Biotinidase Deficiency and Holocarboxylase Synthetase Deficiency

Inherited defects of the biotin cycle and multiple carboxylase deficiency — biotinidase deficiency (BTD; profound and partial, newborn screening, p.Asp444His variant, irreversible deafness and optic atrophy) and holocarboxylase synthetase deficiency (HLCS); biotin dosage and biotin interference with immunoassays

Literature and regulatory information updated: 4 October 2026.

0. Initial Reference Table

1. Introduction

Biotin is a water-soluble B vitamin, present in small amounts in natural foods, mostly bound to proteins, and acts as a coenzyme for five carboxylases involved in gluconeogenesis, fatty acid synthesis and the catabolism of several amino acids [8]. Humans do not synthesise biotin; in addition to dietary intake, some is produced by the intestinal flora, but covalently bound biotin must be released by biotinidase to become available, whether it comes from the diet, intestinal bacteria or degradation of the carboxylases themselves [2]. Two inherited defects of biotin coenzyme function are known: holocarboxylase synthetase deficiency (HCS), in which attachment of biotin to apocarboxylases is impaired, and biotinidase deficiency, in which biotin depletion results from the inability to recycle endogenous biotin and utilise biotin bound to dietary proteins [8]. Both lead to simultaneous deficiency of all biotin-dependent carboxylases, namely multiple carboxylase deficiency (multiple carboxylase deficiency, MCD), with metabolic acidosis, neurological abnormalities and skin disease [8].

The history of these diseases parallels that of the chemistry of organic acidurias. In 1971, Gompertz and colleagues described a child with excretion of 3-methylcrotonylglycine and 3-hydroxyisovaleric acid who responded to biotin, initially interpreted as having an isolated defect of leucine metabolism [3, 21]. Subsequent identification, in Nyhan and Sweetman's laboratory, of methylcitric acid and 3-hydroxypropionic acid in the urine of the same patient led to the demonstration of deficiencies of propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase and, later, pyruvate carboxylase, and the disease became known as “multiple carboxylase deficiency” [3]. In 1981, Burri, Sweetman and Nyhan demonstrated that the fundamental defect in this patient lay in holocarboxylase synthetase, whose affinity for biotin was reduced [3, 22]. In the following years, it became clear that there was a second, later-onset form, with low biotin in blood and urine; in 1983, Wolf and colleagues identified serum biotinidase deficiency in this form, involving the enzyme that cleaves biocytin [2, 23]. The same group developed the colorimetric dried blood spot test and conducted the first pilot newborn screening programme, which identified two patients among the first 81,243 newborns screened and two further previously undiagnosed siblings [2, 24].

The clinical importance of these diseases stems from an asymmetry rarely seen in metabolic medicine: treatment consists of an inexpensive vitamin, administered orally and with no known toxicity, which prevents all manifestations when started before injury occurs, but does not fully reverse established sensorineural deafness, optic atrophy or developmental delay [1, 25]. For this reason, Wolf described biotinidase deficiency as “the inherited metabolic disease to have, if one has to have one”, and newborn screening became the factor that most changed prognosis [25, 26]. A 2023 systematic review, which brought together 1113 individuals described in 144 publications over forty years, confirmed the impact of screening, but also the persistence of late diagnoses and preventable deaths where screening is unavailable [27]. Holocarboxylase synthetase deficiency, in turn, presents mainly as a severe neonatal organic aciduria, usually responsive to biotin, but with a subgroup of patients showing a partial or absent response [8, 20].

This article addresses biotinidase deficiency, holocarboxylase synthetase deficiency and their umbrella entity, multiple carboxylase deficiency, together, with separate subsections for each disease whenever their clinical features, diagnosis or treatment differ. Sodium-dependent multivitamin transporter deficiency (SLC5A6), biotin- and thiamine-responsive basal ganglia disease (SLC19A3) and acquired biotin deficiency are addressed in the differential diagnosis [8].

2. Molecular Genetics and Pathophysiology

2.1 The biotin cycle and biotin-dependent carboxylases

Biotin is covalently attached, through an amide bond, to the ε-amino group of a lysine residue in newly synthesised apocarboxylases, in a two-step reaction catalysed by holocarboxylase synthetase (EC 6.3.4.10): first, biotin is activated to D-biotinyl-5′-adenylate at the expense of ATP; the biotinyl group is then transferred to the apocarboxylase, which only then acquires enzymatic activity [3]. When holocarboxylases are degraded by proteolysis, biotin remains attached to lysine (biocytin) or to small biotinylated peptides; biotinidase (EC 3.5.1.12) hydrolyses these compounds and returns free biotin to the pool available for further biotinylation, and the same enzyme releases biotin bound to dietary proteins [2, 8]. Biotinidase is a ubiquitously expressed sialylated monomeric glycoprotein, with several isoforms resulting from different degrees of sialylation, and has biotinyl hydrolase and biotinyl transferase activities [1].

Four biotin-dependent carboxylases are mitochondrial or associated with mitochondria and one is cytosolic, and each explains part of the biochemical phenotype [3, 8]:

The hypothesis that acetyl-CoA carboxylase deficiency contributes to the dermatosis and alopecia is supported by observation of a patient whose alopecia and skin lesions regressed with oral and topical administration of unsaturated fatty acids [2]. In addition to its classical enzymatic function, holocarboxylase synthetase biotinylates histones and participates, in the cytosol, in a guanylate cyclase-dependent pathway that induces transcription of genes involved in biotin transport and utilisation, and, in the nucleus, in a transcriptional coregulatory complex; the contribution of these functions to human disease remains unclear [8, 10].

2.2 Biotinidase deficiency: the BTD gene, variants and genotype–phenotype correlation

The BTD gene is located at 3p25.1 and has four exons and three introns; there are two possible initiation codons, one in exon 1 and the other in exon 2, separated by an intron, allowing transcripts encoding signal peptides of 41 or 21 residues, possibly with tissue-specific expression [1, 9]. The complementary DNA was cloned in 1994 and the gene was mapped to 3p25 in the same year [2, 28]. The disease mechanism is loss of function [1]. More than 200 pathogenic variants have been described [8], including two large gene deletions detectable only by deletion and duplication analysis and an intronic variant, c.310-15delT, which alters messenger RNA expression and was the first intronic variant described in the gene [1, 2].

A nomenclature peculiarity should be known to those reading laboratory reports: historical numbering is based on transcript NM_000060.4, whereas GeneReviews has adopted transcript NM_001370658.1, in which the classic variant c.1330G>C p.(Asp444His) corresponds to c.1270G>C p.(Asp424His) and variant c.1368A>C p.(Gln456His) corresponds to c.1308A>C p.(Gln436His) [1]. This article uses historical numbering, as it appears in almost all the clinical literature, with the equivalence indicated where relevant.

Genotype–phenotype correlation is not well established, and GeneReviews recommends that treatment decisions be based on enzymatic activity rather than genotype [1]. Nevertheless, there are consistent patterns: null variants (deletions, insertions, nonsense variants) in homozygosity or compound heterozygosity produce profound deficiency, and complete absence of activity is associated with earlier onset [1]. Compound heterozygotes for p.Asp444His and a profound-deficiency allele have approximately 20–25% of mean normal activity, and p.Asp444His homozygotes have 45–50%, similar to carriers of a profound-deficiency allele, and do not require biotin [1]. Swango and colleagues found p.Asp444His on one allele in 18 of 19 randomly selected individuals with partial deficiency and compared this variant with the Duarte variant of galactosaemia [30]. In an Austrian series of 34 patients, four variants accounted for 78% of alleles and p.Asp444His was present in 12 of the 13 patients with mild deficiency [2]. In Turkey, where many patients are homozygous for null variants, all symptomatic children with null variants had hearing loss, except those treated early because they had an affected sibling, whereas the six children homozygous for missense variants had normal hearing even after a period without treatment [2, 34].

Concordance between genotype and biochemical phenotype is also imperfect for non-genetic reasons. In a Brazilian study of 72 individuals with low biotinidase activity, the phenotype expected from the genotype matched that observed in 68.5% of cases; age, prematurity and neonatal jaundice appeared to affect activity, and biochemical classification changed between the first and second sampling in 11 of 22 patients, leading the authors to recommend more than one plasma determination for diagnosis [35].

2.3 Biotinidase deficiency: pathophysiology

The immediate consequence of biotinidase deficiency is urinary loss of biotin, mainly as biocytin, and progressive depletion of stores; biocytin, absent from normal urine, was detected in untreated patients at concentrations of 7.2–28.8 nmol/mmol creatinine, higher than those of biotin [2, 8]. Increased renal clearance of biotin may result from competitive inhibition of its tubular reabsorption by biocytin, and biocytin may compete with biotin as a substrate for holocarboxylase synthetase; however, there is no evidence of direct biocytin toxicity or accumulation during treatment [2, 8]. As newborns are born with presumably normal stores of free biotin, symptoms occur somewhat later than in HCS deficiency, and the timing of onset depends on the amount of free biotin in the diet and the severity of the enzymatic defect [8, 19]. Nevertheless, depletion may develop within a few days after birth, justifying prompt initiation of biotin [8].

The central nervous system is affected earlier and more severely than other organs. Biotinidase activity in the human and rat brain is much lower than in other tissues, and brain biotin concentrations fall earlier and more markedly in biotinidase deficiency [2]. In a patient with a fatal course, carboxylase activities were much more reduced in the brain than in the liver and kidney [2, 8]. This vulnerability explains why lactate and 3-hydroxyisovalerate may be higher in cerebrospinal fluid (CSF) than in plasma, with CSF/plasma lactate ratios up to 3.7, why CSF lactate may be elevated without hyperlactataemia, and why organic aciduria may be absent in patients with neurological symptoms [2, 8]. Pyruvate carboxylase may be the most affected enzyme in the brain, and local accumulation of lactate and organic acids is the proposed mechanism of neurotoxicity [2]. These elevated CSF concentrations are not observed in HCS deficiency, in which neurological injury is secondary to catastrophic decompensations [2]. In a murine model of biotinidase deficiency, sensorineural deafness develops within a defined developmental window, consistent with the irreversibility observed in humans when treatment is late [14, 36].

Cells from patients with biotinidase deficiency have normal carboxylase activities in cultured fibroblasts, whether in biotin-poor or biotin-rich medium, because the culture medium provides free biotin; in fresh lymphocytes, activities are low while depletion persists and normalise within a few hours after an oral dose of biotin [2, 8]. Holocarboxylase synthetase activity is normal [2].

2.4 Holocarboxylase synthetase deficiency: the HLCS gene, variants and genotype–phenotype correlation

The HLCS gene is located at 21q22.13 and the complementary DNA, cloned in 1994 by Suzuki and colleagues and in 1995 by functional complementation of an Escherichia coli auxotroph, encodes a protein of 726 amino acids whose residues 445–701 show homology with the biotin ligases of E. coli and yeast [3, 37]. More than 50 pathogenic variants have been described, approximately two-thirds located in the putative biotin-binding region [8]. In 2005, the review by Suzuki and colleagues counted 30 variants distributed throughout the coding region except exons 6 and 10 (one single-amino-acid deletion, five small insertions or deletions, 22 missense and two nonsense variants), with c.1519+5G>A (IVS10+5G>A; historical numbering NM_000411.8:c.1519+5G>A, = NM_001352514.2:c.1960+5G>A, NC_000021.9:g.36767213C>T) the only intronic variant then known [10]. All this literature numbers variants using the historical transcript NM_000411, which encodes the protein of 726 amino acids; the current MANE Select transcript, NM_001352514.2, starts translation at an upstream codon and adds 147 codons, so positions increase by 441 nucleotides and 147 amino acids: the Polynesian variant c.647T>G p.(Leu216Arg) is recorded in ClinVar as NM_001352514.2:c.1088T>G p.(Leu363Arg), pathogenic/likely pathogenic, and the Japanese variant p.(Leu237Pro) as NM_001352514.2:c.1151T>C p.(Leu384Pro), pathogenic [7, 38]. The following table retains historical numbering, as used in the cited clinical literature.

The relationship between residual HCS activity and clinical response to biotin is consistent: the combination of a null allele with a point variant retaining less than a few per cent of normal activity produces neonatal onset, whereas variants with greater residual activity manifest after the neonatal period and respond well to biotin [10]. Variants in the biotin-binding domain reduce the enzyme's affinity for the cofactor, reflected in elevated Km values for biotin in fibroblasts, and the degree of Km elevation correlates with age at onset and severity: a patient with a Km approximately 70 times normal presented in the first hours of life and had a sibling who died in the neonatal period, patients with a Km 20–45 times normal presented between the first day and seven weeks, and a patient with a Km only three times normal presented at eight months [3, 41]. Variants in the N-terminal region, outside the binding site, are associated with virtually normal Km and reduced Vmax; these patients also usually respond to biotin, but require higher doses and frequently retain residual biochemical and clinical abnormalities [8]. In these cases, the response is thought to result from a positive effect of biotin on HLCS messenger RNA transcription and therefore protein quantity, a mechanism requiring some residual HCS activity itself [8]. Variants p.Leu216Arg and p.Leu237Pro reduce substrate affinity and increase the dissociation rate more than 15-fold, helping to explain the poor response [3].

