Biotinidase biochemical and molecular analyses: Experience at a large reference laboratory.

BACKGROUND: Biotinidase deficiency is caused by absent activity of the biotinidase, encoded by the biotinidase gene (BTD). Affected individuals cannot recycle the biotin, leading to heterogeneous symptoms that are primarily neurological and cutaneous. Early treatment with biotin supplementation can prevent irreversible neurological damage and is recommended for patients with profound deficiency, defined as enzyme activity <10% mean normal (MN). Molecular testing has been utilized along with biochemical analysis for diagnosis and management. In this study, our objective was to correlate biochemical phenotype/enzyme activity to BTD genotype in patients for whom both enzyme and molecular testing were performed at our lab, and to review how the correlations inform on variant severity. METHODS: We analyzed results of biotinidase enzyme analysis and BTD gene sequencing in 407 patients where samples were submitted to our laboratory from 2008 to 2020. RESULTS: We identified 84 BTD variants; the most common was c.1330G>C, and 19/84 were novel BTD variants. A total of 36 patients had enzyme activity <10% of MN and the most common variant found in this group was c.528G>T. No variant was reported in one patient in the profound deficiency group. The most common variant found in patients with enzyme activity more than 10% MN was c.1330G>C. CONCLUSIONS: Although enzyme activity alone may be adequate for diagnosing profound biotinidase deficiency, molecular testing is necessary for accurate carrier screening and in cases where the enzyme activity falls in the range where partial deficiency and carrier status cannot be discriminated.

Recovery of enzyme activity in biotinidase deficient individuals during early childhood.

Deficiency of the biotinidase (BTD) enzyme is an inborn error of biotin metabolism caused by biallelic pathogenic variants in the BTD gene. There are two forms, partial and profound BTD deficiency, which both can be successfully treated with pharmacological doses of biotin, justifying the inclusion of this disorder in the newborn screening in numerous countries. We investigated the BTD deficiency cohort (N = 87) in our metabolic center, as it was detected upon newborn screening since 2005, and aimed to better understand the long-term course of BTD enzyme activity and how it may relate to the patients' genetic background. We observed that individuals with partial BTD deficiency display an elevation of BTD enzyme activity with increasing age in 48% of cases-a recovery which allowed adjustment or stop of biotin supplementation in 20% of all individuals. In addition, we were able to recruit 56 patients (64%) for genetic testing, revealing 19 different variants (2 novel), and constituting 22 different genotypes. Genotype-phenotype correlations revealed that the most abundant allele in our cohort p.(Asp444His) was also the most common variant in patients displaying recovery of BTD enzyme activity. Based on our results, we recommend to retest all patients with partial BTD deficiency at the age of 5 years, as this may result in an impact on therapy. Moreover, genetic testing of BTD deficient individuals can allow prediction of the severity of BTD deficiency and of the likelihood of BTD enzyme activity recovery with age.

Biotinidase deficiency mimicking neuromyelitis optica beginning at the age of 4: A treatable disease.

BACKGROUND: Metabolic and inflammatory conditions may lead to neurological disorders. Neuromyelitis optica spectrum disorders (NMOSDs) refer to a rare group of demyelinating diseases of the central nervous system which essentially involve the optic nerves and spinal cord. METHODS: We report a case of biotinidase deficiency (BD) initially misdiagnosed as NMOSD in a pediatric patient. RESULTS: An 8-year-old girl was initially diagnosed with NMOSD on the basis of optic neuritis (ON) associated with three episodes of longitudinally extensive transverse myelitis (LETM). Intravenous high-dose corticosteroids were effective during the first two episodes of LETM. The third acute episode which resulted in tetraplegia, respiratory distress, and blindness was refractory to corticosteroids, plasmapheresis, and rituximab. The unusual clinical course and persistent high levels of plasma and cerebrospinal fluid (CSF) lactate led to additional metabolic investigations being performed. Acylcarnitine profile revealed increased C5-OH acylcarnitine suggestive of BD. Diagnosis was confirmed by direct assessment of plasma enzyme activity (quantified as 5% of the control value). Genetic analysis revealed two mutations, c.643C>T (p.L215F) and c.1612C>T (p.R538C), in the BTD gene (3p25). Dramatic clinical improvement occurred after long-term oral biotin treatment. CONCLUSION: BD is a treatable condition that may closely mimic the neurological findings of LETM and NMOSD.

Clinical, Biochemical and Genetic Analysis of Biotinidase Deficiency in Iranian Population.

