive care [52].

Course of severe NKH. Children may smile and roll over (developmental age of 6 weeks to 3 months), begin to lose skills at 3–4 months and never sit or grasp; before 6 months progressive spasticity and cortical blindness appear; most have swallowing dysfunction with tube feeding; seizures worsen in the first year and become intractable from the second, despite two or more anticonvulsants, with EEG evolution to hypsarrhythmia or multifocal spikes [23]. Scoliosis and hip dislocation are frequent; about 3% have a retrocerebellar cyst with hydrocephalus [23]. In a series of 12 patients with neonatal NKH (mean age 7 years), all but one had early and progressive neuromuscular scoliosis, all had contractures and talipes equinovarus, 5 hip dislocation and 4 pathological fractures, and 7 died of respiratory complications at a mean age of 8 years and 5 months; Van Hove and colleagues described 4 newborns with acute hydrocephalus requiring shunting, a poor prognostic sign, and Cataltepe and colleagues 2 newborns with classic NKH who died of pulmonary hypertension [20]. Rare systemic manifestations include slow gastric emptying and dysmotility up to pseudo-obstruction, sudden electrolyte disturbances (profound hypokalaemia with cardiac arrest in less than 1%), urinary retention and unexplained prolonged crying [23, 52].

Attenuated NKH. Developmental progress occurs in 15 to 20% of patients with neonatal onset and in 50% of those with onset in infancy [52]. Children with a good attenuated form (developmental quotient, DQ, above 50) have no epilepsy and can attend mainstream school; in the intermediate form (DQ 20–50) they walk, communicate by gestures and have chorea and episodic hyperactivity; in the poor form (DQ below 20) they grasp and sit, with epilepsy and spasticity [23]. Hyperactivity is common and responds poorly to drugs, expressive language is more delayed than receptive language, and febrile illness can trigger episodes of lethargy, chorea, delirium and vertical gaze palsy [23, 46]. An adult with attenuated NKH decompensated on valproate [23].

Variant NKH. It combines a picture similar to severe NKH with mitochondrial features: leukoencephalopathy (often cavitating), optic atrophy, cardiomyopathy, pulmonary hypertension and sometimes severe lactic acidosis [23, 52]. The table summarises the phenotypes by gene according to Van Hove and Van Coster [52].

Transient hyperglycinaemia and forms with minimal symptoms. Transient neonatal hyperglycinaemia has raised plasma and CSF glycine at birth that normalise within 2 to 8 weeks and, at onset, it is clinically and biochemically indistinguishable from typical NKH [20]. In three siblings homozygous for GLDC p.Ala802Val, diagnosed in the first 3 days, no neurological symptoms appeared and development was appropriate despite persistent hyperglycinaemia; atypical AMT forms may present as autism spectrum disorder with epilepsy and normal plasma amino acids [20]. These data justify caution in prognosis before the genotype and the course are known.

GlyT1 deficiency encephalopathy. Six patients described, with hyperekplexia, severe neonatal encephalopathy, arthrogryposis, dysmorphic features and abnormal imaging (thin corpus callosum, ventriculomegaly, cysts), poor respiratory drive and high lethality, usually before 1 year [23, 34]. In the families described in OMIM, prenatal ultrasound showed arthrogryposis (clubfeet with overlapping toes, clenched hands) and, in two fetuses, increased nuchal translucency; most were born preterm because of fetal distress, without respiratory effort, with severe axial hypotonia that evolved into hypertonia with stimulation-induced clonus; CSF glycine was raised with normal or slightly raised serum glycine, and the hyperekplexia tended to disappear in the first months [33].

3.3. Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency

Early-onset MoCD. More than 100 patients have been identified; in the early form, presentation occurs at a median of day 1 of life (1–50 days), usually after an uneventful pregnancy and delivery, although up to 40% have an Apgar score below 7 at one minute, with improvement at 5 and 10 minutes [30]. The absence of a perinatal sentinel event and the interval between birth and symptoms help to distinguish MoCD from HIE [30]. The table reproduces the frequency of signs in the early form [30].

Seizures are the most frequent initial sign, usually in the first 2 weeks, with subtle, clonic, tonic, myoclonic, multifocal or migrating partial semiology, and EEG with burst-suppression, multifocal discharges or hypsarrhythmia; they tend to decrease in frequency over time [30]. Brainstem function is usually preserved; the apnoeas of the first days are central or ictal [30]. Hypertrophic pyloric stenosis has been described in two patients with MoCD and two with ISOD [30]. In the case reported in Nyhan's Atlas, the newborn was well for 2 days, suddenly became cyanotic, mottled and hypotonic, required ventilation on day 2, had status epilepticus and then burst-suppression, cerebral atrophy at 10 days and, by the end of the first year, microcephaly without neurological development [47]. About 75% die in infancy from complications of neurological disability, mainly pneumonia [30].

Late-onset MoCD. Presentation at a median of 1 year (4 months to 23 years), often with acute neurological decompensation during an infection, with appendicular hypertonia (67%), axial hypotonia (58%), ectopia lentis (42%), dystonia (33%), seizures (33%), choreoathetosis (25%) and ataxia (17%) [30]. In a pooled natural history of 82 children with MoCD (Mechler and colleagues, 2015; 70% without a known molecular basis, 15% type A, 10% type B1, 6% type C), median onset was the first day of life, median age at diagnosis 4.5 months (median diagnostic delay of 89 days), the initial signs were seizures (72%), feeding difficulties (25%) and hypotonia (11%), there were affected siblings in 38%, 49% had died at the last assessment and overall median survival was 36 months [26]. The described facies includes a long face with full cheeks, widely spaced eyes, long palpebral fissures, thick lips, a long philtrum and a small nose; ectopia lentis may appear late, preceded by spherophakia, and some patients have macrocephaly due to hydrocephalus instead of microcephaly [26]. In type B2 (MOCS3), OMIM records a mild phenotype in an adolescent with intellectual disability, autism and a negative sulfite test, and a severe neonatal phenotype in a female infant with seizures on day 1, cystic encephalomalacia and death at 12 months, whose brother had died with a diagnosis of HIE [28, 70]. Misko and colleagues, analysing 146 published patients with MoCD or ISOD, separated a class I, with onset between 1 and 50 days with diffuse brain injury and cystic leukomalacia, and a class II, with onset between 30 days and 23 years with movement disorder and selective injury of the basal ganglia and cerebellum, with better survival [71].

Late-onset ISOD. Barbot and colleagues described a mild late-onset form in a 7-year-old Portuguese girl, daughter of first cousins, with ataxia, generalised dystonia, choreoathetosis and minimal language; in mild forms, the sulfite test may be negative and sulfate excretion normal, and clinical distinction from MoCD is not possible [31].

ISOD. The classic form presents in the first hours or days with intractable seizures, feeding difficulties and rapidly progressive encephalopathy (opisthotonus, spastic tetraplegia), progressive microcephaly, profound intellectual disability and usual death in the first months [32]. A fetus with sustained abdominal tremor in the third trimester, interpreted as fetal seizures, has been described [32]. Development stops at the level of brainstem reflexes, the facial dysmorphism is progressive and ectopia lentis appears in a significant proportion of those surviving the neonatal period [32]. The clinical picture and neuroimaging are similar to those of MoCD, except for the absence of xanthine stones [59]. The late form appears between 6 and 18 months, often after a febrile illness, with regression, dystonia, choreoathetosis, ataxia and, rarely, acute hemiplegia [32].

3.4. Neonatal developmental and epileptic encephalopathies

EIDEE. Tonic seizures are frequent, isolated or in clusters of 10 to 20 per day, focal or asymmetric in the newborn and longer than spasms (which last less than 3 seconds); erratic myoclonus is more often associated with a metabolic aetiology; spasms appear more often after the first month; and sequential seizures combine several manifestations [4]. Comorbidities include movement disorders, cortical visual impairment, orthopaedic and feeding problems, and increased early mortality; almost all progress to moderate to profound intellectual disability, except when early treatment is effective (vitamin B6-dependent epilepsies) [4]. EIDEE and West syndrome can evolve into Lennox-Gastaut syndrome [7].

KCNQ2 spectrum. SeLNE manifests as seizures between days 2 and 8, always after a symptom-free interval, in healthy newborns, with spontaneous remission between the 1st and the 6th–12th month and normal development; the seizures appear generalised (apnoea, cyanosis), but video-EEG shows focal onset with tonic stiffening and migration; about 30% will have seizures later in life [8]. KCNQ2-DEE begins in the first week, with encephalopathy from birth, focal tonic seizures with autonomic features several times a day and burst-suppression in more than 60%; the seizures usually stop between 9 months and 4 years, but the encephalopathy persists and the delay is moderate to profound [4, 8]. Neonatal MRI is normal or shows transient hyperintensities in the basal ganglia and globus pallidus, with later volume loss [4, 8]. About 10% (5/48) of infants with epileptic encephalopathy of onset before 3 months and burst-suppression have KCNQ2 variants [8]. In the series of Weckhuysen and colleagues, 8 of 80 patients (10%) with neonatal or early infantile seizures and delay had KCNQ2 variants, two mothers reported abrupt fetal movements in the last 2 months of pregnancy, and in one case the variant was inherited from a father with a mild phenotype and mosaicism; the same variant caused DEE in a son and seizures with remission and normal development in the mother [35]. Gain-of-function variants cause profound encephalopathy at birth, hypotonia, poor respiratory effort and myoclonus without a cortical correlate, with invariant burst-suppression [8]. Severe forms are associated with spasticity, dysphagia, gastro-oesophageal reflux, cortical visual impairment and hearing loss [8].

STXBP1. The median age at seizure onset is 6 weeks (from 6 hours of life to 13 years), with onset in the first year in 84.7 to 89%; in a series of 534 patients, 26% had electroclinical syndromes: infantile spasms (15%), Ohtahara syndrome (9%) and Rett phenotype (2%) [9]. In the review of 147 patients by Stamberger and colleagues, all had intellectual disability (severe to profound in 88%), 95% had epilepsy and 21% presented with Ohtahara syndrome [72]. Neonatal burst-suppression without seizures has been described [9].

SCN2A, CDKL5 and vitamin B6-dependent epilepsies. Early-onset SCN2A-DEE can manifest with uncontrolled seizures from birth and, in the case described in section 10, with prenatal arthrogryposis and polyhydramnios [40, 73]. CDKL5-DEE begins at a median of 6 weeks, with pronounced hypotonia, clusters of spasms and tonic seizures [4]. Pyridoxine-dependent DEE (ALDH7A1, PLPBP) and PLP-dependent DEE (PNPO) often present with neonatal distress, irritability, vomiting, acidosis and a low Apgar score, which leads to a wrong diagnosis of HIE; PNPO deficiency often occurs in preterm infants; intrauterine seizures, a hyperkinetic infant with multifocal myoclonus and spasms, and structural abnormalities on MRI in more than half are frequent [4]. One-third of patients with pyridoxine dependency have an atypical presentation and one-third have features or an initial diagnosis of asphyxia or HIE [2].

