DiagnosisClinical DiagnosisBiotinidase deficiency is suspected in the presence of the following characteristic symptoms and is confirmed by enzymatic testing. Clinical issues and frequently asked questions about biotinidase deficiency have been addressed in a recent review [Wolf 2010]. Children with untreated profound biotinidase deficiency usually exhibit one or more of the following features, which are also observed in children with many other inherited metabolic disorders:SeizuresHypotoniaRespiratory problems such as hyperventilation, laryngeal stridor, and apneaDevelopmental delayHearing lossVision problems, such as optic atrophyMore specific features of profound biotinidase deficiency include the following:Eczematous skin rashAlopeciaConjunctivitisCandidiasisAtaxiaOlder children and adolescents may exhibit limb weakness, paresis, and scotomata.Children with untreated partial biotinidase deficiency (10%-30% of mean normal serum biotinidase activity) may exhibit any of the above symptoms, but usually the symptoms are mild and occur only when the child is stressed, such as with a prolonged infection.TestingNewborn screening. Biotinidase deficiency can be detected in virtually 100% of affected infants in the US if the newborn screening panel for the state in which they are born includes biotinidase deficiency testing (see National Newborn Screening Status Report). The working group of the American College of Medical Genetics Laboratory Quality Assurance Committee has established technical standards and guidelines for the diagnosis of biotinidase deficiency [Cowan et al 2010; click for full text]. Newborn screening utilizes a small amount of blood obtained from a heel prick for a colorimetric test for biotinidase activity [Heard et al 1984, Wolf et al 1985b, Heard et al 1986, Wolf 1991]:False positive test results may occur in premature infants and in samples placed in plastic prior to sufficient drying.Measurement of biotinidase activity in serum/plasma is warranted in infants whose initial screening tests are abnormal.Biotinidase enzyme activity. Biotinidase activity in serum is most commonly determined colorimetrically by measuring the release of p-aminobenzoate from N-biotinyl-p-aminobenzoate, a biocytin analog [Wolf et al 1983a]. Deficient biotinidase activity has also been shown in extracts of leukocytes and fibroblasts [Wolf & Secor McVoy 1983]. Biotinidase activity is also determined fluorimetrically by measuring the release of aminoquinoline from biotinyl-6-aminoquinoline [Wastell et al 1984]. (Other assays for biotinidase activity in serum and tissues measure the hydrolysis of biocytin or other biotinyl derivatives.)Note: It is important that a normal unrelated control sample and samples from the parent(s) accompany the serum/plasma sample from the proband to the diagnostic laboratory for accurate interpretation of enzymatic results [Neto et al 2004]. An increasing problem of enzymatic deterioration (false positives) is almost certainly the result of inadequate storage of samples either prior to shipping to commercial laboratories or at some laboratories [Wolf 2003].Individuals with profound biotinidase deficiency have lower than 10% mean normal serum enzyme activity.Individuals with partial biotinidase deficiency have 10%-30% of mean normal serum biotinidase activity.Note: Individuals with either profound biotinidase deficiency or partial biotinidase deficiency are usually identified by newborn screening in states in which it is offered [McVoy et al 1990, Suormala et al 1990].Plasma biotin concentration. Plasma biotin concentrations may be decreased or in the low normal range in individuals with profound biotinidase deficiency.Other. Most individuals with biotinidase deficiency exhibit metabolic ketolactic acidosis, organic aciduria, and mild hyperammonemia. However, the absence of organic aciduria or metabolic ketoacidosis does not exclude the diagnosis of biotinidase deficiency in a symptomatic child.Carrier detection. Carriers (heterozygotes) usually have serum enzyme activity levels intermediate between those of affected and those of normal individuals [Wolf et al 1983a]. Using serum enzyme activity, heterozygosity can be diagnosed with approximately 95% accuracy [Weissbecker et al 1991].Molecular Genetic TestingGene. BTD is the only gene in which mutations are known to cause biotinidase deficiency.Clinical testingTargeted mutation analysis. Real-time PCR of DNA from the blood spot of a newborn screen card can be used to identify a panel of common BTD mutations (p.Cys33Phefs*36, p.Gln456His, p.Arg538Cys, p.Asp444His, and p.[Ala171Thr;Asp444His]; see Genotype-Phenotype Correlations) [Dobrowolski et al 2003]. These five mutations that cause profound biotinidase deficiency comprise approximately 60% of the abnormal alleles found in symptomatic individuals and in children identified by newborn screening [Pomponio et al 1997a, Norrgard et al 1999]:Two mutations, p.Cys33Phefs*36 and p.Arg538Cys, occurred in both symptomatic individuals and children identified by newborn screening, but occurred in symptomatic individuals at a significantly greater frequency.The other common mutations, p.Gln456His and p.