BACKGROUND: Biotinidase deficiency (BTD) is an autosomal recessive disorder of biotin metabolism. Biotin is a coenzyme that enhances the action of the four enzymes that play an important role in carbohydrates, amino acid, and fatty acid metabolism. Defects in these pathways cause severe metabolic disorder in the body. In general, biotinidase deficiency can be classified into two levels: partial and profound. The incidence of BTD is 1:40,000 to 1:60,000 births in the world, even though no convincing statistical data on the prevalence of this disorder exist in Iran. In this study, we aimed to set up a test for determining biotinidase activity among the Iranian population and report BTD mutations. PATIENTS AND METHODS: The quantitative method for the determination of biotinidase activity was set up in the National Biochemistry Reference Laboratory (NBRL) of Pasteur Institute of Iran in Tehran. To detect mutations in BTD, polymerase chain reaction (PCR) was performed followed by DNA sequencing. RESULTS: The biotinidase activity range values were 3.81 - 8.25 nmol/min/mL. We identified 8 BTD patients out of 47 cases with neurologic signs. We detected two mutations, c.98-104del7ins3 and p.Arg79Cys, in 5 patients with profound BTD, and one p.Asp444His mutation in 3 patients with partial BTD. CONCLUSION: Infants suffering from BTD seem healthy during their first months of life. At present, the screening program for metabolic disorders such as BTD is in progress. The patients that are BTD deficient benefit from the availability of the tests, and consequently receive the Biotin supplements before being clinically affected.

Biotinidase deficiency due to a de novo mutation or gonadal mosaicism in a first child.

Biotinidase deficiency (BD), which is caused by BTD genetic lesions, if untreated, can result in neurological and cutaneous manifestations. Biotin supplementation can improve or prevent symptoms. We herewith present a family, which we studied at biochemical and molecular level, after identifying the proband through a newborn screening programme. BTD gene molecular analysis showed the proband to be compound heterozygous for the c.1330G>C p.(Asp444His) mild known variant, and for the c.1475 C>T p.(Thr492Ile) new variant. Bioinformatic analysis allowed us to confirm the pathogenic role of the newly identified variant. The proband's father, who exhibited low biotinidase (BTD) enzyme activity, was homozygous for the mild variant, whereas the proband's mother, who exhibited borderline BTD values, the BTD mutation carrier status could not be detected. This is the first description of a patient with BD harbouring a variant whose origin is either de novo or the consequence of gonadal mosaicism. BTD molecular analysis and bioinformatic tools for the evaluation of pathogenicity of newly identified variants are necessary for diagnostic purposes (i.e., clarifying borderline enzyme assays and the carrier status of parents), and for genetic counselling.

High incidence of partial biotinidase deficiency cases in newborns of Greek origin.

Biotinidase deficiency (BTD) is an inherited disorder with severe clinical manifestations if not treated early. 63,119 neonates were tested for BTD according to a 3-step protocol. Biotinidase activity was initially estimated through standard colorimetric method on dried blood spots, then the suspected samples were subjected to molecular analysis of the BT gene and determination of BT activity in serum through an HPLC method. 14 infants with partial BTD (incidence 1:4508) were detected. Nine of them were homozygotes (D444H/D444H), and 4 compound heterozygotes carrying D444H combined with Q456H, T532M, C186Y and R157H, respectively. All were asymptomatic and supplemented with 10mg biotin. Although the number of screened neonates is rather small, it may be suggested that the incidence of the partial BTD infants is the highest ever reported. Detection of BTD should be added to the Greek national neonatal screening program.

Development and characterization of a mouse with profound biotinidase deficiency: a biotin-responsive neurocutaneous disorder.

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states.

Profound biotinidase deficiency: a rare disease among native Swedes.

Biotinidase deficiency is an autosomal recessive metabolic disorder included in many newborn screening programmes. Prior to the introduction of screening for biotinidase deficiency in Sweden in 2002, the disorder was almost unknown, with only one case diagnosed clinically. Biotinidase activity was measured in dried blood spots with a semiquantitative method using biotin-6-amidoquinoline as substrate. The cutoff value was set at 25% (later lowered to 20%) of the mean activity of all samples measured on that day. The disorder was confirmed by quantitative determination of biotinidase activity in plasma and DNA analyses. Over a period of 6 years, 13 patients were identified among 637,452 screened newborns and 5,068 adoptive/immigrant children. None of the patients had clinical symptoms at the time of diagnosis. Six patients had profound biotinidase deficiency, with an activity of 0-5% of normal in plasma. Four of these patients were born to parents who were first cousins of Middle Eastern or African origin. Eighteen gene alterations were identified, nine of which have not previously been described: seven mutations p.L83S (c.248T > C), p.R148H (c.443G > A), p.N202I (c.605A > T), p.I255T (c.764T > C), p.N402S (c.1205A > G), p.L405P (c.1214T > C), p.G445R (c.1333G > A) and two silent mutations p.L71L (c.211C > T) and p.L215L (c.645C > T). The predicted severity of the novel mutations was analyzed by sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), predicting p.L83S, p.L405P and p.G445R as severe mutations. Due to the high rate of immigrants since 1990 from non-Nordic countries, the incidence of biotinidase deficiency is similar to that found in many other Western countries.

Profound biotinidase deficiency in a child with predominantly spinal cord disease.

Biotinidase deficiency is an autosomal recessively inherited disorder that manifests during childhood with various cutaneous and neurological symptoms particularly seizures, hypotonia, and developmental delay. Spinal cord disease has been reported rarely. We describe a 3-year-old boy with profound biotinidase deficiency who presented with progressive spastic paraparesis and ascending weakness in the absence of the usual characteristic neurological manifestations. Supplementation with biotin resulted in resolution of paraparesis with persistent mild spasticity in the lower limbs. DNA mutation analysis revealed that he was homozygous for a novel missense mutation (C>T1339;H447Y) in the BTD gene. This case indicates that biotinidase deficiency should be included in the differential diagnosis of subacute myelopathy and emphasizes the importance of a prompt diagnosis to prevent irreversible neurological damage.