3.5. Severe MECP2 neonatal encephalopathy in boys

Affected boys present with a neonatal-onset encephalopathy with abnormal tone, involuntary movements, severe seizures and respiratory abnormalities, with a metabolic-degenerative type of course and death often before 2 years of age [44]. About 30 cases or families have been described [6]. In the review by Kankirawatana and colleagues (4 new boys and 11 from the literature), 7 of the boys had sisters or relatives with Rett syndrome, and all had neonatal encephalopathy, severe delay, respiratory failure, intractable seizures and abnormalities of tone and movement, with usually early death; asymptomatic carrier mothers had, in several families, X-inactivation skewed in favour of the normal allele [43]. Rett syndrome and the MECP2-CDKL5-FOXG1 spectrum are part of the differential diagnosis.

3.6. Neonatal encephalopathy cohorts: what the numbers show

These studies measure different things: proportions of findings in referral series, diagnostic yield in tested subgroups and outcome in therapeutic trials; none estimates the population incidence of genetic NE.

4. Clinical Recognition and Warning Signs

4.1. When to suspect a genetic or metabolic cause

Suspicion should begin at the moment the diagnosis of HIE is made, and not after the other hypotheses have been exhausted. The geneticist should doubt the diagnosis of HIE when the features of an acute perinatal insult are missing (fetal distress, cord acidosis, persistently low Apgar score, multiorgan failure) and when MRI does not show the hypoxic-ischaemic pattern [2]. In HIE, seizures usually begin 6 to 12 hours after birth; a newborn who is born well, feeds well and then deteriorates is more likely to have a metabolic cause [2]. In newborns with NE, the findings that favour a genetic cause are term birth, prominent seizures, MRI without typical hypoxic injury and the absence of a sentinel event; however, genetic diagnoses have been found in newborns with a sentinel event, abnormal blood gases, low Apgar score and hypoxic injury on MRI [68, 69, 76].

4.2. Urgent signs and samples that cannot wait

The therapeutic window of some diseases is measured in hours. In MoCD type A, fosdenopterin must be started within a short window after the first manifestations for maximum benefit, and cPMP was started within the first 24 hours of life in the patients with the best outcome; the distinction between MoCD and HIE is therefore urgent [30, 59]. In neonatal hyperammonaemia, the prognosis depends on the duration of coma and the peak ammonia, and in every newborn with a sepsis-like picture hyperammonaemia should be part of the initial differential diagnosis [16]. Methylmalonic and propionic acidaemia should be considered in the differential diagnosis of neonatal sepsis and of any acute neurological deterioration [77].

Before treatments that alter metabolites, and before a possible death, critical samples should be collected and stored: plasma, urine, blood on filter paper, CSF, DNA, skin biopsy for fibroblast culture and, if the newborn is unlikely to survive, frozen muscle and liver biopsies, clinical photographs and skeletal radiographs [2, 3]. These samples are often the only route to diagnosis and to counselling of the family.

4.3. Newborn screening

Newborn screening by tandem mass spectrometry (MS/MS) detects several metabolic causes in the differential diagnosis (organic acidaemias, some UCD, maple syrup urine disease), but the result may arrive after the neonatal decompensation; in 2015, 7 of 27 European Union countries screened for methylmalonic and propionic acidaemia [77]. Where a full acylcarnitine profile exists, checking the profile of a symptomatic newborn avoids delay [77]. NKH is not, in general, detected by MS/MS screening, and most newborns with very high blood glycine on screening do not have NKH [52]. There is no newborn screening for MoCD, ISOD or DEE in the sources consulted. Genomic newborn screening studies published in 2025 (more than 10,800 newborns in the USA, Belgium and Australia) showed that dried blood spot DNA is usable, with screen-positive rates of 1.6 to 3.7%, but scale, costs, outcomes and equity remain to be demonstrated [78]. The sources consulted do not describe the current panels of the Portuguese National Newborn Screening Programme or of the Brazilian National Newborn Screening Programme.

5. Diagnosis: Criteria, Tests and Algorithms

5.1. Principles

There are no formal diagnostic criteria for neonatal encephalopathy of genetic cause; the diagnosis results from the combination of history, examination, targeted biochemistry, neuroimaging, EEG and genetic testing, carried out in parallel with the investigation of non-genetic causes (infection, meningitis, infarction, haemorrhage, drugs) [2]. With first-line data, therapeutic recommendations should be possible within 2 to 4 hours, without waiting for specialised investigations [3]. For DEE, the ILAE criteria define electroclinical syndromes and do not replace the search for the aetiology [4].

History. Three-generation pedigree with consanguinity, seizures and neonatal or infant deaths; fetal movements (normal, reduced, or periodic bursts of repetitive activity or hiccups, which suggest prenatal seizures); maternal drugs (opiate withdrawal, valproate); third-trimester events (antepartum haemorrhage, death of a monozygotic co-twin); labour, fetal distress, Apgar score, resuscitation and cord gases; normality of the newborn before the symptoms and age at onset [2].

Examination. Head circumference; posture and tone; abnormal movements (tremor, myoclonus); large anterior fontanelle (Zellweger); dysmorphic features; abnormal odour; hepatomegaly or liver failure; cardiomyopathy [2].

5.2. Emergency investigation protocol

The table adapts the emergency protocol of Saudubray and García-Cazorla [3].

A few interpretation rules avoid frequent errors. Acetonuria in a newborn is always abnormal, although serum ketone bodies reach 0.5–1 mmol/L early in life [3]. Hypocalcaemia and glucose abnormalities are frequent in metabolic diseases and should not be taken as the sole cause of marked neurological dysfunction [3]. A normal blood pH does not exclude hyperlactataemia, because neutrality is maintained up to about 6 mmol/L of lactate with bicarbonate above 18 mmol/L [3]. Raised ammonia can induce respiratory alkalosis; propionic, methylmalonic and isovaleric acidaemia can cause neutropenia and thrombocytopenia that may be mistaken for sepsis [3]. At an advanced stage, respiratory acidosis, severe hyperlactataemia and secondary hyperammonaemia obscure the original profile, especially in severe UCD [3]. The dinitrophenylhydrazine test for α-keto acids, still listed in the Oxford Desk Reference, has been largely abandoned according to Saudubray [2, 3].

5.3. Biochemical classification into six types

With first-line results, most newborns with neurological deterioration of metabolic cause can be classified into one of six overlapping types [3]. Reference values used: ammonia <80 µmol/L, lactate <1.5 mmol/L, glucose 3.5–5.5 mmol/L.

Type VI cannot be screened for by the emergency protocol, and for many diseases in this group, in particular those of cellular trafficking, DNA testing is the only diagnostic route [3].

5.4. Markers by neurological presentation and diseases without first-line abnormality

The MetBioNet table of metabolic diagnoses reproduced in the Oxford Desk Reference (2009 guideline, outside the review period) adds glutaric aciduria type 1, in which urinary organic acids and acylcarnitines are not always positive, homocystinuria and remethylation defects (total homocysteine), and tyrosine hydroxylase deficiency (CSF neurotransmitters) [2]. In newborns with dysmorphic features or seizures, the Oxford Desk Reference also recommends plasma biotinidase, VLCFA, transferrin isoelectric focusing, cholesterol and 7-dehydrocholesterol, lysosomal studies, CSF pipecolic acid, urinary α-AASA and CSF PLP, followed by a therapeutic trial of pyridoxine [2].

5.5. Neonatal hyperammonaemia

Severe hyperammonaemia (above 500 µmol/L) is usually due to a UCD (respiratory alkalosis, no ketosis, no myelosuppression) or an organic acidaemia (metabolic acidosis, ketosis, neutropenia and thrombocytopenia) [3]. Plasma glutamine is usually above 1,000 µmol/L in UCD and near normal or low (below 500 µmol/L) in organic acidaemias; citrulline distinguishes mitochondrial from cytoplasmic UCD [3]. Hyperammonaemia with lactic acidosis above 6 mmol/L and hyperketosis suggests pyruvate carboxylase deficiency, multiple carboxylase deficiency or carbonic anhydrase VA deficiency; severe hypoketotic hypoglycaemia with ammonia usually below 250 µmol/L suggests hyperinsulinism-hyperammonaemia syndrome (GLUD1) or a fatty acid oxidation defect [3]. Mild elevations, below 150 µmol/L, also occur in maple syrup urine disease, PDH deficiency and on valproate; in preterm infants there is transient hyperammonaemia of the newborn, with normal glutamine [3, 16]. The 2019 revision of the European UCD guideline, with recommendations graded by the GRADE method (Grading of Recommendations Assessment, Development and Evaluation), indicates that most newborns with UCD have ammonia above 500 µmol/L at presentation, that a normal ammonia practically excludes a UCD in a symptomatic newborn (but not in an older child), that respiratory alkalosis is present in up to 50% of acute UCD and should prompt immediate measurement of ammonia, and that neonatal sepsis is the most frequent misdiagnosis; it strongly recommends considering a UCD in any acute neurological deterioration and in the differential diagnosis of neonatal sepsis (moderate quality of evidence), measuring ammonia as an emergency test taking pre-analytical errors into account (high quality) and, if raised, collecting amino acids and acylcarnitines and requesting urinary organic acids and orotic acid, starting treatment without waiting for the results, which should be available within 24 hours [80]. The same revision suggests an alert in the electronic medical record to measure ammonia in newborns aged 2 to 7 days in whom blood gases are performed [80]. The bedside differential diagnosis of the 2012 edition, extended in 2019 to carbonic anhydrase VA deficiency, HMG-CoA lyase deficiency, TMEM70, phosphoenolpyruvate carboxykinase deficiency and SERAC1, is summarised in the following table [16, 80].

Citrin deficiencies, lysinuric protein intolerance, ornithine aminotransferase deficiency and oxidative phosphorylation defects also cause hyperammonaemia, and the metabolite pattern is more informative than isolated values [80]. The biochemical algorithm for UCD uses plasma amino acids and urinary orotic acid: raised arginine in arginase deficiency; raised argininosuccinic acid in argininosuccinate lyase deficiency; raised citrulline without argininosuccinic acid in argininosuccinate synthetase deficiency; low citrulline and arginine with raised orotic acid in ornithine transcarbamylase deficiency; low citrulline and arginine with low orotic acid in carbamoyl phosphate synthetase 1 or N-acetylglutamate synthase (NAGS) deficiency; raised ornithine with homocitrullinuria in HHH syndrome [16]. The 2019 revision sets out the specific patterns: plasma arginine above 300 µmol/L in arginase 1 deficiency; plasma or urinary argininosuccinic acid in argininosuccinate lyase deficiency; high citrulline without argininosuccinic acid in argininosuccinate synthetase deficiency; high urinary homocitrulline in HHH syndrome; and high plasma ornithine in ornithine aminotransferase deficiency [80]. It strongly recommends genetic testing to confirm the diagnosis and storing DNA, fibroblasts or frozen liver from deceased patients with suspected UCD (moderate quality); enzyme assay is reserved for when genetic testing is negative, and array comparative genomic hybridisation, RNA sequencing and MLPA (multiplex ligation-dependent probe amplification) increase the yield [80]. On newborn screening, it recommends considering it for argininosuccinate synthetase and lyase deficiencies and regards the data as insufficient for NAGS, carbamoyl phosphate synthetase 1, ornithine transcarbamylase and arginase 1 deficiencies and HHH syndrome; in severe neonatal forms, the result often arrives after onset [80].