2.5 Holocarboxylase synthetase deficiency: pathophysiology and enzymatic markers

In HCS deficiency, reduced affinity for biotin and/or reduced maximum velocity decreases formation of the five holocarboxylases at physiological biotin concentrations [8]. Blood and urinary biotin concentrations are normal, unlike in symptomatic biotinidase deficiency [8]. In fibroblasts cultured in medium containing 6 nmol/L biotin, all carboxylases are markedly deficient, and normalise when the cells grow in 100 nmol/L [3]. The first step of the reaction, biotinyl-AMP synthesis, is deficient in all cell lines studied, with activities of 0.3–8% of control in four patients [3]. The following table summarises carboxylase activities measured by Nyhan's group in patients with HCS deficiency, expressed as percentages of control [3]:

Unlike biotinidase deficiency, neurological abnormalities are not an intrinsic component of the disease: they result mainly from initial or repeated decompensations, with cerebral hypoperfusion or hyperammonaemia, and neurological examination may be normal after an episode of hyperammonaemia [3]. In severe forms, such as those of Polynesian patients homozygous for p.Leu216Arg, injury begins in utero, with fetal growth restriction and subependymal cysts [39].

3. Clinical Spectrum and Manifestations by System

3.1 Overview: multiple carboxylase deficiency

The characteristic manifestation of multiple carboxylase deficiency is metabolic acidosis associated with neurological and cutaneous abnormalities, with variable clinical and biochemical expression in the two diseases [8]. Patients with HCS deficiency usually have the typical presentation of an organic aciduria, whereas in biotinidase deficiency the presentation is less consistent, particularly in the early stages: onset may be insidious, with prominent neurological symptoms without metabolic acidosis or markedly abnormal organic acid excretion [8]. Distinction by age at onset — HCS deficiency before three months and biotinidase deficiency after three months — is useful as a guide, but has exceptions in both diseases, and late forms of HCS deficiency are clinically indistinguishable from biotinidase deficiency [1, 3, 8]. Unambiguous distinction requires enzymatic measurement or molecular testing [2].

3.2 Untreated profound biotinidase deficiency: infantile form

Symptoms of untreated profound deficiency usually appear between one week and ten years of age, with a mean of 3.5 months [1]. The 2023 review grouped symptomatic individuals into four presentations: neonatal (<1 month; 7.9%), early childhood (<2 years; 59.2%), juvenile (2–16 years; 25.1%) and adult (>16 years; 7.7%) [27]. The most affected systems were the nervous system (67.2%), skin (53.7%), eye (34.4%), auditory system (26.9%) and respiratory system (17.8%), with multisystem involvement in 82.2% [27]. Some children manifest a single finding, others multiple neurological and cutaneous findings [1]. Gradual progression and periods of remission are characteristic of the disease, possibly related to variations in free dietary biotin [8].

Nervous system. Seizures and hypotonia are the most common neurological manifestations; older children present with ataxia and developmental delay [1]. Seizures occur in more than 70% of patients in some series and may be the only evident symptom; they are usually myoclonic, but may be generalised or focal, frequent, intermittent or exclusively febrile, and infantile spasms may be the first manifestation [1, 2]. In a study of 78 children, 55% had seizures, which were the initial complaint in 70% of these; seizures were poorly controlled with antiseizure medication in 40%, but disappeared with biotin in 75% [2]. Hypotonia, observed in more than half of patients, varies in degree and usually improves with biotin; spasticity also varies and may not reverse [1, 2]. Ataxia may be severe enough to prevent walking, intermittent, and accompanied by intention tremor [2]. Presentations of Leigh syndrome, spastic paraplegia or tetraplegia with or without visual loss, severe acute hypotonia with loss of head control at ten months, psychomotor regression and bulbar symptoms have been described [2]. Developmental delay may be global or specific, often with intellectual disability and speech and language disorders, which may or may not improve with biotin [1]. The disease is included in the differential diagnosis of epileptic encephalopathy beginning in the first year of life and of intermittent ataxia [42]. The 2022 French PNDS, drawing on the 2017 ACMG standard and on reviews, summarises the frequency of manifestations of profound deficiency in infancy as follows: seizures, hypotonia and skin lesions in about 70% of patients; ataxia, developmental delay, recurrent conjunctivitis and visual abnormalities, including optic atrophy, in about 50%; bilateral hearing loss of variable degree in about 75%; heterogeneity is considerable, even within the same family [9, 13]. These proportions are literature syntheses of symptomatic patients and do not derive from a cohort with its own denominator. The most frequently cited seizure types are myoclonic, clonic or tonic–clonic seizures and infantile spasms, with frequent drug resistance and a non-specific, sometimes normal, EEG that may show multifocal spikes, slow waves or hypsarrhythmia [13].

Skin, appendages and mucosae. Skin lesions tend to be patchy, erythematous or exudative and periorificial, unlike the generalised eruption of HCS deficiency, although there may be generalised erythema and scaling, eczematoid dermatitis, a seborrhoeic appearance, perioral fissures, stomatitis, glossitis and perineal dermatitis [2, 8]. Alopecia ranges from thinning of scalp hair, eyebrows or eyelashes to total alopecia [2, 8]. Mucocutaneous candidiasis is a frequent complication, and blepharoconjunctivitis or keratoconjunctivitis may be severe enough to require hospital admission [2]. One of Nyhan's patients was diagnosed with acrodermatitis enteropathica for years, and anhidrotic ectodermal dysplasia was also considered in the differential diagnosis [2]. The eruption and alopecia are the most specific clues, but may appear late or never occur [1, 8].

Hearing and vision. Approximately 76% of untreated symptomatic individuals with profound deficiency have sensorineural deafness which usually neither regresses nor improves with biotin, remaining static [1, 14]. This proportion derives from 33 children diagnosed on the basis of symptoms, none treated from birth, and the authors knew of no deafness in infants diagnosed through screening and treated from the neonatal period [2, 14]. In a retrospective Turkish study from 2026, automated auditory evoked potentials for newborn hearing screening, performed in the first two weeks of life, did not differ between newborns with biotinidase deficiency and controls, or between enzymatic activity groups, supporting the acquired rather than congenital nature of deafness and reinforcing the need for subsequent audiological surveillance despite normal hearing screening [43]. In the Turkish series of patients with profound deficiency, 55% had hearing loss [2]. The Brazilian chapter reports sensorineural hearing loss in 20–30% of symptomatic patients with total deficiency, a value that should be read in light of the proportion of 26.9% with auditory involvement found in the 2023 review, which includes screened and symptomatic patients [6, 27]. Ophthalmological involvement includes optic atrophy, scotomata, optic neuropathy, ocular motility disorders, retinal pigmentary changes and keratoconjunctivitis [1, 6]. A patient diagnosed at ten months and treated with biotin 10 mg/day subsequently developed sensorineural deafness, severe myopia with progressive retinal epithelial dysplasia and optic atrophy [2]. The hypothesis that these lesions resulted from treatment was rejected: they were never observed in HCS deficiency, treated earlier and at higher doses, and were documented before biotin initiation [2].

Respiratory system. Hyperventilation, laryngeal stridor and central apnoea may occur; stridor, which may be the initial manifestation, was interpreted as neurological in origin and regresses with biotin [1, 2].

Immunity. Untreated biotinidase deficiency is associated with cellular and humoral immune dysfunction, with potentially lethal chronic fungal infections, recurrent viral infections and increased susceptibility to bacterial infections [1, 6]. Two patients with extensive chronic mucocutaneous candidiasis lacked in vitro and in vivo responses to Candida antigen; one had IgA deficiency and no response to the pneumococcal polysaccharide vaccine, the other a low percentage of circulating T lymphocytes [2]. One patient was initially treated as having severe combined immunodeficiency with bone marrow transplantation, and manifestations only regressed with biotin [2]. Immune abnormalities disappear with treatment, so GeneReviews does not recommend routine formal immunological investigations [1, 2]. Chronic mucocutaneous candidiasis with normal biotinidase points towards inborn errors of immunity with defective IL-17 immunity.

Metabolism and feeding. Episodic lactic ketoacidosis, sometimes severe, may occur with coma, hypothermia, profound hypotonia and areflexia, compensated chronic acidosis (serum bicarbonate around 15 mEq/L), mild hyperammonaemia and feeding difficulties that regress with biotin [1, 2]. Reported deaths at nine months and three years, and early deaths in the pre-screening era, were due to severe metabolic decompensation with late diagnosis or treatment; a death initially attributed to sudden infant death syndrome was later attributed to the disease [1, 2]. In the 2023 review, 1.6% of reported individuals died because treatment was unavailable or diagnosis was late [27].

Neuroimaging and neurophysiology. Most brain imaging studies are normal; the most characteristic abnormalities are cerebral atrophy and ventricular dilatation, which may improve or regress with biotin [1]. White matter hypodensity consistent with delayed myelination, basal ganglia calcification, leukoencephalopathy and spinal cord hypodensity, demyelination or oedema have also been described [2, 8]. The electroencephalogram is often normal, but may show diffuse slowing or paroxysmal activity, which rapidly normalises with biotin; visual evoked potentials may be abnormal and normalise with treatment [2]. Neuropathology describes superior vermian atrophy with almost complete loss of Purkinje cells, white matter gliosis, subacute necrotising myelopathy and, in a patient with a Leigh presentation, subacute necrotising encephalopathy [2].

3.3 Profound biotinidase deficiency: late adolescent and adult form

Several children with untreated profound deficiency remained asymptomatic until adolescence or adulthood and then developed sudden visual loss due to progressive optic neuropathy, peripheral neuropathy and spastic paraparesis [1]. Patients with late onset present mainly with ophthalmological problems and variable degrees of myelopathy, and are often initially diagnosed with multiple sclerosis or neuromyelitis optica spectrum disorder (NMOSD) [1, 44, 45]. Magnetic resonance imaging shows longitudinally extensive transverse myelitis and T2 hyperintensity of the optic nerves, fornix and mammillary bodies, with negative anti-aquaporin-4 and anti-MOG antibodies and elevated CSF lactate [46, 47]. The first reported adult, a 22-year-old man with extensive myelopathy and bilateral optic neuropathy, had profound deficiency due to a novel missense variant and partially improved with oral biotin [46]. Children and adolescents with these forms often received methylprednisolone, plasma exchange, immunoglobulin and, in some cases, rituximab before diagnosis [47]. The adult chapter in Saudubray includes biotinidase deficiency among the metabolic causes of bilateral optic atrophy, spastic paraparesis and motor neuropathy [48]. After several months of biotin, ocular abnormalities may improve or regress and paraparesis may improve if diagnosis is made within a reasonable time [1, 2].

3.4 Partial biotinidase deficiency

Individuals with untreated partial deficiency (10–30% of mean normal activity) may develop symptoms only under stress, particularly during prolonged infections [1]. An untreated child developed hypotonia, a rash and hair loss during gastroenteritis at around six months, with resolution after biotin [1]. Saudubray records more than 20 children with partial deficiency, identified by screening and untreated, who eventually developed typical symptoms of profound deficiency (hypotonia, rash, alopecia), particularly during moderate infections or gastroenteritis, reversed by biotin in the great majority [8]. In symptomatic patients with partial deficiency, the Brazilian chapter describes chronic fungal infections, recurrent viral infections, increased susceptibility to bacterial infections, hypotonia and facial dermatitis [6]. Conversely, in the Austrian series all patients with partial deficiency followed up remained clinically and neuropsychologically asymptomatic without treatment between 2.5 and 10 years [49].

3.5 Penetrance and asymptomatic individuals

Almost all children with profound deficiency become symptomatic or are at risk of doing so if untreated [1]. However, there are adults with profound deficiency who have never had symptoms and were identified because their children had positive newborn screening, as well as siblings of symptomatic patients who remained asymptomatic; they may become symptomatic under stress [1, 2]. In the Austrian series, only patients with residual activity below 1% (3 of 5) had symptoms in the first weeks of life, whereas five patients with residual activity of 1.2–4.6% developed no symptoms even without treatment up to ages between 3.5 and 21 years [49]. In the experience of the authors of the Saudubray chapter with 61 patients (52 families) with residual activity up to 10%, the risk of biotin depletion was high, supporting treatment of all patients [8].