[Ala171Thr;Asp444His] (see Genotype-Phenotype Correlations), occurred only in the newborn screening group in the Norrgard et al [1999] study.Sequence analysis. Direct sequencing of BTD and its intron/exon junctions is possible [Hymes et al 2001, Wolf 2003].Almost all individuals with partial biotinidase deficiency have the mutation p.Asp444His in one allele of BTD in combination with a mutation for profound deficiency in the other allele [Swango et al 1998].Deletion/duplication analysis. No large deletions have been reported in BTD. Based on the high sensitivity of BTD sequence analysis, a screening test for large deletions/duplications may not be warranted.Table 1. Summary of Molecular Genetic Testing Used in Biotinidase DeficiencyView in own windowGene SymbolTest MethodMutations DetectedMutation Detection Frequency by Test Method ^{1Test Availability BTDTargeted mutation analysisp.Cys33Phefs*36
p.Gln456His
p.Arg538Cys
p.Asp444His
p.[Ala171Thr;Asp444His] 2~60%ClinicalSequence analysisSequence variants 3~99%Deletion/ duplication analysis 4Partial- and whole-gene deletionsUnknown 51. The ability of the test method used to detect a mutation that is present in the indicated gene2. See Genotype-Phenotype Correlations.3. Examples of mutations detected by sequence analysis may include small intragenic deletions/insertions and missense, nonsense, and splice site mutations.4. Testing that identifies deletions/duplications not readily detectable by sequence analysis of the coding and flanking intronic regions of genomic DNA; included in the variety of methods that may be used are: quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and chromosomal microarray (CMA) that includes this gene/chromosome segment.5. No large deletions have been reported in BTD. Based on the high sensitivity of BTD sequence analysis, a screening test for large deletions may not be warranted.Interpretation of test resultsFor issues to consider in interpretation of sequence analysis results, click here.Information on specific allelic variants may be available in Molecular Genetics (see Table A. Genes and Databases and/or Pathologic allelic variants).Testing StrategyTo confirm/establish the diagnosis in a probandA child with clinical features suggestive of biotinidase deficiency or whose biochemical findings are indicative of multiple carboxylase deficiency should have biotinidase enzyme activity determined in serum/plasma. With appropriate controls, this testing is definitive for confirming the diagnosis.A child who is identified as a putative positive for biotinidase deficiency by newborn screening should have testing of biotinidase enzyme activity in serum/plasma to confirm the diagnosis.Molecular genetic testing of BTD is most useful when the results of enzymatic testing are ambiguous, such as in differentiating profound biotinidase deficiency from partial biotinidase deficiency and in differentiating heterozygosity for profound biotinidase deficiency from partial biotinidase deficiency.Note: Because genotype/phenotype correlations in biotinidase deficiency are not well established, decisions regarding treatment should be based on the results of enzyme activity only.Carrier testing for at-risk relatives requires prior identification of the disease-causing mutations in the family. Note: Carriers are heterozygotes for an autosomal recessive disorder and are not at risk of developing the disorder.Prenatal diagnosis for at-risk pregnancies requires prior identification of the disease-causing mutations in the family.Genetically Related (Allelic) DisordersNo other phenotypes are known to be associated with mutations in BTD.}

Natural HistoryIndividuals with biotinidase deficiency who are diagnosed before they have developed symptoms (e.g., by newborn screening) and who are treated with biotin have normal development. Neurologic problems occur only in those individuals with biotinidase deficiency who have recurrent symptoms and metabolic compromise prior to biotin treatment.Profound biotinidase deficiency. Symptoms of untreated profound biotinidase deficiency usually appear between ages one week and ten years, with a mean age of three and one-half months [Wolf et al 1985c].Some children with biotinidase deficiency manifest only a single symptom, whereas others exhibit multiple neurologic and cutaneous findings.The most common neurologic features of individuals with untreated, profound biotinidase deficiency are seizures and hypotonia [Wolf et al 1983a, Wolf et al 1985c, Wastell et al 1988, Wolf 1995]. The seizures are usually myoclonic but may be grand mal and focal; some children have infantile spasms [Salbert et al 1993b]. Some untreated