5.6. Neuroimaging: hypoxic-ischaemic pattern and genetic patterns

MRI is better than cranial ultrasound for cortical abnormalities and intraparenchymal haemorrhage; computed tomography is only justified in birth trauma or high suspicion of haemorrhage, because of the radiation [2]. In the first 24 hours MRI may be normal; the earliest changes are on diffusion, in the first 5 days; on conventional imaging, lesions are best seen between days 5 and 14; atrophy associated with a perinatal insult does not appear before 2 weeks, and its earlier presence suggests a prenatal cause [2]. Acute HIE is associated with lesions of the basal ganglia and thalamus, whereas white matter or cortical changes suggest more chronic injury or a non-hypoxic-ischaemic cause [2].

The Oxford Desk Reference mentions agenesis of the corpus callosum in NKH, but GeneReviews describes the corpus callosum as thin and short rather than absent; gyral malformations and true agenesis suggest variant NKH or PLP disorders [2, 23].

5.7. EEG and amplitude-integrated EEG

Amplitude-integrated EEG (aEEG), with 1 to 2 channels, assesses background activity (loss of sleep-wake cycling, moderate or severe suppression, burst-suppression) and is sensitive for seizures, especially with electroclinical dissociation or under sedation, but the background is influenced by medication [2]. The 2023 ILAE guideline defines three levels of certainty: level 1, conventional EEG; level 2a, aEEG; level 2b, observation by an experienced clinician of focal clonic or tonic seizures, the only ones that can be diagnosed clinically [13]. Burst-suppression consists of high-voltage bursts (150–300 µV) of spikes, sharp and slow waves lasting 1 to 5 seconds, alternating with marked suppression (below 5 µV) of 3 to 10 seconds, in wakefulness and sleep, without reactivity [4]. In KCNQ2-DEE, burst-suppression may only appear in sleep, which justifies video-EEG with sleep recording [8]. Prolonged video-EEG with electromyography distinguishes paroxysmal movement disorders (non-epileptic myoclonus, hyperekplexia) from seizures [4, 30].

5.8. Diagnosis of nonketotic hyperglycinaemia

Biochemistry. Glycine should be measured in plasma and CSF, collected simultaneously, by quantitative amino acid analysis [23]. Raised CSF glycine is the preferred test, with a sensitivity above 99%; plasma glycine is often the first clue, but has incomplete sensitivity and low specificity [52]. The CSF/plasma ratio is a secondary measure, valid only when CSF glycine is raised [23, 52]. Nyhan's Atlas requires raised plasma glycine for the diagnosis; GeneReviews and Van Hove and Van Coster favour CSF glycine [23, 46, 52]. Urine is not suitable for screening for NKH, because excretion may be normal in fasting patients on parenteral fluids [46].

The values come from the series of Swanson and colleagues (124 patients) reproduced in GeneReviews [23, 58]. All patients with severe classic NKH have a ratio above 0.08, and the lowest CSF glycine value in the series of 124 patients was 40 µmol/L [52].

Pitfalls. Contamination of CSF with blood or serum, invisible to the naked eye, falsely raises glycine and the ratio: a normal red cell count and normal protein are required, otherwise the result is invalid [23, 52]. Valproate inhibits the GCS and can raise CSF glycine above 60 µmol/L [23, 52]. CSF threonine should not be raised (its elevation suggests PNPO deficiency), and a normal CSF PLP helps to exclude PLP disorders [23]. There are very rare cases of attenuated NKH with intermittently normal CSF glycine [23]. Plasma glycine also rises with glycine-buffered intravenous immunoglobulin, in disseminated neonatal herpes infection, in severe liver failure (above 2,000 µmol/L) and in organic acidaemias (these with a normal ratio) [23, 52].

Plasma glycine at birth and screening. In a newborn with a prenatal diagnosis, plasma glycine was normal at birth, presumably because of placental clearance, while CSF glycine was very high; a normal plasma glycine in the first hours does not exclude NKH [20]. In the New South Wales newborn screening programme (733,527 newborns over 8 years), only 2 of the 9 newborns with NKH had glycine above the cut-off value, and both were already symptomatic in the first 72 hours [20].

Imaging. The diffusion pattern is always present in the first months and its absence indicates a phenocopy; it does not predict the phenotype [23, 52].

Genetics and enzyme. Confirmation is now mainly molecular, with a panel that includes GLDC, AMT and GCSH, the genes of the differential diagnosis and deletion and duplication analysis; sequencing detects more than 99% of AMT variants and about 80% of GLDC variants, and CNV analysis the remaining 20% of GLDC [23]. Neonatal or infantile epilepsy panels often do not include GLDC and AMT unless specifically requested [23]. GCS activity is measured in liver (100–300 mg of tissue, rapid processing and deep freezing) or trophoblast, and not in fibroblasts; transformed lymphoblasts are not reliable [2, 23, 46]. The glycine exchange reaction identifies the affected subunit, and the 1-¹³C-glycine breath test measures exhaled ¹³CO2 [23, 52]. In variant NKH, glycine is only slightly raised, with increased alanine, lactate, pyruvate and urinary 2-ketoglutarate, and tissue studies show PDH deficiency and reduced lipoylated H protein [52].

5.9. Diagnosis of molybdenum cofactor deficiency and isolated sulfite oxidase deficiency

Confirmation is molecular, by neonatal seizure panel or exome/genome; sequencing detects more than 95% of variants in the four MoCD genes, and for GPHN there is a commonly deleted region in exons 3–5 [30]. In ISOD all described variants have been identified by sequencing and no exonic deletion has been described [32]. Sulfite oxidase activity in fibroblasts is an alternative when molecular testing is not available, with difficulty in distinguishing total from partial loss; xanthine oxidase is not expressed in fibroblasts [30, 47]. When MoCD is suspected with compatible laboratory findings, treatment should begin before genetic confirmation [30]. OMIM stresses that low serum uric acid may be a better clue than the sulfite test, which was negative in described cases, that the absence of urinary urothione (a MoCo degradation product) supports the diagnosis, and that plasma cystine may be absent, as in a newborn presenting with lactic acidosis and hypoglycaemia [26]. In MOCS2, the 2-bp deletion at position 726 accounted for 7 of 14 alleles identified in the series of Reiss and colleagues [27, 81]. OMIM records the SUOX variants in historical protein numbering, offset by 57 residues relative to the MANE transcript: Arg160Gln (historical numbering, = NM_001032386.2:c.650G>A p.(Arg217Gln)), Ala208Asp (historical numbering, = NM_001032386.2:c.794C>A p.(Ala265Asp)) and Gly473Asp (historical numbering, = NM_001032386.2:c.1589G>A p.(Gly530Asp)), ClinVar equivalences confirmed in VariantValidator [82].

5.10. Investigation of EIDEE and of vitamin B6-dependent epilepsies

The ILAE recommends strongly considering metabolic studies, especially when there is no structural abnormality: urinary organic acids and amino acids (including S-sulfocysteine), urinary α-AASA, plasma amino acids, lactate, uric acid, copper and caeruloplasmin, ammonia, acylcarnitines, transferrin isoelectric focusing, VLCFA and, in CSF, glucose, lactate, pyruvate, amino acids and neurotransmitters [4]. On the genetic side, chromosomal microarray, karyotype (ring chromosome 14), gene panel, exome or genome, indicating the phenotypic features to the laboratory [4]. In vitamin B6-dependent DEE, raised α-AASA and pipecolic acid in urine, plasma and CSF should be collected before pyridoxine, without delaying therapy, and usually remain raised after treatment; α-AASA is the most reliable; if only one ALDH7A1 variant is found, look for deletions and duplications by multiplex ligation-dependent probe amplification (MLPA) or microarray; diagnostic withdrawal of pyridoxine is obsolete [4]. The therapeutic trial can begin before the samples are collected without affecting the results, and a partial or transient response to anticonvulsants does not exclude vitamin B6-dependent epilepsy [13].

5.11. Genetic tests

Choice of test. Genome sequencing (GS) is preferred in the neonatal setting because it captures non-coding variants, structural variants (SV), repeat expansions and mitochondrial variants in a single test, as a trio and with a rapid turnaround [1, 12]. The position statement of the Human Genetics Society of Australasia (HGSA, 2024) recommends GS, and not the exome, as the first-line genomic test in critically ill children with a high suspicion of monogenic disease; it recommends trio testing, without excluding testing when the parents are not available; and it regards rapid testing as the standard of care in that context [12]. The American College of Medical Genetics and Genomics (ACMG, 2021) guideline strongly recommends considering the exome or genome as a first- or second-tier test in patients with congenital anomalies with onset before 1 year or with developmental delay or intellectual disability; it is not specific to the NICU or to NE [83]. The strong recommendation is based on a systematic review with formally very low certainty of evidence (observational studies), on the Ontario health technology assessment and on the randomised NSIGHT2 trial [83]. In the meta-analyses, the exome or genome changed short-term clinical management in 8% (95% CI 6–11; 25 studies) and long-term management in 10% (95% CI 7–15; 19 studies) of all patients tested, had reproductive consequences in 9% (95% CI 4–21) and an impact on relatives in 4% (95% CI 2–9); in the Ontario assessment, the diagnostic yield of expanded genomic sequencing was 38%, versus 21% for conventional genetic tests (relative risk 1.76; 95% CI 1.20–2.58), higher with the genome (43%) than with the exome (34%) [83]. In NSIGHT2, rapid genomic sequencing changed management in 57 infants (28%) admitted to intensive care, mainly with the ultra-rapid version and with positive results, and rarely caused confusion or distress in families (6 of 207; 3%) [83]. The guideline stresses that, in patients requiring acute treatment, rapid testing can avoid extensive investigations, make the patient eligible for targeted therapy and clarify the prognosis for decisions on the goals of care; the harms described were rare (misattributed paternity, refusal of transplantation for economic reasons), and genetic counselling before and after the test is considered essential [83]. The Oxford Desk Reference recommends early panel, exome or genome testing in infantile epilepsy, and genomic array in complex epilepsies or those with developmental delay [7]. In typical familial SeLNE, targeted KCNQ2 testing may be justified, but KCNQ3 should be tested at the same time [8].

Smaller studies in NE show yields of 36% (trio exome in 14 newborns from Toronto, with SCN2A, KCNQ2, GNAO1, LIAS and CUL4B), 41% (trio genome in 17 Canadian newborns), 25% (exome or genome in 24 newborns with a diagnosis of HIE, with KIF1A, GBE1, ACTA1 and 15q13.3 deletion among the phenotypes mimicking HIE) and 32.4% (panel in 34 Korean newborns with hypoxic injury, with CACNA1A, KCNQ2, SCN2A, SCN8A, STXBP1 and others) [10, 11, 68, 69]. With a gene panel, the diagnostic rate in epilepsies with onset before 2 months was about 40% in a UK cohort [7].

Limitations of the tests. A negative result does not exclude genetic disease. Panels may not include the relevant genes (in GEMINI, 24.6% of the variants missed by the panel were in genes outside the panel and 25.1% were SV larger than 1 kb) [84]. The exome and single-gene sequencing detect CNV poorly, which make up 20% of GLDC alleles, and the MECP2 duplication, for which array is the first-line test [23, 44]. The GLDC pseudogene can cause alignment artefacts [23]. Variants in the 5′ untranslated region of AMT escape analyses restricted to the coding region [52]. Low-level parental mosaicism in KCNQ2 (allele frequencies of 1 to 30%) requires sufficient read depth [8]. Uniparental disomy explains homozygosity with only one carrier parent in MoCD and ISOD [30, 32]. The yields of the studies are not directly comparable, because they differ in inclusion criteria, trio versus proband-only testing and the inclusion of VUS and CNV [1]. The proportion of variants detectable by a method is not equivalent to the clinical sensitivity of the test for the disease.