3.6 Holocarboxylase synthetase deficiency

Although initially termed early-onset multiple carboxylase deficiency, HCS deficiency has a highly variable age at onset, from a few hours after birth to eight years [8]. In Nyhan's group series, seven patients with a documented enzymatic defect developed symptoms between the first day of life and 18 months, most before six weeks [3]. The typical presentation is a severe acute illness in the first hours or weeks, resembling propionic acidaemia or other classical organic acidurias, with lethargy, hypotonia, vomiting, seizures and hypothermia [3, 8]. The most common initial manifestations are respiratory — tachypnoea or Kussmaul breathing — associated with severe high-anion-gap metabolic acidosis, marked ketosis and hyperammonaemia, which without biotin progress to dehydration, coma and death, sometimes a few hours after birth [3, 8]. Decompensations are precipitated by catabolism during intercurrent infections or by increased protein intake [8]. Thrombocytopenia may occur, and in siblings homozygous for p.Leu216Arg, decompensation in the first 24 hours included metabolic acidosis, lactic acidosis and thrombocytopenia [42, 50]. In the French PNDS, the typical neonatal form accounts for more than half of patients, and the atypical form, presenting in the first years of life, adds developmental delay and neurodevelopmental disorders; epilepsy is less frequent than in biotinidase deficiency, occurs mainly in the neonatal forms, with tonic and myoclonic seizures, and rarely dominates the clinical picture [13].

The classic patient, JR, had recurrent vomiting from birth, an erythematous eruption at six weeks and, at five months, rapid breathing, vomiting and prostration, with ketosis and metabolic acidosis [3]. The dermatosis, an intrinsic component of untreated disease, is a bright-red, erythematous, scaly eruption, usually affecting the whole body and more pronounced in the nappy area and folds, where it may be exudative; it may resemble seborrhoeic dermatitis or ichthyosis, is identical to that of nutritional biotin deficiency and is frequently complicated by candidiasis [3, 8]. Alopecia may progress to total alopecia, with absence of scalp hair, eyebrows, eyelashes and lanugo [3]. Psoriasiform dermatitis has been described as a cutaneous manifestation in adults [8]. Persistent vomiting may lead to growth delay [3]. Unlike biotinidase deficiency, deafness has not been reported [8].

Neurological abnormalities — hypotonia, hypertonia, irritability, athetoid movements, opisthotonus, presentations labelled as cerebral palsy, electroencephalographic and white matter abnormalities — are usually secondary to decompensations [3]. Prenatal and postnatal imaging reveals abnormalities in 86% of 22 patients in a systematic review, including ventriculomegaly, intraventricular haemorrhage and subependymal cysts; one infant's cysts disappeared after six months of biotin [3]. Seven Samoan infants with severe disease, incomplete response to biotin and early death had fetal growth restriction and subependymal cysts [3, 39]. Patients with a less severe defect and late onset may also have recurrent episodes of metabolic acidosis with typical organic aciduria; those who recover without biotin and untreated patients with a less severe defect may develop psychomotor delay, alopecia and skin lesions [8]. Rarely, HCS deficiency presents with predominant neonatal seizures [42]. T- and B-cell immune dysfunction occurs, with reduced circulating T lymphocytes and reduced in vitro response to Candida in a patient with bacteraemia [3].

4. Clinical Recognition and Warning Signs

Clinical recognition relies on combinations of signs which, individually, are relatively non-specific. The following table brings together situations in which these diseases should be actively investigated [1–3, 8, 19, 42]:

Saudubray emphasises that, because of the threat of irreversible brain injury, biotinidase deficiency should be considered in all children with neurological problems, including a therapeutic trial of oral biotin (10 mg/day for 5 days), even without organic aciduria or skin lesions, and that the BTD gene should be included in genetic panels and exome analysis for all patients with neurological symptoms [8]. Absence of metabolic acidosis and organic aciduria in the early stages is the main diagnostic pitfall [8]. In the Oxford Desk Reference, plasma biotinidase is included in additional investigations of infants with neonatal encephalopathy and seizures, with or without dysmorphism, and biotinidase deficiency is listed among treatable causes of early-onset seizures, with usual onset between one week and two years and seizures as the most common initial symptom [19]. In regions without screening, diagnostic delay has improved little over time [8]. The 2023 review recommends considering a therapeutic biotin trial when suspicious signs occur in undiagnosed infants or adults [27].

The French PNDS on vitamin-responsive epilepsies (2022) groups biotinidase deficiency, profound and partial, and isolated HCS deficiency among the vitamin B8-responsive epilepsies and sets broad criteria for seeking them: any child under one year of age whose epilepsy is poorly controlled by antiseizure medication, and any patient, of any age, with a combination of ocular, cutaneous and auditory lesions [13]. In neonatal seizures in a newborn without risk factors, with normal pregnancy, delivery, Apgar score and interictal state and without anoxia, infection, hypoglycaemia or hypocalcaemia on first-line investigation, it recommends a systematic therapeutic trial with a vitamin combination — pyridoxine, biotin and folinic acid, possibly followed by pyridoxal phosphate — started in parallel with sample collection; outside suggestive circumstances, such as a family history, the trial is a second- or third-line measure for persistent seizures, and it should also be discussed for resistant seizures in hypoxic–ischaemic encephalopathy or a brain malformation that do not account for them [13]. At any age, drug-resistant epilepsy, that is, with seizures persisting despite two appropriate antiseizure medications, warrants referral to a rare epilepsies centre, and the PNDS proposes vitamin supplementation in any status epilepticus refractory to the first two lines of treatment, in parallel with metabolic investigation [13]. Blood biotinidase is part of the first-line investigation of refractory neonatal epilepsy of unknown aetiology, and samples should, whenever possible, be collected before the therapeutic trial, because the trial may normalise the specific biochemical profiles [13]. General warning signs of a vitamin-responsive epilepsy in children include onset before one year, polymorphic and drug-resistant seizures, seizures triggered by fasting, infection, vaccination or vomiting, status epilepticus, disturbances of alertness, ataxia, spastic paraparesis, myelopathy, developmental regression, alopecia or eczematous rash, conjunctivitis, oculomotor abnormalities and deafness [13]. Adolescent and young adult forms, rare and usually without epilepsy, have been observed mainly in countries without newborn screening, such as France [13].

5. Diagnosis: Criteria, Tests and Algorithms

5.1 Biotinidase deficiency

No published consensus clinical diagnostic criteria exist [1]. Diagnosis is established in a proband whose newborn screening or biochemical findings suggest multiple carboxylase deficiency, by detecting deficient serum or plasma biotinidase activity, or by identifying biallelic pathogenic variants in BTD when the enzymatic result is ambiguous [1].

Newborn screening. Screening is based on fluorimetric or colorimetric tests of biotinidase activity in dried blood spots, with thresholds defined by each laboratory; confirmation requires measurement of serum or plasma activity [1]. False positives occur in premature infants and in cards placed in plastic before drying sufficiently [1]. Biotin should be started as soon as an abnormal result is received, while investigations are completed, and may only be discontinued after biotinidase deficiency and other biotin-responsive diseases, such as HCS deficiency, have been excluded [1]. The biotinidase assay is specific to biotinidase deficiency and does not identify HCS deficiency, in which serum biotinidase activity is normal [1]. In the Dutch programme, among 304,982 newborns screened, 92 were referred for confirmation: 6 (7%) had profound deficiency, 44 (48%) partial deficiency and 42 (46%) normal activity; all profound cases had screening activity below 15%, leading the authors to propose this threshold [51]. In Brazil, the false-positive rate of a programme involving 225,136 newborns was 0.09%, and discrepancies between activity and genotype were attributed to inappropriate sample handling and storage [52].

Serum biotinidase activity. The American College of Medical Genetics and Genomics (ACMG) working group established technical standards for laboratory diagnosis, defining profound deficiency as activity below 10% of the normal mean and partial deficiency as 10–30% [1, 9]. With appropriate controls, the biochemical test is definitive; an unrelated normal control sample and parental samples should accompany the proband's sample, and enzymatic deterioration due to inappropriate storage or shipping is a growing cause of false positives [1, 52]. Saudubray recommends confirming residual activity in a second sample taken at four months of age or later [8], and the Brazilian study also suggests basing diagnosis on more than one determination [35]. Two recent studies of screened cohorts reinforce this recommendation: in Emilia-Romagna (Italy), among 31 patients (30 with partial deficiency and one with profound deficiency) followed for at least five years, biotinidase activity increased significantly and persistently up to 60 months, particularly in p.Asp444His carriers, and the authors propose reassessing diagnosis and treatment at around 12 months; in a Turkish series of 194 patients, almost all with partial deficiency, the last measured activity was significantly higher than the initial value, with a slower increase in children of consanguineous couples [53, 54]. These observations suggest that some initial classifications of partial deficiency may change with age, but do not establish when it is safe to discontinue biotin, a decision that no guideline defines. Methods use N-biotinyl-p-aminobenzoate (Bratton–Marshall reaction), carbon-14-labelled substrates, including natural biocytin, or the fluorimetric substrate biotinyl-6-aminoquinoline; with the original method, the normal mean was 5.80 nmol/min/mL (range 4.30–7.54) and patients had values between undetectable and 0.18 nmol/min/mL [2]. High-performance liquid chromatography using biocytin enabled subdivision of profound deficiency into residual activity of 0–1% and 1–10%, with clinical significance [49]. Parents have approximately 50% of normal activity [2].

Pre-analytical conditions and molecular confirmation in the French PNDS. Biotinidase is unstable at room temperature and remains stable for less than 24 hours at 4 °C; after centrifugation at 4 °C, serum or plasma is stored at −20 °C (preferably −80 °C) and transported between laboratories on dry ice; storage for more than three months requires −80 °C, and a sample stored for more than three months at −20 °C should not be analysed [13]. Prematurity, transfusion and liver disease bias the result; ampicillin, bilirubin, haemoglobin and glutathione may interfere, depending on the method; and circulating sulphonamides artificially increase measured activity, with a risk of false negatives [13]. Activity is not altered by biotin, so the therapeutic trial does not preclude subsequent measurement; the result usually takes two to four weeks [13]. Because of sample fragility and method dependence, the PNDS requires confirmation by biallelic pathogenic variants in BTD (3p25.1), in the homozygous or compound heterozygous state, requires cases with variants of uncertain significance (ACMG class 3) to be discussed at a multidisciplinary meeting with a specialised centre, and reserves genetic counselling for class 4 and 5 variants [13]. This requirement differs from the GeneReviews position, which considers the biochemical test definitive with appropriate controls and reserves molecular testing for ambiguous results or families with known variants (section 7.7) [1, 13].

Molecular testing. This uses single-gene testing: BTD sequencing (detects approximately 99% of pathogenic variants) followed by deletion and duplication analysis if only one or no variant is found; in individuals of Amish ancestry, c.1270G>C p.(Asp424His) and c.1308A>C p.(Gln436His) may be tested first [1]. Molecular testing is useful to distinguish profound from partial deficiency and heterozygosity for profound deficiency from partial deficiency, but biallelic variants of uncertain significance neither establish nor exclude diagnosis [1]. Saudubray considers whole-gene analysis rarely necessary, as it has no therapeutic consequences [8].

Supporting investigations. Ketolactic metabolic acidosis, organic aciduria (lactate, 3-hydroxyisovalerate, 3-methylcrotonic acid, 3-methylcrotonylglycine, 3-hydroxypropionate, methylcitrate) and hyperammonaemia, usually mild, up to a few hundred µmol/L, support diagnosis [1]. 3-Hydroxyisovalerate may be the only abnormal metabolite, and urinary organic acids may be normal in symptomatic patients: in 20% of untreated children, excretion was normal despite symptoms [1, 8]. In the 2023 review, metabolic acidosis was present in 42.4% of symptomatic individuals and characteristic metabolites in 57.1% [27]. Plasma and urinary biotin are usually reduced in symptomatic patients, provided the method does not detect biocytin, but have little diagnostic value and mainly serve to assess adherence [1, 8]. Lymphocyte carboxylase activities are low when biotin depletion is present and normalise within hours after a biotin dose; in cultured fibroblasts they are normal [8].

5.2 Holocarboxylase synthetase deficiency

Suspicion arises in the presence of characteristic organic aciduria, with excretion of 3-hydroxyisovaleric acid — usually greater than that of 3-methylcrotonylglycine and up to 200 times normal — and lactic acid, often massive, accompanied by methylcitric acid, 3-hydroxypropionic acid and tiglylglycine, in a sample collected during a crisis [3, 8]. The profile may be less evident if urine is collected after intensive fluid therapy [3]. In severe cases, an unpleasant urinary odour (“cat urine”) may suggest the defect [8]. In plasma acylcarnitines, C5-OH is elevated, with C3 and C5:1 to a lesser degree [8]. Tandem mass spectrometry newborn screening should identify most cases, because patients excrete the metabolites during the neonatal period; in the Faroe Islands, acylcarnitine analysis detected 5 of 6 patients [1, 12]. In a child born to a mother with HCS deficiency treated with biotin, screening was positive despite the infant being biochemically normal [3]. A patient with a novel variant was initially diagnosed as having 3-methylcrotonyl-CoA carboxylase deficiency [3].