Novel mutation causing partial biotinidase deficiency in a Syrian boy with infantile spasms and retardation.

We report a case of partial biotinidase deficiency (plasma biotinidase levels: 1.30 nm/minute/mL) in a 7-month-old boy who presented with evidence of perinatal distress followed by developmental delay, hypotonia, seizures, and infantile spasms without alopecia or dermatitis. His neurologic symptoms improved markedly on biotin supplementation and antiepileptic drug therapy. DNA mutational analysis revealed that the patient was homozygous for a novel E64K mutation and his parents were heterozygous for the same mutation. Whereas preexisting perinatal distress probably contributed to the severity of the patient's symptoms, the described mutation is novel and is possibly responsible for at least some of his clinical manifestations.

Biotinidase deficiency: novel mutations and their biochemical and clinical correlates.

Biotinidase deficiency is a defect in the recycling of the vitamin biotin. Biotin supplementation can markedly improve the neurological and cutaneous symptoms of affected children and prevent symptoms in children identified by newborn screening or treated since birth. We have determined thirteen novel mutations in children with the disorder. Two nonsense mutations, eight single missense mutations, three allelic double missense mutations, and two are polymorphisms were identified in the biotinidase gene (BTD). One of the missense mutations, c.734G>A (p. C245Y), is the first to be reported that alters the cysteine in the putative location crucial for ester formation and binding of the biotinyl-moiety in the active site of the enzyme. These mutations add to the growing list of mutations that are helping to delineate structure/function relationships of the enzyme.

Outcome in patients with profound biotinidase deficiency: relevance of newborn screening.

Profound biotinidase deficiency (PBD) is an autosomal recessively inherited disorder of biotin metabolism, which can be detected by newborn screening and treated with biotin supplementation. Children were investigated in whom PBD was detected by newborn screening and who were treated presymptomatically, or who were not screened but were diagnosed and treated after experiencing initial clinical symptoms (symptomatic children). In a follow-up of our study group, differences in development, social and behavioural adaptation, and signs of residual impairment were examined. Parents and physicians of children with PBD completed questionnaires which included the Child Behavior Checklist and Vineland Adaptive Behavior Scales. Information was obtained for 37 children (24 males, 13 females; median age at recruitment 6 years 8 months, range to 6 months-20 years; median length of follow-up 6 years 6 months, range 5 months to 18 years 3 months). All 11 symptomatic children had residual enzyme activity of <1%, or variants of the Michaelis-Menten constant which were not detected by newborn screening. Some symptomatic children showed residual impairments: hearing impairment (n=2), optic atrophy (n=2), both hearing impairment and optic atrophy (n=2). In addition, symptomatic children had a higher risk of delayed motor and speech development. No child with PBD detected by newborn screening (n=25) had auditory or visual loss; and milestones of speech development and motor skills were reached at an appropriate age. There was no significant difference in social adaptation or behavioural problems between symptomatic and asymptomatic children. Symptomatic children often have developmental delay and are at risk of irreversible damage to auditory, visual, or central nervous functions; whereas children with PBD (established presymptomatically following newborn screening) treated with biotin supplementation, do not experience these effects.

Molecular characterisation and neuropsychological outcome of 21 patients with profound biotinidase deficiency detected by newborn screening and family studies.

UNLABELLED: Early recognition by newborn screening and oral biotin supplementation may prevent clinical and neurological deficits in profound biotinidase deficiency (residual plasma biotinidase activity <10%). In order to evaluate possible correlations of molecular characteristics, onset and continuation of treatment and clinical outcome, we investigated 21 patients detected by newborn screening and consecutive family investigations. In 18 patients found by newborn screening, the range of biotinidase activities was 0%-9% residual activity. Application of a sensitive HPLC assay enabled us to discriminate five patients with residual biotinidase activities <1%. Two patients with zero activities were homozygous for the G98:d7i3 mutation and three patients with activities <1% carried mutations G98:d7i3, R157H, and Q456H. The mutation spectrum of the remaining patients included T532M, A171T+D444H, V62M,C432W, and D444H. Evaluation of clinical and neuropsychological outcome showed that only patients with biotinidase activities <1% exhibited characteristic clinical symptoms within the first weeks of life whereas five patients with residual activities of 1.2%-4.6% did not develop clinical symptoms even when not treated until 3.5-21 years. In all patients treated with biotin within the first weeks of life, neuropsychological outcome was normal whereas abnormal in three out of five patients tested for IQ and treated after the age of 3.5 years. CONCLUSION: The clinical and molecular spectrum of profound biotinidase deficiency is heterogeneous. Early onset of symptoms is predicted by the presence of zero residual activity as measured by sensitive assays and by homozygosity for the G98:d7i3 mutation. In patients with higher residual activities and variable mutational spectrum, correlation with the onset and severity of symptoms cannot be made.