Report and interpretation. The report should state the gene, the transcript and its version (preferably MANE Select), the variant in HGVS nomenclature, the zygosity, the phase (cis or trans) when known and the class according to the criteria of the ACMG and the Association for Molecular Pathology (ACMG/AMP). A VUS neither confirms nor excludes the diagnosis; it should be interpreted together with biochemistry and imaging (for example, CSF glycine and the diffusion pattern in NKH) and, when possible, by family segregation or functional studies [12, 23, 52]. In NKH, more than 90% of missense alleles in new cases had already been described, and the Leiden Open Variation Database (LOVD) brings together more than 500 families [52]. Pre-test counselling should explain the possible results (pathogenic variant, possibly of a life-limiting disease; VUS; no diagnosis; incidental finding), and post-test counselling should include natural history, management, inheritance, recurrence risk and, in the face of a negative result, the limitations of the test [12]. Consent should be explicit, ideally with counselling by a genetic counsellor and without implicit coercion, and the result should be delivered with a written summary in plain language [12].

5.12. Diagnosis after death

In a level IV NICU in Boston, among 170 children admitted between 2011 and 2015 who died before 5 years of age, 28% had a confirmed genetic diagnosis, and 30% of those diagnoses were made after death [86]. In the USA, among 517 infants who died before 1 year (2011–2017), 22% had a genetic diagnosis, but only 53% of these had the underlying cause coded under the ICD-10 codes for congenital malformations and chromosomal abnormalities (Q00–Q99), which underestimates mortality from genetic disease, especially monogenic disease, in vital statistics [87]. When the newborn is unlikely to survive, DNA, blood on filter paper, frozen plasma and urine, CSF, fibroblasts, muscle and liver biopsies frozen at −70 °C, photographs and radiographs should be stored; for NKH, a liver biopsy taken immediately after death and rapidly frozen in liquid nitrogen allows the GCS to be measured [2]. Autopsy is essential, including in fetal loss with suspected IEM, and the concept of the "metabolic autopsy" allows fluids and tissues to be collected for biochemical confirmation [3].

5.13. Practical algorithm

6. Differential Diagnosis

6.1. Comparative table of the main causes

The sources for this table are those listed by entity in sections 2 to 5 and the differential diagnosis tables of the GeneReviews entries on NKH, MoCD and ISOD, of the Oxford Desk Reference and of the ILAE [2, 4, 23, 30, 32, 34]. The features of cholesterol biosynthesis defects (cleft palate in 37–52%, heart disease in 36–38%, hypospadias or cryptorchidism in 90–100% of boys, syndactyly of toes 2–3 in more than 95%) come from the Oxford Desk Reference [2].

6.2. HIE and genetic mimics

The distinction between HIE and genetic encephalopathy is not dichotomous. Genetic diseases can reduce tolerance to labour and worsen a real hypoxic-ischaemic insult [2, 45]. Therefore, the presence of a sentinel event, cord acidosis or a low Apgar score does not exclude a genetic cause, and genetic diagnoses have been made in newborns with isolated HIE and without comorbidities [68]. In the HEAL trial, infants with HIE and a genetic or congenital anomaly had encephalopathy severity, EEG and MRI similar to the others, but a worse outcome at 2 years [75]. The features that should prompt the search are: MRI without the hypoxic-ischaemic pattern or with signs of prenatal injury; prominent seizures out of proportion to the injury; atypical course after hypothermia; dysmorphism or malformations; and family history [2, 30, 76]. Therapeutic hypothermia, if started, can be stopped before 72 hours when the diagnosis of HIE is excluded (section 7.1) [2].

6.3. Hyperglycinaemia: NKH, phenocopies and other causes

The most important phenocopy is transient glycine encephalopathy due to hypoxic-ischaemic or haemorrhagic injury, through disruption of the blood-brain barrier; the follow-up described ranges from normal development to mild intellectual disability at school age [23, 46]. The absence of the diffusion pattern typical of NKH points to a phenocopy [52]. A slight elevation of CSF glycine has also been described in vanishing white matter disease [23].

6.4. MoCD, ISOD and vitamin B6-dependent epilepsies

MoCD and ISOD are clinically identical in the neonatal period and are distinguished by serum uric acid (low in MoCD, normal in ISOD) and by urinary xanthine and hypoxanthine (raised only in MoCD) [30, 32]. Both are part of the differential diagnosis of vitamin B6-dependent epilepsies, with the added complication that α-AASA may be raised in some patients with MoCD or ISOD [30, 59]. In ALDH7A1 epilepsy, MRI may show a thin corpus callosum and mega cisterna magna, but not diffuse restricted diffusion; in PNPO deficiency there is raised lactate, hypoglycaemia and normal to raised glycine and threonine; in PLPBP deficiency, raised lactate and metabolic acidosis [30]. NKH is distinguished by glycine and by restricted diffusion in the areas undergoing myelination at birth [32]. In late-onset MoCD, the differential diagnosis includes cerebral palsy, juvenile Huntington disease, early-onset Parkinson disease, Lesch-Nyhan syndrome, progressive supranuclear palsy and Wilson disease [30].

6.5. Self-limited neonatal seizures, DEE and hyperekplexia

In SeLNE there is always a symptom-free interval, seizures begin between days 2 and 7, development is normal and the EEG background is normal or mildly abnormal, without burst-suppression; the exclusion of spasms, myoclonic seizures and generalised tonic seizures helps to separate it from KCNQ2-DEE [4, 8]. The family history may require interviewing older relatives [8]. When onset is in the first days of life of a term newborn, KCNQ2 is the most frequent gene; if seizures begin later in the neonatal period, the probability of other genes increases [8]. Treatable neurometabolic diseases causing early epileptic encephalopathy should always be considered before accepting a DEE without aetiology: vitamin B6-dependent epilepsies, biotinidase deficiency, GLUT1, creatine deficiencies, holocarboxylase synthetase deficiency and serine deficiencies [9].

Hereditary hyperekplexia manifests as generalised stiffness soon after birth, which increases with handling and disappears in sleep, excessive startle to unexpected stimuli and a short period of stiffness after the startle, with head retraction on tapping the tip of the nose, hernias and congenital hip dislocation; it can be confused with seizures and there is a risk of sudden death during the episodes [7, 34]. Inheritance is usually AD with almost complete penetrance; GLRA1 detects about 80% of cases and, without a dominant history, the panel detects about 20%, mainly recessive; the diagnosis is clinical and supported by the response to clonazepam [34]. In MoCD, hyperekplexia (9%) with tonic spasms can cause apnoea without an electrographic seizure, which makes video-EEG necessary [30].

7. Treatments and Risk Management Strategies

7.1. General principles, therapeutic hypothermia and intensive care

The treatment of neonatal encephalopathy of genetic cause combines common measures (ventilatory and haemodynamic support, seizure control, correction of metabolic abnormalities, nutrition) with specific therapies that depend on the diagnosis and, in several cases, on time. When an IEM is suspected, supportive measures and investigations proceed simultaneously; early referral to a specialised metabolic centre is recommended in all intoxication disorders and in MoCD [3, 16, 30, 77].

Therapeutic hypothermia for 72 hours is the standard of care in term HIE and should begin as early as possible; in cooled newborns, other causes should be considered, especially if the picture is not typical [2]. If the diagnosis of HIE is excluded, it is reasonable to rewarm before 72 hours; if there are signs of an additional hypoxic-ischaemic insult, cooling should continue; there is no evidence of benefit or harm from cooling in non-hypoxic-ischaemic causes [2]. The ILAE considers that hypothermia may reduce the seizure burden in term newborns with HIE (weak evidence) [13]. In preterm infants of 33 to 35 weeks with moderate or severe HIE, the trial of the Neonatal Research Network of the National Institute of Child Health and Human Development (NICHD) (168 newborns, 2015–2022) showed no reduction in death or disability with hypothermia (35% vs 29%; adjusted relative risk 1.11; 95% credible interval 0.74–2.00), with a Bayesian probability of 87% of increased mortality [88].

7.2. Treatment of neonatal seizures

ILAE recommendations (2023). The guideline of the Task Force on Neonatal Seizures (systematic review, GRADE and Delphi) is the reference for neonatal doses [13].

The ILAE considers that features such as the absence of another aetiology, tonic or sequential seizures, normal ultrasound and MRI and certain EEG patterns suggest a channelopathy; it warns that sodium channel blockers may increase seizures in rare neonatal-onset channelopathies due to SCN1A loss of function [13]. Brivaracetam, oxcarbazepine and lacosamide have no controlled studies in the newborn and their use is not recommended [13]. Each neonatal unit should have a standardised protocol, and parents should be informed about the seizures, the possible aetiologies and the options [13].

Neonatal doses. The table reproduces the doses suggested by the ILAE (approved by Delphi with 84% agreement) and compares them with the labels where these exist [13, 89–91].

The Sezaby® label data come from the NEOLEV2 trial, and the pyridoxine precautions include those of the ILAE and of the ILAE 2022 (rare neuropathy with doses up to 200 mg/day in prolonged treatment) [4, 13, 89]. The ILAE table refers to the term and preterm newborn and only sets maximums for phenytoin (7.5 mg/kg per dose) and midazolam (5 µg/kg/min); for phenobarbital, levetiracetam, lidocaine (fixed-duration infusion schedule, 28 hours in total), pyridoxine and PLP there is no maximum dose defined in the source, and the Sezaby® label limits the total phenobarbital loading dose to 40 mg/kg [13, 89]. The response to pyridoxine or PLP may be delayed, and the ILAE recommends continuing the trial for at least 3 to 5 days before concluding that it is ineffective; other authors repeat doses up to 500 mg in total [13]. Vitamin B6-dependent DEE have their own maintenance dose and may require folinic acid.

Channelopathies: the gene changes the choice of drug. In KCNQ2-DEE, seizures typically do not respond to phenobarbital, and the ILAE recommends considering sodium channel blockers early [4]. In the series of Pisano and colleagues, 15 newborns with KCNQ2-DEE and seizure onset between days 1 and 4 received several drugs before becoming seizure-free: 6 (40%) were controlled within 2 weeks of carbamazepine and 5 (33%) with phenytoin; the two patients whose seizures stopped within a few days had only mild cognitive impairment [37]. In the systematic review by Kuersten and colleagues (52 studies, 217 patients), 92.5% of self-limited forms and 65.5% of early encephalopathies became seizure-free (p=0.003), with spontaneous remission without medication in 30.1% of self-limited forms; phenobarbital and sodium channel blockers were those that most often led to seizure freedom [92]. In 19 newborns with familial neonatal epilepsy (14 with KCNQ2), 17 (88%) became seizure-free within a few hours of oral carbamazepine or oxcarbazepine, without adverse effects, and earlier initiation was associated with a shorter hospital stay [93]. In an Australian series of 37 children with KCNQ2 neonatal epilepsy, the 6 diagnosed in less than 30 days all received a sodium channel blocker in the neonatal period (versus 48% of the others) and were all seizure-free at 12 months (versus 54%; p=0.049), with no demonstrated effect on neurodevelopment [94]. In SCN2A-DEE and SCN8A-DEE, response to sodium channel blockers is frequent, often at high doses [4]. In contrast, in rare neonatal-onset channelopathies due to SCN1A loss of function, sodium channel blockers may increase seizures, which makes rapid genetic testing a direct determinant of the therapeutic choice [13].