Confirmation is achieved by molecular testing of HLCS or by demonstration, in fibroblasts cultured in low-biotin medium (0.1 nM), of severely reduced activity of at least two mitochondrial carboxylases, which normalises or increases in medium supplemented with high biotin concentrations (0.2–10 µM) [8]. Lymphocyte carboxylase activities are reduced and may remain low even during prolonged supplementation [8]. A rapid method to distinguish the two diseases consists of measuring carboxylases in fresh lymphocytes before and after pre-incubation with biotin [2]. Direct measurement of HCS activity requires a protein substrate (apocarboxylase or a fragment containing the biotinylation site) and is not routinely performed [8]. Serum biotinidase activity is normal [1, 6]. In HCS deficiency, the French PNDS also requires confirmation by biallelic pathogenic variants in HLCS (21q22.13), with multidisciplinary discussion of variants of uncertain significance, and notes that lactate and urinary organic acids fall within a few days of biotin in most cases, making it essential to collect urine and blood before the therapeutic trial; acylcarnitines are available in two to four days and organic acids in days to weeks [13].

5.3 Integrated algorithm

After diagnosis through screening in an asymptomatic infant, GeneReviews recommends consultation with a metabolic disease specialist and dietitian, genetic counselling, growth measurement, neurological assessment (with electroencephalography if seizures are suspected) and audiological assessment by audiometry, emphasising that newborn hearing screening does not replace it [1]. In symptomatic patients, nutritional and swallowing assessment (with possible gastrostomy), ophthalmological (optic atrophy, scotoma, conjunctivitis), skin and mucosal, respiratory, developmental and infection history assessments are added [1]. The Brazilian chapter recommends ophthalmological and hearing assessment after confirmation and objective neuropsychological assessment between 6 and 7 years [6].

After confirmation, the French PNDS adds verification of follow-up by a paediatric neurologist or neurologist at a reference or competence centre, referral to a metabolic physician and to a genetics clinic, to dermatology when skin lesions are present, to otorhinolaryngology and ophthalmology in both diseases, to physical and rehabilitation medicine according to motor development, and a neurodevelopmental assessment (neuropsychology, occupational therapy, speech and language therapy), repeated as needed, with additional assessment of vision and hearing [13].

Table 5.1 — Therapeutic trial of biotin in suspected vitamin B8-responsive epilepsy (French PNDS, appendix 6)

Source: [13]. The Saudubray trial, 10 mg/day for five days, has a fixed duration, whereas the PNDS trial continues until the results are available (section 7.7) [8, 13].

5.4 Genetic tests: coverage, limitations and reporting

In biotinidase deficiency, BTD sequencing detects approximately 99% of pathogenic variants, but does not identify the two reported large deletions, which require deletion and duplication analysis (MLPA, quantitative PCR or a targeted array) when only one or no variant is found [1]. Panels restricted to exons and junctions may not capture intronic variants such as c.310-15delT, the first intronic variant described in the gene, and approaches targeting only common variants, such as testing for the two Amish variants, do not exclude the disease outside that context [1, 2]. In HCS deficiency, the Scandinavian variant c.1519+5G>A (IVS10+5G>A) is intronic, outside canonical splicing positions, and should be explicitly covered and interpreted; confirmation may require RNA testing or enzymatic testing in fibroblasts [8, 10]. Mosaicism and uniparental isodisomy should be considered when the proband's variant is not found in one parent [1]. In both diseases, a negative molecular result does not exclude diagnosis when enzymatic activity or the biochemical profile is compatible, and treatment decisions in biotinidase deficiency rely on enzymatic activity rather than genotype [1, 8].

The report should state the gene, reference transcript with version, HGVS nomenclature at DNA and protein level, zygosity, phase (cis/trans, confirmed by parental testing), classification according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria and technical limitations [7, 55]. Phase is particularly important in BTD: the double allele c.[511G>A;1330G>C], with p.(Ala171Thr) and p.(Asp444His) in cis, behaves as a profound-deficiency allele, whereas isolated p.(Asp444His) is a partial-deficiency allele, so without defined phase the same combination of variants may be classified as profound or partial deficiency [1, 8, 31]. The MANE Select transcripts are NM_001370658.1 for BTD and NM_001352514.2 for HLCS; both change numbering relative to the literature (20 fewer codons in BTD, so p.(Asp444His) becomes p.(Asp424His); 147 more codons in HLCS, so p.(Leu216Arg) becomes p.(Leu363Arg)), and a report using standardised numbering should also provide the historical name to allow comparison with the literature [1, 7, 38]. A variant of uncertain significance does not become pathogenic through clinical or biochemical response to biotin or partial segregation; low enzymatic activity may contribute as supporting evidence in classification, but reclassification requires formal weighing of criteria, and segregation or functional studies should be described as research [1, 55].

6. Differential Diagnosis

Vomiting, hypotonia and seizures with ketolactic acidosis or mild hyperammonaemia are common to many metabolic diseases, and biotinidase deficiency was confused with isolated carboxylase deficiencies before correct diagnosis [1]. The most characteristic manifestations (eruption, alopecia) also occur in nutritional biotin deficiency, HCS deficiency, zinc deficiency and essential fatty acid deficiency [1].

Organic aciduria enables differentiation of isolated carboxylase deficiencies from multiple deficiencies, and demonstration of deficient activity of one of the three mitochondrial carboxylases in leucocytes or fibroblasts cultured in biotin-poor medium, with the other two normal, confirms an isolated deficiency [1, 6]. In isolated deficiencies, the phenotype ranges from neonatal forms with severe neurological involvement to asymptomatic adults [6]. A therapeutic biotin trial helps distinguish biotin-responsive from non-responsive deficiencies [1]. Serum biotin and biotinidase measurements distinguish biotin deficiency from biotinidase deficiency [6]. In an 11-year-old boy with intellectual disability, biotin deficiency resulted from a prescribed diet containing raw eggs, rich in avidin [8].

7. Treatments and Risk Management Strategies

7.1 General principles and regulatory status of biotin

Except for some cases of HCS deficiency, both diseases are effectively treated with pharmacological doses of oral biotin, provided treatment begins before irreversible neurological injury, such as deafness in biotinidase deficiency, develops [8]. Biotin (Portuguese common name biotina; biotin in English) acts as a cofactor: in biotinidase deficiency, administered free biotin bypasses the inability to recycle and release protein-bound biotin; in HCS deficiency, high plasma concentrations compensate for the mutant enzyme's reduced affinity and, in variants with reduced Vmax, increase HLCS transcription [8]. It should be used in free rather than protein-bound form [1]. In more than 30 years of experience in biotinidase deficiency, no adverse effects of treatment have been observed, and there is no accumulation of biocytin in body fluids, previously considered a theoretical risk [8]. GeneReviews states that there are no known adverse effects of pharmacological doses [1]. Protein restriction is unnecessary except in very severe cases of HCS deficiency [1, 8].

From a regulatory perspective, biotin has no medicinal product label in the United States with an indication for biotinidase or HCS deficiency (openFDA database query yielded no results for these indications), and there is no centrally authorised European Medicines Agency (EMA) medicinal product containing biotin for these diseases [56]. Its use therefore involves a pharmacological-dose vitamin supported by patient series and natural history studies rather than randomised trials [1, 20]. In Brazil, biotin for biotinidase deficiency was incorporated into the Unified Health System (SUS) by Ordinance no. 34 of 27 September 2012, following a favourable recommendation from the National Commission for the Incorporation of Technologies (Conitec); biotin alone was registered with the National Health Surveillance Agency (Anvisa) only in 2016, in 2.5 mg capsules under the brand name Untral® (Aché Laboratórios Farmacêuticos); the Clinical Protocol and Therapeutic Guidelines (PCDT) for biotinidase deficiency were published in 2018, biotin entered the Specialised Component of Pharmaceutical Assistance in August 2018 and first dispensing occurred in 2020 [57].

7.2 Biotinidase deficiency: dosage

All individuals with profound and partial deficiency should receive lifelong free oral biotin [1]. Doses published by reference sources are as follows:

None of the sources in the table defines a maximum dose or a dose per kilogram of body weight for biotinidase deficiency: GeneReviews refers to infants and children and notes that data are lacking for older children, and Saudubray indicates 5–10 mg/day for children diagnosed early with profound deficiency and 2.5–5 mg/day for partial deficiency, with no defined upper limit [1, 8]. The French PNDS states explicitly that the dose is not modified with weight gain and likewise defines no maximum [13].

No official label consulted indicates biotin for biotinidase or HCS deficiency, so all doses in the table, including maintenance doses, are off-label. In the United States, biotin products in the openFDA database are unapproved over-the-counter medicines, supplements and multivitamin combinations, without dosing for these diseases [56, 59]. In Portugal, a search for the substance biotin in Infomed returned only one authorised and marketed multivitamin combination (Becozyme forte®, 0.15 mg of biotin per tablet, indicated for the prophylaxis of vitamin deficiencies), with no single-ingredient biotin medicine [60]. In France, the only single-ingredient biotin labels (Biotine Bayer® 5 mg tablet and Biotine Bayer® 0.5% solution for intramuscular injection, 5 mg/1 mL) are authorised only as adjunctive treatment of diffuse alopecia in adults, at 15 mg/day orally for 6 weeks or 5–10 mg intramuscularly three times a week; no intravenous form is authorised in the labels consulted, and the intravenous route mentioned in Saudubray is likewise off-label [58, 61]. These labels provide the warning about interference with biotin–streptavidin immunoassays (section 7.4), but their indication and duration are not analogous to those of these diseases, and the reference dose remains that of the clinical sources in the table [1, 8, 61].

Two girls with profound deficiency developed alopecia in adolescence, which resolved after increasing the dose from 10 mg/day to 15 or 20 mg/day; Saudubray reports similar situations in puberty and adulthood [1, 8]. Perspectives on partial deficiency differ: some centres treat only in the first months of life and others do not treat, whereas the GeneReviews author recommends lifelong treatment because of the absence of known toxicity and the reported symptomatic cases [1]. Saudubray considers it prudent to supplement patients with 10–30% residual activity, as some untreated children develop symptoms and conclusive evidence is lacking [8]. In late forms with myelopathy and optic neuropathy, case reports used doses above usual maintenance doses — for example, 200 mg/day in a 9-year-old child — without a defined dose for this situation [47]. Heterogeneity in doses in practice was documented in a 2026 Italian national survey covering the 22 centres responsible for patients detected through screening (included in the Italian programme since 2017): in profound deficiency, 13.6% of centres prescribed 5 mg/day, 40.9% 10 mg/day and 45.5% 20 mg/day, as a single dose in 45.5% and in two doses in 50.0%; in partial deficiency, 68.2% prescribed 5 mg/day and 31.8% 10 mg/day, and lifelong maintenance, intensity of follow-up and diagnostic reassessment varied even more; most centres recorded no symptoms in treated patients, and the authors advocate harmonised recommendations [62]. These values describe centre practice and are not dose recommendations.

Administration requires practical attention, particularly in infants, to ensure a uniform complete dose [1, 63]. Biotin is usually dispensed in tablets or capsules, in which the vitamin quantity is minute relative to the excipient; for infants or young children, the crushed tablet or capsule contents are mixed with breast milk or formula in a spoon, dispenser or syringe [1]. Powder should not be placed in a feeding bottle, where it adheres to the walls or fails to pass through the teat, and solutions or syrups are not recommended, as they are suspensions that settle (particularly in the refrigerator), may support bacterial growth and do not ensure a constant dose [1]. Treatment decisions should be based on enzymatic activity rather than genotype [1].

Symptomatic patients require supportive treatment. Metabolic decompensation is managed with hydration, usually intravenous, and bicarbonate for acidosis, bearing in mind that biotin corrects the metabolic abnormality and many symptoms within hours to days [1]. Developmental delay warrants speech and language therapy, occupational therapy, physiotherapy and educational support; optic atrophy requires ophthalmological follow-up and low-vision services; deafness may benefit from hearing aids and, in selected cases, cochlear implantation [1]. Raw eggs should be avoided because egg-white avidin binds biotin and reduces its bioavailability; thoroughly cooked eggs pose no problem, because heat inactivates avidin [1, 8].