Discontinuation of ASM. In the prospective cohort of the Neonatal Seizure Registry (284 newborns with acute symptomatic seizures, 9 US centres, born in 2015–2018), discontinuation of ASM before discharge was not associated with worse functional development or more post-neonatal epilepsy up to 8 years of age (adjusted hazard ratio 0.93; 95% CI 0.48–1.80) [95]. This finding applies to acute symptomatic seizures and not to neonatal-onset genetic epilepsies, in which the duration of treatment depends on the aetiology.

7.3. Other therapeutic trials and corrections

A pyridoxine trial is justified in all intractable early-onset seizures, whatever the suspected cause [2]. In the undiagnosed patient with hyperammonaemia, the 2012 European guideline, maintained in the 2019 revision, suggests considering levocarnitine 100 mg/kg/day IV, hydroxocobalamin 1 mg/day intramuscularly or IV and biotin 10 mg/day IV or orally [16, 80]. Congenital magnesium malabsorption, a cause of seizures through severe hypomagnesaemia and hypocalcaemia, responds to magnesium supplementation [2, 3]. Serine synthesis deficiencies respond to serine, GLUT1 deficiency to the ketogenic diet, biotinidase deficiency and holocarboxylase synthetase deficiency to biotin, and congenital hyperinsulinism to treatment of the hypoglycaemia [3]. Some patients with SLC13A5 respond to acetazolamide, and some with KCNQ2 to vitamin B6 [3].

7.4. Neonatal hyperammonaemia

The prognosis depends on the duration of coma and the peak ammonia; treatment cannot be postponed and paediatric hospitals should have the first-line drugs and written protocols [16]. Before transfer to a specialised centre: stop protein (24 hours, maximum 48), give 10% glucose IV (10 mg/kg/min in the newborn), start the first-line drugs and collect plasma and urine without delaying treatment [16]. The 2019 revision strongly recommends immediately starting the measures that reverse catabolism and promote ammonia detoxification (moderate quality) and transferring without delay to a specialised centre; the action thresholds and consensus doses remain those of the 2012 edition (grade D), with the additions indicated in the tables [16, 80]. Ammonia above 1,000 µmol/L, coma lasting more than 3 days and significant intracranial hypertension indicate a very poor prognosis, but ammonia alone is not an absolute criterion: there are patients with a normal outcome despite initial values above 1,000 µmol/L [80].

In severe decompensations, benzoate and phenylbutyrate or phenylacetate are given in parallel; in less severe cases, one can start with benzoate and add the second scavenger if hyperammonaemia persists; the initial doses are then adapted to ammonia and amino acids; after a scavenger bolus, ondansetron 0.15 mg/kg can be given to prevent vomiting (no maximum dose defined in the source); the equimolar benzoate and phenylacetate solution, registered by the FDA, is available in the European Union only on a named-patient basis [80]. The sources for this table are the 2012 and 2019 guidelines and the FDA and EMA labels [16, 80, 96–99]. The European label of carglumic acid includes isovaleric acidaemia and allows doses up to 250 mg/kg/day in organic acidaemias, whereas the US label sets 150 mg/kg/day for up to 7 days and does not include isovaleric acidaemia [97, 98]. In methylmalonic and propionic acidaemias, the 2021 guideline advises against sodium phenylbutyrate as the primary scavenger (it reduces glutamine and anaplerosis), recommends avoiding catabolism (strong), testing responsiveness to parenteral vitamin B12 in every suspected methylmalonic acidaemia (strong) and considering carglumic acid, which is tolerated but of unproven efficacy [77]. The enteral emergency regimens proposed by age are, between 0 and 1 year, 10% glucose polymer and 3.5% lipid emulsion (71.5 kcal/100 mL) [77]. The FDA labels of Ammonul® warn of hypokalaemia, sodium overload (30.5 mg/mL), extravasation necrosis and phenylacetate neurotoxicity [96].

Extracorporeal detoxification and nutrition in the 2019 revision. The response to medical treatment is assessed after 4 hours, the time estimated to alert the dialysis team and obtain vascular access; if insufficient, continuous kidney replacement therapy is started [80]. The guideline strongly recommends preparing extracorporeal detoxification in parallel with medical treatment in every patient with severe neurological symptoms and starting it as soon as possible, unless there is sufficient improvement with initial treatment (moderate quality); it recommends continuous venovenous haemodialysis or haemodiafiltration as the method of choice in the newborn, peritoneal dialysis only as a bridge when haemodialysis is not available or during transfer, and strongly recommends against exchange transfusion (high quality) [80]. Intermittent haemodialysis, when feasible in the newborn, is safe and effective, although technical and haemodynamic complications are frequent; after discontinuation there may be a relapse of hyperammonaemia [80]. A systematic review of published neonatal cases showed no improvement in outcome with dialysis in current practice, and the authors advocate starting all options, dialysis included, as early as possible; Kido and colleagues propose haemodialysis with an initial peak above 180 µmol/L, a position cited by the guideline without adopting it as a threshold [80]. In acute nutrition, the guideline strongly recommends high-dose glucose ± insulin with sodium and potassium, lipids (1–2 g/kg/day) as soon as fatty acid oxidation defects have been excluded and protein-free nutrition for no more than 24 (–48) hours; if the enteral route is not possible, IV amino acids start at 0.5 g/kg/day and increase by 0.5 g/kg/day up to at least the safe intake for age, with energy of about 120% of requirements (moderate quality) [80].

7.5. Nonketotic hyperglycinaemia

There are no formal management guidelines for NKH, nor randomised trials; the evidence is observational (cohorts and sibling studies) [23, 100]. The Clinical Genome Resource (ClinGen) classified clinical actionability in glycine encephalopathy as limited in the paediatric setting [100]. Treatment rests on two principles, lowering plasma glycine and blocking the NMDA receptor, and on symptomatic care [23, 52]. In severe NKH, no treatment changes the natural history, even if started at birth, but lowering glycine reduces the frequency and severity of seizures, improves alertness and resolves neonatal apnoea; in attenuated NKH, early and intensive treatment of children with variants with residual activity improved neurodevelopment and reduced the propensity to epilepsy in two independent studies [23].

Sodium benzoate. Benzoate conjugates with glycine to form hippurate, which is excreted in the urine; it acts in the liver and kidney, does not normalise CSF glycine and does not require serial CSF measurements [23, 46]. The doses differ between sources; that of GeneReviews, which also defines the target and the titration, is used as the reference.

The sources of the table are GeneReviews, Van Hove and Van Coster and Nyhan's Atlas [23, 46, 52]. Benzoate toxicity carries high morbidity and mortality: the early signs are hypocalcaemia and low plasma glycine (below 150 µmol/L); overdose causes nausea, vomiting, lethargy, coma, seizures, acidosis, hypernatraemia and hypokalaemia; treatment is to stop benzoate, give glycine and, if necessary, haemodialysis [23, 52]. Gastritis and oesophagitis are frequent with 500–750 mg/kg/day, and Van Hove and Van Coster recommend a prophylactic proton pump inhibitor; benzoate causes carnitine loss, which should be measured and supplemented [23, 52]. The reports collected in OMIM show the limits of early treatment: a newborn with a prenatal diagnosis treated from birth with benzoate and ketamine had no neonatal hypotonia or apnoea, but at 11 months had intractable seizures and severe delay; another, treated from 65 hours, died at 5 months despite dextromethorphan up to 40 mg/kg/day; and episodes of lethargy, coma and worsening seizures can result both from an insufficient dose of benzoate and from toxicity due to excess [20]. The dose should be reduced when starting a ketogenic diet, which reduces the glycine pool [23, 52]. In some countries there is a saliva-resistant granulated form with about 75% of the benzoate content of the regular form [23]. Oral benzoate has no approved indication in NKH; the only formulation with an FDA label is the injectable combination with phenylacetate for hyperammonaemia in UCD, at a dose of 250 mg/kg of each component in infants up to 20 kg [96]. In the neonatal phase, exchange transfusion, peritoneal dialysis or benzoate may allow spontaneous breathing to resume [46].

NMDA receptor antagonists. Dextromethorphan blocks the glutamate site on the NMDA receptor; the dose of GeneReviews and of Van Hove and Van Coster is 3 to 15 mg/kg/day, and the GeneReviews authors start at 10 mg/kg/day in newborns, 5 mg/kg/day in children and 3 mg/kg/day in adolescents and adults, in 3–4 doses or 2 doses with the extended-release form (for example, Delsym®) [23, 52]. Nyhan's Atlas indicates 5 to 22 mg/kg/day and a therapeutic level of 50–100 ng/mL, whereas GeneReviews considers that the therapeutic level is not defined and that measurement is for research only [23, 46]. Metabolism is highly variable (CYP2D6), and drugs that slow it, such as cimetidine, cause toxicity (drowsiness and increased movements) [23, 46]. Dextromethorphan is more useful in attenuated NKH; in the severe form, the effect is doubtful and it may increase pneumonias by reducing cough [23, 52]. Oral ketamine is an alternative, at 15 mg/kg/day in newborns, reduced to 9 mg/kg/day in infants (range 1–32 mg/kg; no maximum dose defined in the source), with better cognitive outcome with early treatment of carriers of attenuated variants [52]. Dextromethorphan and ketamine are used off-label, and strychnine has been abandoned because of serious effects with prolonged use [23].

Seizures and spasms. Control of severely disturbed EEG backgrounds (burst-suppression, hypsarrhythmia) is essential for any progress [23]. For myoclonus in the newborn and infant, benzodiazepines are the most effective, and clobazam is currently the preferred first-line drug; GeneReviews gives no dose, and its use in the newborn is off-label [23, 52]. Phenytoin has limited efficacy; phenobarbital has a variable effect in the newborn and is often useful in older children; levetiracetam and topiramate have a variable response; felbamate, an NMDA blocker, was effective in some children, with hepatic and haematological monitoring; the ketogenic diet improves seizure control without changing hypsarrhythmia; and vagus nerve stimulation was effective in older children with severe NKH [23, 52]. In attenuated NKH, first-line treatment is benzoate with dextromethorphan, with which many patients become seizure-free [23].

Drugs to avoid. Valproate is contraindicated, because it inhibits residual GCS activity and raises brain glycine, with encephalopathy, chorea, paradoxical increase in seizures and coma [23, 52]. Vigabatrin caused rapid loss of skills in West syndrome associated with NKH, and adrenocorticotrophic hormone was ineffective and associated with worsening, including coma [23, 52].

Dietary restriction. The dietary contribution of glycine is small. Van Hove and Van Coster accept a glycine- and serine-restricted diet in severe patients on high doses of benzoate and recommend always avoiding gelatine and high protein intake; GeneReviews considers that the benefits rarely outweigh the complexity and the risk of protein malnutrition, and that increasing benzoate is often sufficient [23, 52].