7.3 Holocarboxylase synthetase deficiency: dosage and acute treatment

The biotin dose depends on the severity of the enzymatic defect and must be assessed individually [8]. Most patients respond to 10–20 mg/day, but some require 40–200 mg/day or more [8]. The French PNDS indicates 5–40 mg/day orally, with a favourable response in most patients, and allows an increase up to 200 mg/day in the rare cases of partial response; when the condition is suspected, the therapeutic trial uses 10–40 mg/day [13]. The initial dose used by Nyhan's group was 10 mg/day, effective in most patients; in some, residual metabolites present at 10 mg/day disappeared at 40 mg/day; a patient with a very high Km retained skin lesions, metabolite excretion and low carboxylase activities at 60 mg/day, and another achieved resolution of skin lesions only at 100 mg/day, retaining small quantities of organic acids [3]. In a patient with p.Gln379*/p.Tyr663His, 100 mg/day improved blood and CSF biochemical parameters, clinical stability and neurodevelopment [3]. In Polynesian patients with p.Leu216Arg, 25–50 mg/kg/day was necessary to obtain a clinical response, and two homozygous siblings received 1.2 g/day orally, the highest published dose, without requiring readmission for decompensation [3, 50]. Even with apparent clinical recovery, some patients continue to excrete metabolites, particularly 3-hydroxyisovalerate, inversely related to lymphocyte carboxylase activity; a patient with c.1519+5G>A (IVS10+5G>A, historical numbering; = NM_001352514.2:c.1960+5G>A) in trans with a truncating variant retained, on 200 mg/day, lymphocyte carboxylase activities below 50% and elevated 3-hydroxyisovalerate in urine, plasma and CSF [8, 64].

Pharmacokinetic measurement supports dose adjustment. A three-month-old infant receiving 10 mg/day reached approximately 100 ng/mL blood biotin, a two-year-old child on the same dose reached 703 ng/mL and a newborn on 20 mg/day reached 660 ng/mL, values above the altered Km range of the patients studied; a patient with a poor response to 10 mg/day responded to 100 mg/day, with plasma concentrations above 500 nM, exceeding the enzyme's Km for biotin (164 nM) [3]. The usual clinical response is rapid: ketosis, acidosis and hyperammonaemia disappear, lactate and pyruvate normalise, lethargy, hypotonia and ataxia regress, skin lesions disappear and hair grows; leucocyte carboxylase activities normalise within a few days, but established developmental delay and ventricular dilatation do not regress [3]. No patient receiving biotin had acute ketoacidosis in the Atlas series [3].

Patients with acute decompensation require general emergency treatment for organic acidurias, with correction of acidosis and hyperammonaemia, in addition to biotin [8]. In an infant with lactic acidosis, if organic acid analysis is not rapidly available, a therapeutic biotin trial is justified, retaining urine for subsequent analysis [3]. Moderate protein restriction may reduce metabolite accumulation in patients with a less complete response [3]. Death from ketoacidosis and disseminated intravascular coagulation was documented even after biotin had been started, and an infant treated in utero without diagnosis, whose biotin was stopped after birth while awaiting enzymatic diagnosis, developed severe ketoacidosis, lactic acidaemia and shock, subsequently responding to biotin [3]. Gene therapy and epigenetic intervention are only research directions, with no published clinical results [20].

7.4 Biotin interference with immunoassays

The pharmacological biotin doses used in these diseases interfere with laboratory tests using biotin–streptavidin technology, and methods should be confirmed with laboratories to avoid this interference [1, 16]. In a non-randomised trial in six healthy adults, biotin 10 mg/day for seven days — the same dose used in biotinidase deficiency — produced potentially relevant interference in 9 of 23 biotinylated immunoassays (39%) and in none of the 14 non-biotinylated assays; 5 of 8 competitive assays gave falsely elevated results and 4 of 15 sandwich assays gave falsely low results [17]. Analytes assessed included thyroid-stimulating hormone, total and free thyroxine and triiodothyronine, parathyroid hormone, prolactin, N-terminal pro-B-type natriuretic peptide, 25-hydroxyvitamin D, prostate-specific antigen and ferritin [17]. The French PNDS gives as clinical examples the underestimation of troponin and the overestimation of thyroid hormones, and notes that alternative analytical methods exist [13].

Interference is proportional to dose, making the problem more relevant in HCS deficiency and other biotin-responsive diseases. Wolf illustrated this variation with a child with partial biotinidase deficiency treated with 5 mg/day, a child with HCS deficiency treated with 20–100 mg/day and a child with biotin- and thiamine-responsive basal ganglia disease treated with more than 300 mg/day [18]. In practice, patients, families and physicians should inform the laboratory that the sample comes from someone receiving high-dose biotin, specifying the daily dose, any interruption before sampling and the reason for treatment [18]. Methods for removing biotin from samples (streptavidin microbeads and commercial adsorption devices) can be performed in less than an hour in most laboratories, and there is no agreed duration for the alternative of withholding biotin before sampling [65]. The authors consider the alternatives of using another analytical platform without biotin–streptavidin immobilisation or repeating sampling after interruption less practical, and note that recognising and communicating these interferences are the responsibility of the clinical laboratory, whereas Wolf emphasises that removal will only be applied if the laboratory knows the sample comes from a patient receiving high-dose biotin [18, 65].

7.5 Risk management in relatives and special situations

A newborn whose older sibling has biotinidase deficiency should start biotin at birth while awaiting enzyme activity or molecular testing, if prenatal diagnosis has not been performed; the status of older siblings, even if asymptomatic, and of any relative with compatible symptoms, including late-onset symptoms, should be clarified by enzyme activity or molecular testing [1]. Investigation of all relatives of a patient is important to detect asymptomatic individuals at risk of developing symptoms at any age [8]. In HCS deficiency, treatment of at-risk children immediately after birth may be sufficient, although prenatal treatment has been used [8]. In pregnancies of women with profound biotinidase deficiency treated with biotin, the pregnancies and the offspring were normal, and the only particular consideration for a pregnancy with an affected or at-risk fetus is possible maternal biotin supplementation, the optimal dose of which has not been determined [1, 26]. The French PNDS asks that a pregnancy with suspected biotinidase or HCS deficiency in the fetus be followed with advice from a reference centre for rare epilepsies and for inherited metabolic diseases, so that the newborn is treated as early as possible, considers that maternal treatment during pregnancy may be discussed, and provides for temporary adjustment of the patient's dose during intercurrent illness or pregnancy [13].

7.6 Regulatory status, evidence interpretation and use outside approved indications

In October 2026, there is no medicinal product in the United States or European Union with an approved indication for biotinidase or HCS deficiency: the search of FDA databases identified no biotin label with these indications, and the EMA medicines dataset includes no centrally authorised biotin medicinal product [56]. In the United States, biotin is marketed mainly as a dietary supplement, without regulatory assessment of efficacy for these diseases [56]. The only registered interventional drug trial in these diseases, the open-label single-arm phase 2 study NCT03269045 (Orpha Labs), evaluated a product called ORL-1B in 20 patients aged up to 18 years with biotinidase deficiency, with seizure frequency at 12 months as the primary outcome; it was completed in 2018, with no results published in the registry or publication identified [66]. In September 2025, biotin received an orphan designation in the European Union for biotin- and thiamine-responsive basal ganglia disease (EU/3/25/3172, sponsor Lydian Limited), one of the differential diagnoses; designation is not an authorisation and does not cover biotinidase or HCS deficiency [67].

Table 7.1 — Regulatory status of biotin in these diseases (October 2026)

Sources: [56, 57, 60, 67].

In France, in 2022, biotin was available as a 5 mg tablet and as a 5 mg/1 mL ampoule for intramuscular use, without reimbursement by the social security system; the PNDS identifies it as use outside the marketing authorisation for these indications, with a duty to inform the patient [13]. Reimbursement may be requested through the long-term illness scheme for inherited metabolic diseases (ALD 17), decided case by case by a medical adviser, or, if this is refused, through an exceptional application for a rare disease, dependent on the family's means; the PNDS recommends that the metabolic physician support the application by letter [13]. When the commercial form is unsuitable, especially in paediatrics or in dysphagia, compounded preparations are prescribed from authorised pharmacies, with a lead time of 48–72 hours for outpatients, reimbursed only with the statement “compounded preparation in the absence of a suitable pharmaceutical form”; hospital discharge should be prepared with doses for the first few days and with direct contact between the centre's pharmacy and the community pharmacy [13].

There are no randomised trials in these diseases, and the evidence supporting treatment comes from natural history studies and patient series:

Table 7.2 — Studies supporting biotin treatment

These sources do not allow determination of an optimal dose or comparison of regimens. Good outcomes in screened patients derive from comparison with symptomatic historical controls rather than concurrent groups; the systematic review pools published reports, subject to publication bias, and its percentages refer to reported individuals rather than a defined population; in partial deficiency, absence of symptoms in treated patients does not demonstrate that treatment is necessary in all, nor does the Austrian series of untreated asymptomatic patients with partial deficiency demonstrate the opposite, because neither study was designed to compare strategies [8, 27, 49, 68]. The absence of known toxicity is based on absence of adverse effects reported in series rather than formal safety studies [1, 8].

Evidence hierarchy. The recommendations in this article rely on peer-reviewed articles (cohorts, series, reviews, the Italian survey), reference texts (GeneReviews, Saudubray, Atlas) and the 2017 ACMG laboratory technical standard, which addresses laboratory diagnosis rather than treatment [1, 8, 9]. The 2022 French PNDS is a national protocol developed by expert consensus according to the HAS method, based on the literature, without formal grading of evidence [13]. Registered trials without results (NCT03269045) and ongoing observational studies, such as the Bologna newborn screening cohort (NCT06723925, recruiting, with estimated completion in December 2026), do not constitute evidence of efficacy [66]. Orphan designation of biotin for another disease is neither an opinion on efficacy nor an authorisation, and incorporation into the Brazilian SUS is an access decision, distinct from marketing registration [57, 67].

Use outside approved indications and extrapolation. As biotin has no medicinal product label with these indications in the United States and European Union, its use in these diseases is always outside a label, supported by expert consensus and series; in Brazil, where registration and a PCDT exist, prescribing within the protocol is use provided for by the health system [1, 56, 57]. Evidence extrapolation is a separate issue: maintenance doses were studied mainly in infants and children, and their application to adolescents and adults, high doses in late forms with myelopathy (for example, 200 mg/day), very high doses in HCS deficiency with partial response (up to 1.2 g/day) and maternal supplementation during pregnancy with an at-risk fetus are extrapolations based on case reports, without a defined optimal dose [1, 3, 47, 50].

7.7 Divergences between the French PNDS and other sources

Source for the PNDS column: [13]. In the absence of comparative trials, none of these positions takes precedence over the others on the basis of evidence; the chapter keeps the GeneReviews and Saudubray doses as the main reference and presents those of the PNDS as French national guidance, both off-label [1, 8, 13].

8. Reproductive Options and Family Counselling

Both diseases are inherited in an autosomal recessive manner [1, 8]. Parents of a patient are presumed heterozygotes; if the proband has undergone molecular testing, testing the parents is recommended to confirm heterozygosity and allow reliable risk calculation, and if the variant is found in only one parent, a de novo variant in the proband, parental mosaicism or uniparental isodisomy should be considered [1]. With both parents heterozygous, each sibling has, at conception, a 25% probability of being affected, 50% of being an asymptomatic carrier and 25% of being neither affected nor a carrier [1, 6]. Heterozygotes are asymptomatic, are not at risk of developing the disease and do not require biotin, whether they carry a profound-deficiency or partial-deficiency allele [1]. The parents' siblings have a 50% probability of being carriers [1].

The French PNDS adds guidance for the family [13]. The diagnosis is disclosed at a dedicated consultation by the physician who knows the disease or who requested the genetic test, ideally with a psychologist and after discussion at a multidisciplinary meeting, and covers the natural history, prognosis, treatment, recurrence risk and referral to a genetics clinic, which should be offered early [13]. In couples with an affected child, the parents' status should be known; with ACMG class 4 or 5 variants in the proband and the parents, prenatal or preimplantation diagnosis is possible, after prior discussion at a multidisciplinary prenatal diagnosis centre [13]. For adult patients planning a family, consanguineous unions are discouraged and systematic testing of the spouse is not indicated, although genetic counselling should be revisited over time; in siblings, presymptomatic testing of minors is subject to specific regulation in France and is decided with the geneticist, the reference centre and the specialised laboratory, and the siblings' results may help interpret those of the proband; in second-degree or more distant relatives, outside consanguinity, the risk of affected offspring is low and systematic testing is not proposed, but a genetics clinic appointment is offered to those who request it [13].

With a carrier frequency of approximately 1:120 in the general population, the empirical risk of an individual with biotinidase deficiency having an affected child is approximately 1:240; otherwise, offspring are obligate carriers [1, 11]. Carrier detection by serum activity has approximately 95% accuracy, because heterozygotes have intermediate activity, but molecular testing is preferred when familial variants are known [1]. Carrier testing in the reproductive partner of a known carrier should be considered, particularly in consanguinity or shared ethnic origin, for example in Amish communities with two founder variants [1]. In the Faroe Islands, the carrier frequency for HCS deficiency was estimated at approximately 1:20 [12]. The ideal time to assess genetic risk and discuss prenatal or preimplantation diagnosis is before pregnancy, and DNA storage is recommended for probands without a confirmed molecular diagnosis [1]. Counselling includes detection of other affected family members, recurrence risk, the possibility of prenatal diagnosis and referral to patient associations [6].

Prenatal and preimplantation diagnosis in biotinidase deficiency. When familial variants are identified, prenatal diagnosis by amniocentesis or chorionic villus sampling and preimplantation genetic testing are possible, particularly to enable treatment from birth; biotinidase activity can also be measured in cultured amniocytes and amniotic fluid, but molecular testing is preferred [1, 2]. Saudubray considers prenatal diagnosis possible but unjustified, because prenatal treatment is unnecessary [8]. Different professional and family perspectives on use of prenatal diagnosis should be discussed [1].

Prenatal diagnosis and prenatal treatment in HCS deficiency. Prenatal diagnosis is possible by molecular testing when the index case variants are known, by enzymatic studies in chorionic villi or cultured amniocytes (with markedly elevated Km for biotin as a diagnostic finding) or by measuring amniotic fluid metabolites by isotope dilution [3, 8]. Quantification of 3-hydroxyisovaleric acid in amniotic fluid, more reliable than methylcitric acid, is the best rapid chemical method, but may be equivocal, and in mild forms organic acid analysis may fail to identify an affected fetus, requiring molecular or enzymatic confirmation [3, 8]. Prenatal biotin treatment (10 mg/day to the mother) was performed in at least four at-risk pregnancies, without maternal or fetal adverse effects, with affected newborns clinically well and with normal urinary organic acids [3, 69]. The Atlas considers it prudent, because delivery itself may be sufficiently catabolic to render an affected newborn irreversibly moribund within a few hours, whereas Saudubray considers it uncertain whether it is essential [3, 8].

9. Special Considerations: Population Diversity and Epidemiology

9.1 Biotinidase deficiency

Based on the 1991 worldwide newborn screening survey, incidence is 1:137,401 for profound deficiency, 1:109,921 for partial deficiency and 1:61,067 for both combined, with a carrier frequency of approximately 1:120 [1, 11]. Incidence is higher in populations with high consanguinity, such as Turkey and Saudi Arabia, appears increased in the Hispanic population and may be lower in the African American population [1, 6]. Recent screening data show wide geographical variation, partly dependent on thresholds and inclusion of partial deficiency:

For Brazil, the Brazilian chapter presents frequencies of 1:9,000 for combined deficiency, 1:14,192 for profound deficiency and 1:9,000 for partial deficiency, and for the general population 1:112,000 for profound deficiency and 1:130,000 for partial deficiency, emphasising that national data are scarce and discordant [6]. In Brazil, allelic diversity is broad, with novel variants identified in screening studies and genotype–biochemical phenotype concordance of only 68.5% [35, 71]. Incidences in the table come from screening programmes and depend on thresholds and inclusion of partial deficiency, which dominates the higher European figures (Italy, Netherlands); clinical series, such as the Turkish series of 194 patients or the Brazilian series of 72 individuals, describe followed patients and do not allow incidence calculation [35, 53, 54].

Newborn screening for biotinidase deficiency is widespread in the United States and many other countries [1]. In a European survey published in 2021 (2020 data), biotinidase deficiency was screened in Austria, Belgium, the Czech Republic, Denmark, Germany, Hungary, Italy, Latvia, the Netherlands, Norway, Poland, Sweden, Switzerland and Turkey, was included in pilot or regional programmes in Spain, Ukraine and Uzbekistan, and was not screened in Portugal, France, the United Kingdom, Ireland, Greece and several other countries; screening in Spain was scheduled to start in 2021 [72]. India has no universal newborn screening, which favours late presentations [47]. The 2022 French PNDS confirms that biotinidase deficiency was not then part of the French newborn screening programme and links this absence to the observation of late-onset forms in adolescence and young adulthood [13]. No population incidence data for Portugal, or population-based data for most of Africa, were found in the literature consulted.

9.2 Holocarboxylase synthetase deficiency

HCS deficiency appears rarer than biotinidase deficiency, and Orphanet records its prevalence as unknown [4, 8]. The French PNDS cites an estimated prevalence of 1:200,000 live births, taken from a 2016 review, without stating the source population; this is a literature estimate and not a population screening result [13]. The exception is the small isolated population of the Faroe Islands, with a calculated incidence of 1:1200 newborns and a carrier frequency of approximately 1:20, associated with the Scandinavian founder variant c.1519+5G>A [8, 10, 12]. In Japanese patients, p.Leu237Pro and c.780delG predominate, probably founder variants; in Polynesian patients, particularly Samoans, p.Leu216Arg is associated with the most severe form, with antenatal injury and incomplete biotin response; p.Arg508Trp and p.Val550Met occur in several ethnic groups [3, 10, 39]. Consanguinity has been documented in both diseases [2, 3].

10. Illustrative Clinical Case

10.1 Late-onset profound biotinidase deficiency mimicking neuromyelitis optica

A 9-year-old boy with age-appropriate development, seen at a reference centre in Bangalore (India), presented with urinary frequency and urgency of two months' duration, followed by sequential weakness of the lower and then upper limbs, a mid-thoracic sensory level and painless bilateral visual loss, with continuous progression [47]. On admission, he had light perception, optic disc pallor, spastic tetraparesis, generalised hyperreflexia, bilateral Babinski signs, sensory loss up to C3, fluctuating blood pressure and a modified Rankin scale score of 5 [47]. Magnetic resonance imaging showed longitudinally extensive transverse myelitis from the cervicomedullary junction to the conus medullaris and hyperintensity of the optic nerves, mammillary bodies, periaqueductal grey matter, dorsal midbrain, fornix and tectal plate [47]. Anti-aquaporin-4 and anti-MOG antibodies were negative, CSF had 15 cells/mm³, oligoclonal bands and lactate of 80 mg/dL, and acylcarnitines showed mild elevation of C3 and C5-DC/C6-OH; visual and somatosensory evoked potentials were absent and brainstem auditory evoked potentials were normal [47]. Before diagnosis, he received methylprednisolone, intravenous immunoglobulin and plasma exchange [47].

Serum biotinidase was 0.52 nmol/min/mL (normal >5 nmol/min/mL), and exome sequencing identified two BTD variants in compound heterozygosity: an exon 3 splicing acceptor site variant and the deletion–insertion c.98_104delinsTCC [47]. Treated with high-dose biotin (200 mg/day), alongside other measures, at six months and one year he walked with support, his Rankin score fell to 3 and visual acuity improved from light perception to 20/200 [47]. The case magnetic resonance images (hyperintensity of the fornix, dorsal midbrain, periaqueductal grey matter and mammillary bodies) appear in figure 1 of the original open-access article (https://pmc.ncbi.nlm.nih.gov/articles/PMC10666890/figure/F1/) [47]. The case illustrates three points: the late form presents as opticomyelopathy and is confused with seronegative NMOSD; elevated CSF lactate, acylcarnitines and fornix and mammillary body hyperintensity are diagnostic clues; and recovery is only partial when treatment is late [1, 46, 47].

10.2 Severe holocarboxylase synthetase deficiency due to homozygous p.Leu216Arg

Two siblings followed in Hawaii, homozygous for p.Leu216Arg — a variant observed in individuals of Polynesian ancestry and considered one of the most severe and biotin-unresponsive — both had cerebral haemorrhage and subependymal cysts on perinatal imaging and metabolic decompensation in the first 24 hours of life, with metabolic acidosis, lactic acidosis and thrombocytopenia [50]. The biochemical profile was compatible with HCS deficiency and genetic testing confirmed homozygosity [50]. After the neonatal crisis, the biotin dose was titrated in outpatient care mainly according to dermatitis control, and the older sister received 1.2 g/day orally, a dose higher than any other published for this disease [50]. Neither sibling was readmitted for acute decompensation, and at 7 years the older sister, the oldest reported patient homozygous for p.Leu216Arg at that time, had mild intellectual disability [50]. The case contrasts with the series of seven Samoan infants with fetal growth restriction, subependymal cysts, partial biotin response and poor prognosis, and shows that, with specialist neonatal care and very high-dose biotin, prognosis for this variant may be less severe than initially described [39, 50].

10.3 Biotinidase deficiency with relapse due to poor adherence

A 27-month-old Saudi girl with biotinidase deficiency lost all her scalp hair and eyebrows at 20 days of life; hair regrew, but disappeared again at eight months, indicating lack of adherence to biotin [2]. She had reddish periocular and perioral dermatitis, with fissures at the mouth corners, extensive perineal dermatitis and spastic tetraparesis [2]. After six months of biotin, tetraparesis disappeared — an exception to the rule that established spasticity persists — dermatitis regressed and hair grew; six months later, another treatment interruption caused alopecia to recur, again responding to biotin, but the child had almost total hearing loss [2]. The case encapsulates the dissociation between reversible manifestations (skin, hair, tone) and irreversible manifestations (hearing), and the importance of daily adherence [1, 2].

11. Prognosis and Long-Term Follow-Up

11.1 Biotinidase deficiency

Children diagnosed before symptoms, particularly through newborn screening, and treated with biotin have normal development [1]. In Michigan, 142 children identified through screening over 25 years (22 with profound and 120 with partial deficiency), treated shortly after birth and with good adherence, had apparently normal physical and cognitive development; the authors recommend that, when symptoms occur in a treated patient, after addressing adherence and dose, other causes should be sought [68]. A survey of 44 individuals with profound deficiency identified through screening, with a mean age of 23.1 years, showed that all completed secondary education and many higher education; several developed symptoms when stopping biotin for days or weeks, which regressed when it was resumed, and five treated women had nine uneventful pregnancies and deliveries [26]. In Austria, all patients with activity below 10% and biotin in the first weeks had normal neuropsychological development, whereas three of five treated after 3.5 years had an altered intelligence quotient [49]. In the 2023 review, biotin led to clinical stability or improvement in 89.2% of individuals [27].

In symptomatic patients, biochemical abnormalities and seizures cease rapidly after biotin initiation, followed by improvement in skin lesions, and hair regrows over weeks to months [1, 6]. Optic atrophy and deafness may resist treatment, particularly if a long interval elapsed between onset and treatment; some children rapidly regain developmental milestones and others retain delay, and sequelae tend to become static without complete regression [1]. Intellectual disability and ataxia have been observed as long-term complications in those diagnosed late [8]. Life expectancy is normal, both in initially symptomatic patients and in those identified through screening, provided they continue biotin lifelong [1].

Follow-up recommended by GeneReviews is as follows [1]:

The Brazilian chapter adds objective neuropsychological assessment between 6 and 7 years [6]. ClinGen classifies actionability as definitive in the paediatric setting and limited in adults, reflecting that benefit centres on treatment from birth, without this justifying denial of biotin to an adult with optic neuropathy or myelopathy, which may respond [1, 15].

The French PNDS proposes a more frequent schedule in the first year, common to both diseases and to be adapted to the clinical situation of patients whose treatment is already adjusted [13]:

The two schedules diverge: GeneReviews proposes annual visits in profound deficiency and every two years in partial deficiency and reserves organic acids for recurrence of symptoms, whereas the PNDS proposes regular biochemical surveillance in both forms (section 7.7) [1, 13].

11.2 Holocarboxylase synthetase deficiency

The prognosis for most surviving, well-treated patients appears good, except for those with partial or absent response to biotin; irreversible auditory or visual sensorineural deficits such as those in biotinidase deficiency have not been described, and long-term follow-up studies are needed [8]. A 2026 review reports that early diagnosis through newborn screening, combined with standardised biotin supplementation, enables biochemical normalisation and symptom resolution in more than 85% of patients [20]. Patients homozygous for c.1519+5G>A (IVS10+5G>A, historical numbering) have a slow and, in some, only partial response, and those homozygous for p.Leu216Arg usually retain an incomplete response, although favourable outcomes on very high doses have been described [8, 50]. In the Faroe Islands, all symptomatic patients responded to biotin, but only when treated early [12]. Follow-up should include monitoring of urinary organic acids (particularly 3-hydroxyisovalerate), lymphocyte carboxylase activities where available, neurodevelopment and skin, and possibly plasma biotin concentrations for dose adjustment [3, 8]. The French PNDS stresses that untreated HCS deficiency can cause severe metabolic acidosis, cerebral oedema, coma and death, that early treatment allows complete resolution of seizures and other symptoms, and that delayed treatment may leave irreversible neurological damage [13].

11.3 Reference networks and patient associations

In the European Union, both diseases are managed by MetabERN (European Reference Network for Hereditary Metabolic Disorders) centres, the European reference network for inherited metabolic diseases [73]. Patient associations covering these diseases include Metabolic Support UK in the United Kingdom, APOFEN (Associação Portuguesa de Fenilcetonúria e outras Doenças Metabólicas) in Portugal and, in Brazil, Instituto Vidas Raras, a national rare disease association; the Brazilian chapter recommends referring families to patient associations [6, 74].