Variant NKH. There is no effective treatment: benzoate and dextromethorphan have no apparent effect, high-dose lipoate is disappointing, the ketogenic diet can cause massive acidosis, and avoiding catabolism prevents regressive episodes in BOLA3 deficiency; exogenous ketones and triheptanoin have positive preliminary indications [52]. In GlyT1 deficiency encephalopathy, benzoate and ketamine brought no improvement [34].

Supportive care. Early gastrostomy in swallowing dysfunction; treatment of reflux (sometimes Nissen fundoplication) and constipation; physiotherapy; treatment of scoliosis and hip dislocation weighing quality of life; respiratory secretion clearance, because recurrent pneumonia is often the terminal complication; ventriculoperitoneal shunt in hydrocephalus with a retrocerebellar cyst [23, 52]. The hyperactivity of attenuated NKH responds poorly to drugs and better to applied behaviour analysis [52].

Decisions on intensive care. Up to 80% of newborns with symptomatic NKH develop apnoea requiring ventilation in the first week; in the second or third week spontaneous breathing resumes, even without glycine-lowering treatment, and thereafter apnoea rarely recurs [23]. After the apnoeic phase, some untreated infants die within the next 2 years, but many live several years [23]. Given the prognosis of the severe form, some families choose to withdraw intensive support during the apnoeic phase; correct distinction between the severe and attenuated forms (CSF glycine, ratio, genotype, imaging) supports that decision [23, 52]. Once breathing has resumed, children with classic NKH rarely die suddenly, so adequate supportive care must be ensured [52].

7.6. Molybdenum cofactor deficiency type A: fosdenopterin

Mechanism and indication. Fosdenopterin (Nulibry®) is synthetic cPMP; it replaces the intermediate that patients with MOCS1 do not produce and is converted into molybdopterin and then into MoCo, restoring sulfite oxidase [14, 30]. It therefore acts only in type A: in type B (MOCS2) and type C (GPHN) the block is downstream of cPMP, and there are no efficacy data in these types [14, 30]. The FDA label indicates it "to reduce the risk of mortality" in MoCD type A; the European SmPC indicates it "for the treatment" of MoCD type A [14, 15]. Both allow treatment to be started on a confirmed or presumptive diagnosis and require discontinuation if type A is not confirmed by genetic testing; in the EU, treatment is initiated and supervised in hospital by a professional experienced in IEM [14, 15]. Treatment must begin within a very short window after the manifestations, because neurological injury prior to therapy, including prenatal injury, is not reversible [15, 30].

The doses are identical at the FDA and the EMA and are based on actual weight; the maximum recommended dose is 0.9 mg/kg/day, and the maximum tolerated dose has not been established [14, 15]. Each vial contains 9.5 mg of lyophilised powder, reconstituted with 5 mL of water for injections (1.9 mg/mL); the daily IV infusion is given at 1.5 mL/min, with a di(2-ethylhexyl) phthalate (DEHP)-free set and a 0.2 µm filter, or as a slow IV bolus over 1–2 minutes for volumes below 2 mL, and must be completed within 4 hours of reconstitution; a missed dose is given as soon as possible, with at least 6 hours until the next [14]. Home administration by the caregiver, through a long-term catheter, is allowed when the professional considers it appropriate [14, 30]. There is no published dose for any other condition, because the drug has only this indication.

Efficacy. Efficacy was established by comparing 13 patients with genetically confirmed MoCD type A, treated with fosdenopterin or recombinant cPMP (Study 1, NCT02047461, n=8; Study 2, NCT02629393, n=1; Study 3, retrospective, n=4), with a genotype-matched untreated natural history cohort (n=18): deaths 15% vs 67%; 1-year survival 92% (95% CI 57–99%) vs 67% (40–83%); at 3 years 84% (49–96%) vs 55% (30–74%); hazard ratio for death 0.18 (95% CI 0.04–0.72) [14]. Median gestational age was 39 weeks; 10 patients received the first dose by 14 days (5 on the first day of life) and 3 between 32 and 69 days [14]. Urinary S-sulfocysteine fell from 89.8 µmol/mmol creatinine (baseline in one patient) to means of 11 to 7 µmol/mmol between months 3 and 48 [14]. In the European SmPC, the analysis of 15 treated patients shows survival of 93% at 1 year and 86% at 3 years, and treated patients were more likely to walk, feed orally, gain weight, progress in development and maintain head circumference close to that of their peers [15]. European authorisation is under exceptional circumstances, with annual reassessment, because the rarity of the disease prevented complete information from being obtained [15].

Safety. The most common adverse reactions (more than 25%) in the 9 patients of studies 1 and 2 were catheter complications (89%), pyrexia (78%), viral infections (56%), pneumonia, otitis media, vomiting and cough (44% each), gastroenteritis, diarrhoea and bacteraemia (33% each) [14]. Animal studies showed phototoxic potential: direct sunlight and ultraviolet (UVA or UVB) phototherapy should be avoided, with protective clothing, a hat, sunglasses and broad-spectrum sunscreen from 6 months of age, and medical and dermatological assessment should be sought in case of photosensitivity [14].

Earlier experience with cPMP. Six patients with MoCD type A diagnosed in utero or between days 2 and 20 received daily IV cPMP (80–240 µg/kg), with normalisation or near-normalisation of urinary markers within days, greater alertness and cessation of seizures; two siblings with the same variant, treated from days 5 and 7, had a different course at 15 and 18 months (satisfactory development in one and dystonic cerebral palsy with cystic encephalopathy in the other) [47]. To avoid irreversible damage, treatment generally has to begin within the first 24 hours of life [59]. According to OMIM, which summarises the consensus of Schwahn and colleagues (2024) on MoCD and other sulfite intoxication disorders, daily IV cPMP requires very early initiation, within the first hours or ideally in the prenatal period, complications of central catheters are frequent and even very early postnatal treatment may leave neurological sequelae; in the patient described, treated with recombinant cPMP and then with fosdenopterin from 29 months, there were mild to moderate learning difficulties at 5 years and 11 months [26]. The detailed recommendations of that consensus are not included in the sources consulted.

Access. In the United Kingdom, the National Institute for Health and Care Excellence appraisal (NICE TA1078, 25 June 2025) was terminated because Sentynl Therapeutics withdrew from the process: NICE states that it cannot make a recommendation on fosdenopterin in MoCD type A in people of all ages, without clinical or cost-effectiveness judgement, and will review the decision if the company makes a submission (page reviewed on 25 June 2025) [101]. The sources consulted do not describe the reimbursement situation in Portugal or registration in Brazil.

Other measures in MoCD. In all subtypes a cysteine-restricted diet is used (low-protein, with restriction of natural protein; for example, 1.75 g/kg/day of protein in the newborn, reduced with age, and protein-free formula), with limited evidence and monitoring of essential amino acids [30]. Thiamine supplementation is recommended, because sulfite degrades thiamine: 1.2 mg/day in infants and from 50 mg once daily to 100 mg twice daily in children and adolescents [30]. Pyridoxine may be considered (raised α-AASA in some patients; mixed results) [30]. Seizures are treated with standard ASM, avoiding valproate, because sulfite intoxication impairs mitochondrial function [30]. Before attributing apnoeas to a central cause, prolonged EEG should exclude seizures, and polysomnography confirms central apnoea; tracheostomy is rarely needed, because the brainstem is preserved [30]. Spasticity and dystonia are treated with baclofen, tizanidine, botulinum toxin, orthoses and physiotherapy; headaches with magnesium and migraine prophylaxis [30]. Molybdate has been used without success [47]. In an at-risk newborn sibling without a prenatal diagnosis, metabolic treatment should begin immediately at birth and continue until the diagnosis is excluded [30].

7.7. Isolated sulfite oxidase deficiency

There is no definitive treatment [32]. Management is multidisciplinary: ASM and antispastic agents, early gastrostomy, treatment of reflux and aspiration pneumonia, chest physiotherapy, sleep studies and monitoring for scoliosis [32]. A low-protein diet restricted in cysteine and methionine improved two patients with the late-onset form clinically and biochemically, with no benefit in the classic form; betaine, thiamine, cysteamine and penicillamine have been tried with minimal success; GeneReviews gives no doses [32, 59]. In the two late-onset patients of Touati and colleagues, the diet provided 130–150 mg/day of methionine from natural protein and 50 g/day of an amino acid mixture free of cystine and methionine; after 2 years, urinary thiosulfate and S-sulfocysteine fell markedly, and both grew normally without neurological deterioration, at the cost of hypoaminoacidaemia with low plasma methionine and cystine [31]. In a patient with the p.Gly362Ser variant (protein maturation defect), molybdate increased sulfite oxidase activity in fibroblasts, which suggests a possible therapy for variants of this type, without clinical data [31]. Sulfhydryl-containing agents (for example, N-acetylcysteine, cysteamine, mesna) and some antibiotics give false positives in the urinary sulfite test, which should be considered in monitoring [32].

7.8. Targeted therapies in DEE

There is no approved specific therapy for KCNQ2-DEE [42]. Ezogabine (retigabine; Trobalt® in the EU, Potiga® in the USA), a KCNQ channel opener approved in 2011 as adjunctive treatment in focal seizures, was discontinued after June 2017 because of blue-grey retinal and mucocutaneous pigmentation after prolonged use [8]. The phase 3 trial of the paediatric formulation XEN496 in KCNQ2-DEE (NCT04639310) and its extension (NCT04912856) were terminated with 8 participants by sponsor decision, unrelated to safety [42]. In STXBP1-DEE there are no guidelines; the most used ASM are phenobarbital, valproate and vigabatrin; about 20% need more than one ASM and about 25% are refractory [9].

7.9. Comparative regulatory status

The package leaflets of the Brazilian Health Regulatory Agency (ANVISA) were not consulted, so the situation in Brazil is not described.

7.10. Trials and studies supporting the decisions

These results should not be compared with one another. The efficacy of fosdenopterin rests on a comparison with historical controls and not on a randomised trial; the relative reduction in the risk of death (82%) corresponds to 15% versus 67% deaths in very small samples, with a wide confidence interval [14]. NEOLEV2 assessed a short-term outcome and not neurodevelopment [13]. The EMBRAVE result is a press release on nine patients, not published in a peer-reviewed journal [41]. The hypothermia trial in late preterm infants is negative, and the paediatric ezogabine trial in KCNQ2-DEE was terminated without published efficacy results [42, 88]. There are no randomised trials in NKH, MoCD types B and C or ISOD.

7.11. Developments in the last 12 months

The phase 1/2 EMBRAVE trial (NCT05737784) is active, not recruiting, with primary completion in April 2026 and no results in the registry [42]. A French registry of neonatal-onset epileptic encephalopathies (NCT04802135) is ongoing, recruiting until 2029 [42].

7.12. Special situations

Pregnancy and lactation. A pregnancy has been described in a woman with attenuated NKH, with no obvious teratogenic effect and a child of normal intelligence [23]. In subsequent pregnancies in families with pyridoxine-dependent epilepsy, the ILAE mentions prenatal testing and maternal pyridoxine as options to consider; high doses of pyridoxine in pregnancy can cause neonatal dependency seizures, according to the Portuguese SmPC [4, 102]. The sources consulted do not describe fosdenopterin in pregnancy or lactation, and patients with MoCD and ISOD have not reproduced to date [30, 32].