In France, the PNDS was developed by the rare epilepsy reference centres of Lille and Marseille and involves the rare epilepsy reference and competence centres (CRéER, DéfiScience network) and the inherited metabolic disease centres (G2M network), with follow-up preferably in joint clinics [13]. There is no patient association specific to these diseases in France; the PNDS suggests the Maladies Rares Info Services forum, Epilepsie France and the Feux Follets association, for inherited metabolic diseases treated with diet [13]. Transition to adulthood includes renewed information for the patient about the disease, a dedicated transfer consultation (joint, with a detailed letter to the general practitioner, or after a multidisciplinary meeting), review of sequelae, therapeutic education on adherence and treatment effects, and topics such as contraception, pregnancy, genetic counselling and employment; in adulthood, follow-up passes to the neurologist, the adult metabolic physician and the geneticist [13]. For travel, the PNDS recommends a medical certificate for emergencies and for carrying medication in the cabin, a supply for the entire stay, the prescriptions, and temporarily giving up pill organisers that do not identify the medicines; medico-social measures include the long-term illness scheme, entitlements through the departmental centre for people with disabilities (MDPH) and individualised school support plans [13].

12. Summary and Final Considerations

Biotinidase deficiency and holocarboxylase synthetase deficiency are the two inherited causes of multiple carboxylase deficiency, both autosomal recessive and both treatable with oral biotin. In biotinidase deficiency, inability to recycle biotin leads to progressive depletion affecting the central nervous system first; the disease usually manifests after three months, with seizures, hypotonia, ataxia, periorificial dermatitis, alopecia, keratoconjunctivitis, infections and respiratory abnormalities, and, in adolescence or adulthood, with optic neuropathy and myelopathy mimicking multiple sclerosis or NMOSD. Organic aciduria and acidosis may be absent, so diagnosis depends on serum biotinidase measurement with appropriate controls, and classification as profound (<10%) or partial (10–30%) deficiency guides treatment; the p.Asp444His variant, in trans with a severe allele, explains almost all partial deficiency. Newborn screening and biotin from birth (5–10 mg/day in profound deficiency and 2.5–10 mg/day in partial deficiency, according to GeneReviews) produce normal development, whereas sensorineural deafness, optic atrophy and developmental delay established before treatment do not fully regress.

Holocarboxylase synthetase deficiency usually presents as severe neonatal organic aciduria with generalised dermatitis, is not detected by the screening biotinidase assay but by acylcarnitines (C5-OH), and responds to biotin doses generally between 10 and 20 mg/day, with some patients requiring 40–200 mg/day or more and a subgroup, associated particularly with p.Leu216Arg and N-terminal variants, showing a partial response. The correlation between Km for biotin, residual activity and severity supports individual dose adjustment, aided by pharmacokinetics. In both diseases, pharmacological-dose biotin interferes with biotin–streptavidin-based immunoassays, and laboratories should be informed of the dose. In the United States and the European Union, biotin has no medicinal product label for these indications, and treatment rests on natural history studies and case series, without randomised trials; in Brazil, it is registered and incorporated into the SUS with its own PCDT. The principal gaps are the absence of formal international clinical guidelines (national guidance exists, such as the Brazilian PCDT and the 2022 French PNDS, which diverge on doses and surveillance), of comparative dose trials and of epidemiological data for countries without screening, including Portugal.