Perioperative context and intercurrent illness. In attenuated NKH and late-onset MoCD, febrile illness and catabolism trigger decompensations; avoiding catabolism is the preventive measure in intoxication IEM and in BOLA3 deficiency [23, 30, 52, 77]. Valproate should also be avoided in NKH and MoCD in the acute setting [23, 30].

Palliative care. In severe NKH, MoCD types B and C, classic ISOD, GlyT1 encephalopathy, the neonatal forms of variant NKH and male MECP2 encephalopathy, the vital and functional prognosis is very guarded, and ongoing assessment of the need for palliative care and home nursing is part of management [23, 30]. In hyperammonaemia with a peak above 1,000 µmol/L and prolonged coma, the European guideline accepts the decision for palliative care [16]. In NICU mortality series, redirection of care was similar between infants with and without genetic assessment, and about one-third of genetic diagnoses arrived after death, which limits the use of the diagnosis in end-of-life decisions [86]. No sequencing study in NE has systematically assessed the use of the results in palliative decisions [1].

Transition. Patients with attenuated NKH can live several decades and adults need benzoate dosed by body surface area (maximum 16.5 g/m²/day) [23, 52]. The sources consulted do not describe specific transition programmes.

7.13. Divergences between sources and guidelines

8. Reproductive Options and Family Counselling

8.1. Without an aetiological diagnosis

When the investigation does not reach a diagnosis, the possibility of a genetic disease with a recurrence risk of up to 25% should be considered; even without a precise diagnosis, an exhaustive investigation usually gives a clue to the aetiological group [2]. If the diagnosis of HIE is secure, the recurrence risk is low, and the family should be referred to fetal medicine for monitoring of the next pregnancy and delivery [2]. Without a known diagnosis, prenatal diagnosis is not possible [2]. Therefore, banking DNA and samples from the proband, especially when the proband dies without a molecular diagnosis, is decisive for future counselling and for reanalysis of genomic data [2, 23, 30].

8.2. Risks by mode of inheritance

In NKH, carrier detection is only possible by molecular testing, because biochemical methods are not reliable [23]. Severity (severe or attenuated) is consistent in families with more than one affected member, although the degree of progress in the attenuated form may vary; in siblings with the attenuated form, early treatment was associated with better development [23]. In DEE, the phenotypic variability when a variant is inherited from a parent with few signs should be explained before prenatal diagnosis [7].

8.3. At-risk newborn siblings

In MoCD, if there was no prenatal diagnosis, metabolic treatment should begin at birth and continue until the diagnosis is excluded by molecular testing (known familial variants) or, failing that, by serum uric acid and urinary sulfite, S-sulfocysteine, xanthine, hypoxanthine and uric acid [30]. In NKH, newborn siblings should undergo biochemical testing for early diagnosis and treatment, especially when there is a risk of the attenuated form, followed by molecular testing if the variants are known [23]. In families with pyridoxine-dependent epilepsy, prenatal testing and maternal pyridoxine are options to consider [4]. In families with UCD, the delivery and treatment plan for the at-risk newborn should be prepared with the metabolic centre [16]. The 2019 revision, adapting the protocol of the British Inherited Metabolic Disease Group (BIMDG), distinguishes two scenarios [80]. If the previous sibling had neonatal onset: planned delivery in a hospital with a metabolic unit, immediate transfer of the newborn to the neonatal unit, 10% glucose IV (4 mL/kg/h; 6–8 mg/kg/min) with electrolytes within the first 30 minutes, protein-free feeding (breast milk is expressed and stored), sodium benzoate and L-arginine 50 mg/kg each every 6 hours, ammonia at 6 hours and then every 6 hours if below 80 µmol/L, repeated in 4 hours if between 80 and 150 µmol/L and, above 150 µmol/L or if the newborn becomes unwell, immediate remeasurement, suspension of feeding and action according to the thresholds in section 7.4; quantitative plasma amino acids at 12 hours [80]. If the sibling had late onset: IV glucose only if the delivery was complicated, first infant formula with up to 6 g of protein per day (about 130 mL/kg/day in a 3.5 kg newborn), breastfeeding with supplements on days 1 and 2, ammonia and amino acids at 24 hours (reassessment in 24 hours if ammonia is below 60 µmol/L, in 12 hours if 60 to 150 µmol/L) and, if ammonia at 48 hours is below 80 µmol/L, formula every 3 hours up to 150 mL/kg/day [80]. Molecular testing on chorionic villi or amniocytes is the prenatal method of choice in all UCD (strong recommendation, high quality); in a female fetus, the OTC genotype only excludes the disease and does not predict the phenotype, because of lyonisation [80].

8.4. Prenatal and preimplantation diagnosis

With the familial variants identified, molecular prenatal diagnosis and preimplantation genetic testing are possible in NKH, MoCD and ISOD [23, 30, 32]. In NKH, if only one variant is known, intragenic linkage analysis with polymorphisms can be used, with material from the proband and both parents [23, 52]. Assay of the GCS in uncultured chorionic villi is reserved for when molecular testing is not possible, because it has a false-negative rate of at least 1%, especially in T protein deficiencies in attenuated forms, owing to overlap of activity between affected individuals and carriers [23, 52]. Applegarth and colleagues recorded 3 false negatives in 290 at-risk pregnancies, and Kure and colleagues, in 28 families, 2 inconclusive results and one false positive, with a healthy newborn despite very low activity in two samples [20]. The Oxford Desk Reference still recommends confirming GCS activity in a post-mortem liver biopsy before offering prenatal diagnosis, a recommendation that reflects the era before molecular diagnosis [2]. In MoCD and ISOD, sulfite oxidase activity in uncultured chorionic villi allowed prenatal diagnosis before the molecular era [2, 47]. In methylmalonic and propionic acidaemia, prenatal diagnosis should preferably be molecular [77]. Perspectives on prenatal diagnosis differ between families and should be discussed in counselling [30].

8.5. Content of counselling

Counselling after a severe NE of genetic cause includes explaining the diagnosis and its certainty (including VUS), the natural history and prognosis, the mode of inheritance and the recurrence risk, the reproductive options (prenatal diagnosis and preimplantation genetic testing), cascade testing of relatives and, when the proband died without a diagnosis, the use of the stored samples [12, 23, 30]. Family planning should take place before pregnancy, and young adult carriers or those with attenuated NKH should receive their own counselling [23]. The sources consulted do not address carrier testing in healthy minors; in a newborn sibling at risk of MoCD or NKH, the test is not a carrier test but a diagnostic one, with immediate clinical utility [23, 30].

9. Special Considerations: Population Diversity and Epidemiology

9.1. The finding: neonatal encephalopathy and mortality from genetic disease

NE affects 1 to 6 per 1,000 term live births (1 to 7 per 1,000 in other estimates), and HIE occurs in up to 7 per 1,000 births and accounts for almost a quarter of neonatal deaths worldwide [10, 11, 68]. These figures measure the syndrome and not the genetic fraction, for which there is no population estimate; the sequencing yields of 23.5% to 53.1% measure the proportion of diagnoses in selected and tested series, not prevalence [1]. In the USA, 22% of infants who died before 1 year in a cohort of 517 had a confirmed genetic diagnosis, and only half of these had the genetic cause coded on the death certificate; vital statistics therefore underestimate mortality from genetic disease [87].

9.2. Incidence and prevalence by entity

Nyhan's Atlas states that NKH is the second most frequent amino acid IEM after phenylketonuria; this statement is not supported by population studies in the sources consulted [46]. The prevalence of 0.4 per 100,000 for West syndrome given in the Oxford Desk Reference is implausible in view of the ILAE incidence of infantile spasms (30 per 100,000) and should not be used [4, 7].

9.3. Founder populations and consanguinity

NKH has founder effects in Finland, the United Kingdom (GLDC p.Arg515Ser), the Netherlands (AMT p.Ile106Thr), New Zealand (GLDC p.Ser132Leu) and Arab villages in Israel (GLDC c.2T>C and AMT p.His42Arg described in an Israeli Arab family) [23, 46]. In MoCD, the recurrent MOCS1 variants are geographically concentrated in Europe and a MOCS2 allele is prevalent in the Roma community [47, 59, 60]. Consanguinity increases the incidence of recessive IEM: in southern Israel, the incidence of IEM was much higher in the consanguineous Bedouin population than in the Jewish population [108]. In ISOD, the incidence may be higher in consanguineous populations [32].

9.4. Portugal and Brazil

In Portugal, the only population-based data in the sources is the Coimbra series (2004–2010), with 3 cases of NKH diagnosed in a paediatric hospital and an incidence of 1:47,455 [107]. The authors stress that early diagnosis does not change the outcome of the severe form, but allows genetic counselling and prenatal diagnosis [107]. One of the first late-onset mild forms of ISOD was described in a Portuguese child of first-cousin parents [31]. The sources consulted do not include Brazilian data on NE of genetic cause, NKH, MoCD or ISOD, which does not mean that such data do not exist.

9.5. Special populations

Preterm infants deserve attention: PNPO deficiency often occurs in preterm infants; the fosdenopterin dose is adjusted to gestational age; hypothermia showed no benefit in newborns of 33 to 35 weeks; and transient hyperammonaemia of the preterm newborn should be distinguished from UCD [4, 14, 16, 88]. In boys, X-linked forms should be considered [2]. In classic NKH, more girls than boys died in the neonatal period (mean age at death under 1 month in girls and 2.6 years in boys), according to the surveys of Hoover-Fong and Hennermann cited in the Atlas [46]. Access to rapid sequencing is unequal, and the HGSA recommends that testing be equitable [1, 12].

9.6. European networks and patient associations

The relevant European Reference Networks are MetabERN (hereditary metabolic disorders), EpiCARE (rare and complex epilepsies) and ITHACA (rare malformation syndromes and intellectual disability) [17–19]. The E-IMD network maintains a European registry of intoxication-type metabolic diseases (UCD and organic acidaemias) [109]. Patient associations with pages confirmed in October 2026 include NKH Crusaders (https://www.nkhcrusaders.com/), the KCNQ2 Cure Alliance (https://www.kcnq2cure.org/), the FamilieSCN2A Foundation (https://www.scn2a.org/), the STXBP1 Foundation (https://www.stxbp1disorders.org/), the PDE online consortium for pyridoxine-dependent epilepsy (https://www.pdeonline.org/) and, in Portugal, the Associação Portuguesa de Fenilcetonúria e outras Doenças Hereditárias do Metabolismo (APOFEN, https://www.apofen.pt/). GeneReviews lists for NKH Joseph's Goal (United Kingdom), NKH Deutsches Familien Netzwerk and the NKH International Family Network, and for ISOD Metabolic Support UK; these pages were not verified in 2026 [23, 32].

10. Illustrative Clinical Case

10.1. SCN2A developmental and epileptic encephalopathy with onset at birth, treated with an antisense oligonucleotide

Wagner and colleagues described, in Munich, a female newborn born at 29 weeks and 4 days, weighing 1,400 g [73]. The pregnancy was complicated by polyhydramnios; the 27-week ultrasound showed constantly flexed arms with hyperextended wrists and clenched hands, and fetal MRI at 29 weeks and 2 days showed prominent basal ganglia, normal gyration and myelination and overlapping fingers, raising the suspicion of arthrogryposis [73]. Prenatal trio exome sequencing identified the de novo SCN2A variant NM_001040142.2:c.3986C>A p.(Ala1329Asp), in the intracellular S4–S5 linker of domain III; NM_001040142.2 is the MANE Select transcript, and the genomic coordinate is NC_000002.12:g.165374698C>A [55, 73].

On the first day of life, the newborn went into status epilepticus, with sequential automotor and tonic seizures confirmed by aEEG, refractory to phenobarbital, levetiracetam, pyridoxine, lacosamide, oxcarbazepine, midazolam infusion, carbamazepine and lidocaine; phenytoin interrupted the status epilepticus only for about 20 minutes, even with levels above 40 µg/mL [73]. Postnatal MRI showed hypoplasia of the corpus callosum, bilateral focal frontotemporal T2 hyperintensities, thin thalami and enlarged lateral ventricles [73]. The status epilepticus lasted 7 weeks, with 20 to 25 seizures per hour [73]. Functional studies (voltage clamp and dynamic action potential clamp) showed impaired inactivation and increased persistent current, with increased firing frequency and reduced rheobase, that is, gain of function, which underpinned the choice of a therapy reducing SCN2A messenger RNA [73].

At 7 weeks, the child received intrathecal elsunersen under individual compassionate use, approved by the ethics committee of Ludwig-Maximilian University [73]. Eight days after the first dose, the status epilepticus began to be intermittently interrupted and ended with the subsequent doses; after a cumulative 2.5 mg in three doses and reintroduction of phenytoin, the mean seizure frequency fell from 19.7 (±4.2) per hour in the first 12 weeks to 6.6 (±4.3) between weeks 13 and 35, a reduction of 67% [73]. She received 7 doses (30.5 mg) between March and September 2023 and then 10 monthly doses of 8 mg; worsening of seizures 4 to 6 weeks after each dose led to a pharmacokinetic model suggesting intervals of 4 to 6 weeks [73]. There were no serious adverse events in 19 administrations over 20 months and no hydrocephalus [73].

The neurological course was severe: optic disc atrophy at 2.5 months; at 8 months, severe hypotonia without head control, tube feeding despite preserved swallowing and minimal visual responses; MRI at 5, 17 and 36 weeks with progressive symmetrical cortical atrophy, sparing the basal ganglia, thalamus and cerebellum [73]. At 22 months she still had 5 to 7 seizures per hour (a reduction of more than 60%), and phenytoin was withdrawn at 14 months without worsening [73]. The authors attribute the outcome to the initial severity and to the approximately 60,000 seizures of the first 8 months; because this is a single patient, causality of the improvement has not been demonstrated [73].

The figures of the article, whose legends were consulted, show the three-dimensional model and the functional effect of the variant (https://pmc.ncbi.nlm.nih.gov/articles/PMC12283366/figure/Fig1/), the clinical course with the drug regimen and seizure frequency (https://pmc.ncbi.nlm.nih.gov/articles/PMC12283366/figure/Fig2/), the fetal MRI (https://pmc.ncbi.nlm.nih.gov/articles/PMC12283366/figure/Fig3/) and the progressive cortical atrophy (https://pmc.ncbi.nlm.nih.gov/articles/PMC12283366/figure/Fig8/) [73].

The case illustrates several points of this article: the prenatal clues (polyhydramnios, arthrogryposis) to a severe DEE; the refractoriness of a channelopathy to phenobarbital and levetiracetam, with a partial and transient response to phenytoin, consistent with the ILAE recommendation to use sodium channel blockers when a channelopathy is likely; the genetic diagnosis before birth, which allowed early therapeutic decisions; and the importance of distinguishing gain from loss of function. Elsunersen has no marketing authorisation, the reported use was compassionate and does not constitute current practice; the controlled data available are those of EMBRAVE Part A, in children aged 2 to 12 years, known from a sponsor press release [41, 42].

10.2. Molybdenum cofactor deficiency with an HIE-like presentation

Nyhan's Atlas describes a newborn born after an uneventful pregnancy and delivery, who was well for 2 days and then suddenly became cyanotic, mottled and hypotonic, with apnoea requiring ventilation on day 2 [47]. The EEG showed status epilepticus and then burst-suppression, with a poor response to anticonvulsants, and there was cerebral atrophy at 10 days [47]. The MOCS1 variants p.R67W (novel, exon 1) and p.G126D (exon 2, described in several European and American patients) were identified, which in ClinVar correspond to NM_001358530.2:c.199C>T p.(Arg67Trp) and c.377G>A p.(Gly126Asp) [47, 62]. The newborn was weaned from the ventilator, but by the end of the first year had microcephaly with overriding sutures and no neurological development [47]. The figure of the case was not viewed. The symptom-free interval of 2 days and the absence of a sentinel event are the signs that, according to GeneReviews, distinguish MoCD from HIE and that would today trigger urgent testing of uric acid, total homocysteine and S-sulfocysteine and, in type A, fosdenopterin [14, 30].

11. Prognosis and Long-term Follow-up

11.1. Prognosis by entity

Neonatal encephalopathy in general. In a level IV NICU, 10% of newborns with NE not attributed to HIE died before discharge and 48% of survivors were discharged with medical equipment [74]. In the HEAL trial, death or neurodevelopmental impairment at 2 years occurred in 75% of infants with HIE and a genetic or congenital anomaly versus 50% in the others, with cerebral palsy in 32% vs 13% of survivors [75]. In the Chinese cohort of 366 newborns with NE, a genetic diagnosis, seizures and a severely abnormal EEG were associated with developmental delay [76].

NKH. The median age at death in classic NKH, in two surveys with about 150 patients, was 31.5 months, with survival possible into the third decade; most attenuated forms survive, often for several decades [46, 52]. At least 15 to 30% die in the neonatal period, often through withdrawal of intensive care [52]. Individual prediction of survival in the severe form is not possible [52]. Predictors of outcome are summarised in the table [23, 52, 58].

The diffusion pattern on MRI does not predict the phenotype, and neonatal age at presentation alone is not sufficiently predictive [23, 58]. In the Swanson series, severe patients needed several anticonvulsants and patients with a DQ above 30 needed none [58]. Homozygotes for GLDC p.(Ala802Val) treated early and intensively in the first two years had normal intelligence [23]. In variant NKH, death usually occurs in the first 2 years in the BOLA3, NFU1 and ISCA2 forms, and in the first months in the neonatal forms [52].

MoCD and ISOD. Without treatment, about 75% of patients with early-onset MoCD die in infancy, mainly from pneumonia; in the natural history cohort of the fosdenopterin label, the median survival of untreated type A patients was 48 months (95% CI 10–99), and with treatment survival was 92% at 1 year and 84% at 3 years [14, 30]. Injury prior to treatment, including prenatal injury, is not reversible [15]. In classic ISOD death usually occurs in the first months [32]. The late-onset forms (Misko class II) have better survival, but the long-term prognosis is poorly characterised [30, 71].

DEE. In KCNQ2-DEE, more than half of patients become seizure-free within a few months to several years, but development is moderately to severely impaired; an abnormal neonatal neurological examination and a markedly abnormal EEG background predict abnormal development (100% vs 46%) [4, 94]. In SeLNE, development is normal, but about 30% have seizures later [8]. In STXBP1-DEE, intellectual disability is severe to profound in 88%, with no correlation between seizure severity and the severity of the impairment [72]. In EIDEE, almost all have moderate to profound intellectual disability and early mortality is increased [4]. Boys with MECP2 encephalopathy often die before 2 years of age [44].

11.2. Surveillance

The sources do not set numerical intervals for MRI, EEG and orthopaedic assessment in NKH, or for surveillance in ISOD; the frequency depends on the clinical course.

11.3. Follow-up of the family

Genetic follow-up should be offered to all families after rapid genomic testing, whatever the result; when there was no diagnosis, reanalysis of the data and alternative investigations should be planned [12, 51]. In families with NKH or MoCD, the plan for a subsequent pregnancy includes prenatal diagnosis or immediate treatment of the at-risk newborn [23, 30].

12. Summary and Final Considerations

Severe neonatal encephalopathy is a syndrome, not a diagnosis. In more than half of term newborns no cause is identified, and genomic sequencing finds a diagnosis in a quarter to a half of selected newborns with NE or suspected HIE. HIE remains the most frequent cause, but the absence of a sentinel event, a symptom-free interval after birth, seizures out of proportion to the injury, an MRI without the hypoxic-ischaemic pattern, dysmorphism and family history require a search for a genetic or metabolic cause, without excluding the coexistence of the two.

The approach must be simultaneous and time-driven. In the first hour the critical samples are collected and stored and glucose, blood gases, lactate, ammonia, uric acid and urinary sulfite are measured. In the first hours the picture is classified into one of the six biochemical types and hyperammonaemia, hypoglycaemia and seizures are treated. In the first 24 hours plasma and CSF amino acids are measured simultaneously, together with total homocysteine, S-sulfocysteine and α-AASA, and a trial of pyridoxine and PLP is given when seizures are refractory. MRI with diffusion, video-EEG and rapid trio genome sequencing complete the diagnosis. Sample collection and autopsy allow counselling when the newborn dies.

Three diseases illustrate the spectrum. Nonketotic hyperglycinaemia is diagnosed by CSF glycine collected together with plasma and without contamination, and by restricted diffusion in the brainstem and cerebellum. The severe form keeps a very guarded prognosis with any treatment; in the attenuated form, benzoate and dextromethorphan started early improve development. Molybdenum cofactor deficiency mimics HIE and is recognised by low uric acid, very low homocysteine and raised S-sulfocysteine; type A has had, since 2021 in the USA and 2022 in the EU, a replacement therapy with fosdenopterin that reduces mortality compared with historical controls, provided it is started before irreversible injury. Isolated sulfite oxidase deficiency shares the clinical and biochemical features of MoCD, except for normal uric acid, and has no effective treatment.

In neonatal-onset DEE, the gene changes the choice of drug. Phenobarbital is the general first-line treatment for neonatal seizures, but in KCNQ2-DEE and in SCN2A and SCN8A DEE sodium channel blockers control seizures better, and diagnosis in less than 30 days was associated with more seizure freedom. Antisense oligonucleotides for SCN2A are in a phase 3 trial, with preliminary results from a sponsor press release and published individual cases.

Counselling depends on the diagnosis. Without a diagnosis, the recurrence risk can reach 25%. In recessive diseases, parental carrier status should be confirmed molecularly. In de novo DEE, parental mosaicism should be investigated. In siblings at risk of MoCD, treatment begins at birth, until the diagnosis is excluded.

13. References

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2. Firth HV, Hurst JA. Neonatal encephalopathy and intractable seizures. In: Oxford Desk Reference: Clinical Genetics and Genomics. 2nd ed. Oxford: Oxford University Press; 2017. p. 246–251.
3. Saudubray JM, García-Cazorla Á. Clinical approach to inborn errors of metabolism in paediatrics. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 1, sections 1.1–1.3.10 e 1.4.16.
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5. Orphanet. Early infantile developmental and epileptic encephalopathy (ORPHA:1934). https://www.orpha.net/en/disease/detail/1934. Accessed 28 Oct 2026.
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