13. References

1. Wolf B. Biotinidase Deficiency. 2000 Mar 24 [updated 2023 May 25; revised 2026 Feb 19]. In: Adam MP, Bick S, Mirzaa GM, Pagon RA, Wallace SE, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2026. PMID: 20301497. Available at: https://www.ncbi.nlm.nih.gov/books/NBK1322/
2. Nyhan WL, Hoffmann GF, Al-Aqeel AI, Barshop BA. Atlas of Inherited Metabolic Diseases. 4th ed. Boca Raton: CRC Press; 2020. Chapter 7, Multiple carboxylase deficiency/biotinidase deficiency; p. 52–61.
3. Nyhan WL, Hoffmann GF, Al-Aqeel AI, Barshop BA. Atlas of Inherited Metabolic Diseases. 4th ed. Boca Raton: CRC Press; 2020. Chapter 6, Multiple carboxylase deficiency/holocarboxylase synthetase deficiency; p. 45–51.
4. Orphanet. Biotinidase deficiency (ORPHA:79241); Holocarboxylase synthetase deficiency (ORPHA:79242); Multiple carboxylase deficiency (ORPHA:148) [Internet]. Paris: INSERM; [accessed 2026 Oct 4]. Available at: https://www.orpha.net/en/disease/detail/79241, https://www.orpha.net/en/disease/detail/79242 and https://www.orpha.net/en/disease/detail/148
5. Online Mendelian Inheritance in Man (OMIM). #253260 Biotinidase deficiency; *609019 Biotinidase (BTD); #253270 Holocarboxylase synthetase deficiency; *609018 Holocarboxylase synthetase (HLCS) [Internet]. Baltimore (MD): Johns Hopkins University; [accessed 2026 Oct 4]. Available at: https://omim.org/entry/253260 and https://omim.org/entry/253270
6. Aires CMP, Corrêa MB, Ribeiro EM. Deficiência de biotinidase. In: Ribeiro EM, coord. Síndromes Genéticas: Reconhecer para Tratar. Fortaleza: EdUnichristus; 2021. p. 315–317.
7. Morales J, Pujar S, Loveland JE, Astashyn A, Bennett R, Berry A, et al. A joint NCBI and EMBL-EBI transcript set for clinical genomics and research. Nature. 2022;604(7905):310-5. doi:10.1038/s41586-022-04558-8. PMID: 35388217. MANE.GRCh38.v1.5.summary.txt file available at: https://ftp.ncbi.nlm.nih.gov/refseq/MANE/MANE_human/current/.
8. Froese DS, Baumgartner MR. Biotin-Responsive Disorders. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 27. doi:10.1007/978-3-662-63123-2_27
9. Strovel ET, Cowan TM, Scott AI, Wolf B. Laboratory diagnosis of biotinidase deficiency, 2017 update: a technical standard and guideline of the American College of Medical Genetics and Genomics. Genet Med. 2017;19(10). doi:10.1038/gim.2017.84. PMID: 28682309.
10. Suzuki Y, Yang X, Aoki Y, Kure S, Matsubara Y. Mutations in the holocarboxylase synthetase gene HLCS. Hum Mutat. 2005;26(4):285-90. doi:10.1002/humu.20204. PMID: 16134170.
11. Wolf B. Worldwide survey of neonatal screening for biotinidase deficiency. J Inherit Metab Dis. 1991;14(6):923-7. doi:10.1007/BF01800475. PMID: 1779651.
12. Lund AM, Joensen F, Hougaard DM, Jensen LK, Christensen E, Christensen M, et al. Carnitine transporter and holocarboxylase synthetase deficiencies in The Faroe Islands. J Inherit Metab Dis. 2007;30(3):341-9. doi:10.1007/s10545-007-0527-9. PMID: 17417720.
13. Centres de référence des épilepsies rares (CRMR Lille et Marseille); Filière DéfiScience. Protocole National de Diagnostic et de Soins (PNDS): Épilepsies vitamino-sensibles. Déficits en antiquitine, en pyridoxamine-5' phosphate oxydase (PNPO), en PLP binding protein (PLPBP), déficits en biotinidase, en holocarboxylase synthétase, déficit cérébral en folates lié à une mutation du gène FOLR1. Avril 2022. Saint-Denis La Plaine: Haute Autorité de Santé; mis en ligne le 2 août 2022 [accessed 6 Nov 2026]. Available from: https://www.has-sante.fr/jcms/p_3358937
14. Wolf B, Spencer R, Gleason T. Hearing loss is a common feature of symptomatic children with profound biotinidase deficiency. J Pediatr. 2002;140(2):242-6. doi:10.1067/mpd.2002.121938. PMID: 11865279.
15. Clinical Genome Resource (ClinGen). Actionability summary report AC098: Biotinidase deficiency (pediatric and adult contexts); Gene-disease validity: HLCS – holocarboxylase synthetase deficiency [Internet]. [accessed 4 Oct 2026]. Available from: https://actionability.clinicalgenome.org/ac/ and https://search.clinicalgenome.org/kb/genes/HGNC:4976
16. Wolf B. High doses of biotin can interfere with immunoassays that use biotin-strept(avidin) technologies: implications for individuals with biotin-responsive inherited metabolic disorders. Mol Genet Metab. 2019;127(4):321-4. doi:10.1016/j.ymgme.2019.07.003. PMID: 31320189.
17. Li D, Radulescu A, Shrestha RT, Root M, Karger AB, Killeen AA, et al. Association of biotin ingestion with performance of hormone and nonhormone assays in healthy adults. JAMA. 2017;318(12):1150-60. doi:10.1001/jama.2017.13705. PMID: 28973622.
18. Wolf B. Response to comment on "High doses of biotin can interfere with immunoassays that use biotin-strept(avidin) technologies: implications for individuals with biotin-responsive inherited metabolic disorders". Mol Genet Metab Rep. 2019;21:100511. doi:10.1016/j.ymgmr.2019.100511. PMID: 31516832.
19. Firth HV, Hurst JA. Oxford Desk Reference: Clinical Genetics and Genomics. 2nd ed. Oxford: Oxford University Press; 2017. Neonatal encephalopathy and intractable seizures; p. 248–250.
20. ZeZhao J, Ablimit A. Holocarboxylase synthetase deficiency: pathogenesis, clinical features, diagnosis, treatment, and research prospects. Eur J Pediatr. 2026;185(7):512. doi:10.1007/s00431-026-07038-7. PMID: 42324336.
21. Gompertz D, Draffan GH, Watts JL, Hull D. Biotin-responsive beta-methylcrotonylglycinuria. Lancet. 1971;2(7714):22-4. doi:10.1016/s0140-6736(71)90009-2. PMID: 4103667.
22. Burri BJ, Sweetman L, Nyhan WL. Mutant holocarboxylase synthetase: evidence for the enzyme defect in early infantile biotin-responsive multiple carboxylase deficiency. J Clin Invest. 1981;68(6):1491-5. doi:10.1172/jci110402. PMID: 6798072.
23. Wolf B, Grier RE, Allen RJ, Goodman SI, Kien CL. Biotinidase deficiency: the enzymatic defect in late-onset multiple carboxylase deficiency. Clin Chim Acta. 1983;131(3):273-81. doi:10.1016/0009-8981(83)90096-7. PMID: 6883721.
24. Heard GS, Wolf B, Jefferson LG, Weissbecker KA, Nance WE, McVoy JR, et al. Neonatal screening for biotinidase deficiency: results of a 1-year pilot study. J Pediatr. 1986;108(1):40-6. doi:10.1016/s0022-3476(86)80766-1. PMID: 3944695.
25. Wolf B. Biotinidase deficiency: "if you have to have an inherited metabolic disease, this is the one to have". Genet Med. 2012;14(6):565-75. doi:10.1038/gim.2011.6. PMID: 22241090.
26. Wolf B. Successful outcomes of older adolescents and adults with profound biotinidase deficiency identified by newborn screening. Genet Med. 2017;19(4):396-402. doi:10.1038/gim.2016.135. PMID: 27657684.
27. Tankeu AT, Van Winckel G, Elmers J, Jaccard E, Superti-Furga A, Wolf B, et al. Biotinidase deficiency: what have we learned in forty years? Mol Genet Metab. 2023;138(4):107560. doi:10.1016/j.ymgme.2023.107560. PMID: 37027963.
28. Cole H, Reynolds TR, Lockyer JM, Buck GA, Denson T, Spence JE, et al. Human serum biotinidase. cDNA cloning, sequence, and characterization. J Biol Chem. 1994;269(9):6566-70. doi:10.1016/S0021-9258(17)37409-4. PMID: 7509806.
29. Pomponio RJ, Hymes J, Reynolds TR, Meyers GA, Fleischhauer K, Buck GA, et al. Mutations in the human biotinidase gene that cause profound biotinidase deficiency in symptomatic children: molecular, biochemical, and clinical analysis. Pediatr Res. 1997;42(6):840-8. doi:10.1203/00006450-199712000-00020. PMID: 9396567.
30. Swango KL, Demirkol M, Hüner G, Pronicka E, Sykut-Cegielska J, Schulze A, et al. Partial biotinidase deficiency is usually due to the D444H mutation in the biotinidase gene. Hum Genet. 1998;102(5):571-5. doi:10.1007/s004390050742. PMID: 9654207.
31. Norrgard KJ, Pomponio RJ, Swango KL, Hymes J, Reynolds T, Buck GA, et al. Double mutation (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. Hum Mutat. 1998;11(5):410. doi:10.1002/(SICI)1098-1004(1998)11:5<410::AID-HUMU10>3.0.CO;2-8. PMID: 10206677.
32. Funghini S, Tonin R, Malvagia S, Caciotti A, Donati MA, Morrone A, et al. High frequency of biotinidase deficiency in Italian population identified by newborn screening. Mol Genet Metab Rep. 2020;25:100689. doi:10.1016/j.ymgmr.2020.100689. PMID: 33312878.
33. Cowan TM, Kazerouni NN, Dharajiya N, Lorey F, Roberson M, Hodgkinson C, et al. Increased incidence of profound biotinidase deficiency among Hispanic newborns in California. Mol Genet Metab. 2012;106(4):485-7. doi:10.1016/j.ymgme.2012.05.017. PMID: 22698809.
34. Sivri HS, Genç GA, Tokatli A, Dursun A, Coşkun T, Aydin HI, et al. Hearing loss in biotinidase deficiency: genotype-phenotype correlation. J Pediatr. 2007;150(4):439-42. doi:10.1016/j.jpeds.2007.01.036. PMID: 17382128.
35. Borsatto T, Sperb-Ludwig F, Lima SE, Carvalho MRS, Fonseca PAS, Camelo JS Jr, et al. Biotinidase deficiency: genotype-biochemical phenotype association in Brazilian patients. PLoS One. 2017;12(5):e0177503. doi:10.1371/journal.pone.0177503. PMID: 28498829.
36. Maheras KJ, Pindolia K, Wolf B, Gow A. Developmental window of sensorineural deafness in biotinidase-deficient mice. J Inherit Metab Dis. 2017;40(5):733-44. doi:10.1007/s10545-017-0049-z. PMID: 28516283.
37. Suzuki Y, Aoki Y, Ishida Y, Chiba Y, Iwamatsu A, Kishino T, et al. Isolation and characterization of mutations in the human holocarboxylase synthetase cDNA. Nat Genet. 1994;8(2):122-8. doi:10.1038/ng1094-122. PMID: 7842009.
38. National Center for Biotechnology Information. ClinVar [Internet]. Bethesda (MD): National Library of Medicine; [accessed 4 Oct 2026]. Records NM_001352514.2(HLCS):c.1088T>G (p.Leu363Arg) e NM_001352514.2(HLCS):c.1151T>C (p.Leu384Pro). Available from: https://www.ncbi.nlm.nih.gov/clinvar/.
39. Wilson CJ, Myer M, Darlow BA, Stanley T, Thomson G, Baumgartner ER, et al. Severe holocarboxylase synthetase deficiency with incomplete biotin responsiveness resulting in antenatal insult in Samoan neonates. J Pediatr. 2005;147(1):115-8. doi:10.1016/j.jpeds.2005.03.006. PMID: 16027709.
40. Morrone A, Malvagia S, Donati MA, Funghini S, Ciani F, Pela I, et al. Clinical findings and biochemical and molecular analysis of four patients with holocarboxylase synthetase deficiency. Am J Med Genet. 2002;111(1):10-8. doi:10.1002/ajmg.10532. PMID: 12124727.
41. Burri BJ, Sweetman L, Nyhan WL. Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency. Am J Hum Genet. 1985;37(2):326-37. PMID: 3920902.
42. 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.
43. Alan MA, Ugur C, Kahraman AB, Agyar B. Auditory function at birth in infants with biotinidase deficiency. Int J Pediatr Otorhinolaryngol. 2026;204:112818. doi:10.1016/j.ijporl.2026.112818. PMID: 41955783.
44. Wolf B. Biotinidase deficiency should be considered in individuals exhibiting myelopathy with or without and vision loss. Mol Genet Metab. 2015;116(3):113-8. doi:10.1016/j.ymgme.2015.08.012. PMID: 26358973.
45. Wolf B. Biotinidase deficiency should be considered in individuals thought to have multiple sclerosis and related disorders. Mult Scler Relat Disord. 2019;28:26-30. doi:10.1016/j.msard.2018.11.030. PMID: 30551056.
46. Bottin L, Prud'hon S, Guey S, Giannesini C, Wolf B, Pindolia K, et al. Biotinidase deficiency mimicking neuromyelitis optica: initially exhibiting symptoms in adulthood. Mult Scler. 2015;21(12):1604-7. doi:10.1177/1352458515596457. PMID: 26203071.
47. Ravikumar NA, Dhar D, Mahale R, Reddy AM, Shreedevi AU, Prabhu S, et al. Longitudinally extensive transverse myelitis with optic neuritis related to profound biotinidase deficiency: NMOSD mimic! Ann Indian Acad Neurol. 2023;26(5):795-8. doi:10.4103/aian.aian_697_23. PMID: 38022461.
48. Mochel F, Sedel F. Inborn errors of metabolism in adults: a diagnostic approach to neurological and psychiatric presentations. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 2.
49. Möslinger D, Stöckler-Ipsiroglu S, Scheibenreiter S, Tiefenthaler M, Mühl A, Seidl R, et al. Clinical and neuropsychological outcome in 33 patients with biotinidase deficiency ascertained by nationwide newborn screening and family studies in Austria. Eur J Pediatr. 2001;160(5):277-82. doi:10.1007/s004310100740. PMID: 11388594.
50. Slavin TP, Zaidi SJ, Neal C, Nishikawa B, Seaver LH. Clinical presentation and positive outcome of two siblings with holocarboxylase synthetase deficiency caused by a homozygous L216R mutation. JIMD Rep. 2014;12:109-14. doi:10.1007/8904_2013_252. PMID: 24085707.
51. Wiltink RC, Kruijshaar ME, van Minkelen R, Onkenhout W, Verheijen FW, Kemper EA, et al. Neonatal screening for profound biotinidase deficiency in the Netherlands: consequences and considerations. Eur J Hum Genet. 2016;24(10):1424-9. doi:10.1038/ejhg.2016.65. PMID: 27329734.
52. Neto EC, Schulte J, Rubim R, Lewis E, DeMari J, Castilhos C, et al. Newborn screening for biotinidase deficiency in Brazil: biochemical and molecular characterizations. Braz J Med Biol Res. 2004;37(3):295-9. doi:10.1590/s0100-879x2004000300001. PMID: 15060693.
53. Ortolano R, Menabò S, Candela E, Biasucci G, Bortolamedi E, Montanari G, et al. Enzymatic evolution and longitudinal recovery in biotinidase deficiency: genotypic and clinical insights from the follow-up of a newborn-screened cohort in Emilia-Romagna, Italy. Metabolites. 2025;15(9):605. doi:10.3390/metabo15090605. PMID: 41002989.
54. Gülbahçe A, Muderrisoglu A, Güneş H, Erdoğan M, Dündar M, Kardaş F. Assessment of biotinidase activity changes over time in biotinidase deficient patients. Front Pediatr. 2026;14:1694031. doi:10.3389/fped.2026.1694031. PMID: 41847504.
55. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-24. doi:10.1038/gim.2015.30. PMID: 25741868.
56. U.S. Food and Drug Administration. openFDA drug label API: search for labels with the indication “biotinidase” [Internet]; and European Medicines Agency. Medicines data (centrally authorised medicines) [Internet]. [accessed 4 Oct 2026]. Available from: https://api.fda.gov/drug/label.json and https://www.ema.europa.eu/en/medicines
57. Moraes SALM, Soprani GFA, Cata-Preta J, Santos BO, Borges BB, Melo SS, et al. Implementação de tecnologias no Sistema Único de Saúde: o caso da biotina para o tratamento da deficiência de biotinidase [abstract ID 135]. Epidemiol Serv Saude. 2025;34(supl 1). PMCID: PMC12806684.
58. Morris AAM, Jones S. Medications used in the treatment of inborn errors of metabolism. In: Saudubray JM, Baumgartner MR, García-Cazorla Á, Walter J, editors. Inborn Metabolic Diseases: Diagnosis and Treatment. 7th ed. Berlin: Springer; 2022. Chapter 45, Table 45.1.
59. U.S. Food and Drug Administration. openFDA drug label API: search openfda.generic_name “biotin” (30 labels: unapproved over-the-counter medicines, supplements and multivitamin combinations) [Internet]. [accessed 26 Oct 2026]. Available from: https://api.fda.gov/drug/label.json?search=openfda.generic_name:%22biotin%22
60. INFARMED. Infomed: search for the substance “biotina” (authorised and marketed medicines) and Summary of Product Characteristics of Becozyme forte, combination, coated tablets (approved 18-11-2022) [Internet]. [accessed 26 Oct 2026]. Available from: https://extranet.infarmed.pt/INFOMED-fo/
61. Agence nationale de sécurité du médicament et des produits de santé (ANSM). Base de données publique des médicaments: Résumé des caractéristiques du produit — BIOTINE BAYER 5 mg, comprimé (mis à jour le 30/05/2024; CIS 66001124) and BIOTINE BAYER 0,5 %, solution injectable I.M. (mis à jour le 15/01/2026; CIS 61890004) [Internet]. Bayer HealthCare SAS. [accessed 26 Oct 2026]. Available from: https://base-donnees-publique.medicaments.gouv.fr/medicament/66001124/extrait
62. Tagliaferri F, Candela E, Biasucci G, Bonati MT, Bordugo A, Concolino D, et al. Current management of biotinidase deficiency following newborn screening in Italy: evidence from a clinical nationwide survey. Mol Genet Metab. 2026;149(3):110264. doi:10.1016/j.ymgme.2026.110264. PMID: 42759219.
63. Wolf B. Revisiting the administration of biotin to children with biotin-responsive disorders. Mol Genet Metab. 2022;137(1-2):225-7. doi:10.1016/j.ymgme.2022.07.004. PMID: 35843775.
64. Santer R, Muhle H, Suormala T, Baumgartner ER, Duran M, Yang X, et al. Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects. Mol Genet Metab. 2003;79(3):160-6. doi:10.1016/s1096-7192(03)00091-x. PMID: 12855220.
65. Favresse J, Gruson D, Gheldof D. Comment on "High doses of biotin can interfere with immunoassays that use biotin-strept(avidin) technologies: implications for individuals with biotin-responsive inherited metabolic disorders". Mol Genet Metab Rep. 2019;21:100506. doi:10.1016/j.ymgmr.2019.100506. PMID: 31467852.
66. ClinicalTrials.gov. NCT03269045 (An open-label, single-arm, phase 2 study of ORL-1B in patients with biotinidase deficiency; Orpha Labs; last update 2018 Jan 17) and NCT06723925 (Neonatal screening of biotinidase deficiency: genotype-phenotype correlation and clinical follow-up, Bologna; last update 2026 Mar 12) [Internet]. Bethesda (MD): National Library of Medicine; [accessed 4 Oct 2026]. Available from: https://clinicaltrials.gov/study/NCT03269045 and https://clinicaltrials.gov/study/NCT06723925.
67. European Medicines Agency. EU/3/25/3172: Orphan designation for the treatment of biotin-thiamine-responsive basal ganglia disease (biotin); sponsor Lydian Limited; date of designation 2025 Sep 12 [Internet]. [accessed 4 Oct 2026]. Available from: https://www.ema.europa.eu/en/medicines/human/orphan-designations/eu-3-25-3172.
68. Jay AM, Conway RL, Feldman GL, Nahhas F, Spencer L, Wolf B. Outcomes of individuals with profound and partial biotinidase deficiency ascertained by newborn screening in Michigan over 25 years. Genet Med. 2015;17(3):205-9. doi:10.1038/gim.2014.104. PMID: 25144890.
69. Packman S, Cowan MJ, Golbus MS, Caswell NM, Sweetman L, Burri BJ, et al. Prenatal treatment of biotin responsive multiple carboxylase deficiency. Lancet. 1982;1(8287):1435-8. doi:10.1016/s0140-6736(82)92452-7. PMID: 6123722.
70. Jezela-Stanek A, Suchoń L, Sobczyńska-Tomaszewska A, Czerska K, Kuśmierska K, Taybert J, et al. Molecular background and disease prevalence of biotinidase deficiency in a Polish population — data based on the national newborn screening programme. Genes (Basel). 2022;13(5):802. doi:10.3390/genes13050802. PMID: 35627187.
71. Lara MT, Gurgel-Giannetti J, Aguiar MJ, Ladeira RV, Carvalho NO, Del Castillo DM, et al. High incidence of biotinidase deficiency from a pilot newborn screening study in Minas Gerais, Brazil. JIMD Rep. 2015;24:103-7. doi:10.1007/8904_2015_447. PMID: 25967232.
72. Loeber JG, Platis D, Zetterström RH, Almashanu S, Boemer F, Bonham JR, et al. Neonatal screening in Europe revisited: an ISNS perspective on the current state and developments since 2010. Int J Neonatal Screen. 2021;7(1):15. doi:10.3390/ijns7010015. PMID: 33808002.
73. MetabERN — European Reference Network for Hereditary Metabolic Disorders [Internet]. [accessed 4 Oct 2026]. Available from: https://metab.ern-net.eu/.
74. Metabolic Support UK (https://www.metabolicsupportuk.org/); APOFEN — Associação Portuguesa de Fenilcetonúria e outras Doenças Metabólicas (https://www.apofen.pt/); Instituto Vidas Raras (https://vidasraras.org.br/) [Internet]. [accessed 4 Oct 2026].