children have exhibited spinal cord involvement characterized by progressive spastic paresis and myelopathy [Chedrawi et al 2008]. Older affected children often have ataxia and developmental delay.Sensorineural hearing loss and eye problems, such as optic atrophy, have also been described in untreated children [Wolf et al 1983b, Taitz et al 1985, Salbert et al 1993a, Weber et al 2004]. Approximately 76% of untreated symptomatic children with profound biotinidase deficiency have sensorineural hearing loss that usually does not resolve or improve but remains static with biotin treatment [Wolf et al 2002b].Many symptomatic children with biotinidase deficiency exhibit a variety of central nervous system abnormalities on MRI or CT of the brain [Wolf et al 1983b, Wastell et al 1988, Salbert et al 1993b, Lott et al 1993, Grunewald et al 2004]. These findings may improve or become normal after biotin treatment.Cutaneous symptoms include skin rash, alopecia, and recurrent viral or fungal infections caused by immunologic dysfunction. Respiratory problems, such as hyperventilation, laryngeal stridor, and apnea can occur. One death initially thought to be caused by sudden infant death syndrome was subsequently attributed to biotinidase deficiency [Burton et al 1987].A number of children with profound biotinidase deficiency were asymptomatic until adolescence, when they developed sudden loss of vision with progressive optic neuropathy and spastic paraparesis [Ramaekers et al 1992, Ramaekers et al 1993, Lott et al 1993]. After several months of biotin therapy, the eye findings resolved and the spastic paraparesis improved. In other individuals with enzyme deficiency, paresis and eye problems have occurred during early adolescence [Tokatli et al 1997, Wolf et al 1998]. Several reports describe adults with profound biotinidase deficiency who have offspring who also have profound biotinidase deficiency identified by newborn screening, but who have never had symptoms [Wolf et al 1997, Baykal et al 2005].Partial biotinidase deficiency. One child with partial biotinidase deficiency who was not treated with biotin exhibited hypotonia, skin rash, and hair loss during an episode of gastroenteritis at approximately age six months. When treated with biotin, the symptoms resolved. Individuals with partial biotinidase deficiency may develop symptoms only when stressed, such as during infection.Outcome with biotin treatment. An outcome study of children with biotinidase deficiency indicates that biotin treatment is effective in preventing symptoms [Möslinger et al 2001, Weber et al 2004]. Möslinger et al [2003] stated that children with profound deficiency who have less than 1% biotinidase activity should be treated with biotin, but those with greater than 1% to 10% biotinidase activity may not need treatment. A child with 1% to 10% biotinidase activity may be just as likely to develop symptoms as one with total loss of enzyme activity [Wolf 2002]. It is therefore strongly recommended that all children with profound biotinidase deficiency, regardless of the residual biotinidase enzyme activity, be treated with biotin.

Genotype-Phenotype CorrelationsGenotype/phenotype correlations are not well established. Deletions, insertions, or nonsense mutations usually result in complete absence of biotinidase enzyme activity, whereas missense mutations may or may not result in complete loss of biotinidase enzyme activity. Those with absence of all biotinidase enzyme activity are likely to be at increased risk for earlier onset of symptoms. Regardless of their molecular genetic test results, all individuals with deficient biotinidase enzyme activity require biotin treatment.Although genotype-phenotype correlations are not well established, in one study, children with symptoms of profound biotinidase deficiency with null mutations were more likely to develop hearing loss than those with missense mutations, even if not treated for a period of time [Sivri et al 2007].Certain genotypes correlate with partial biotinidase deficiency and others with complete biotinidase deficiency:Most mutations in BTD cause complete loss or near-complete loss of biotinidase enzyme activity. These alleles are considered profound biotinidase deficiency alleles; a combination of two such alleles, whether homozygous or compound heterozygous, results in the individual having profound biotinidase deficiency. Such individuals are likely to develop symptoms if not treated with biotin.Individuals with one profound biotinidase deficiency allele and a normal allele are heterozygotes or carriers of profound biotinidase deficiency. Parents of children with profound biotinidase deficiency are in this group. No heterozygous parents of children with profound or partial biotinidase deficiency have ever exhibited symptoms [B Wolf, personal observation]. Such individuals do not need biotin therapy.Individuals who are compound heterozygotes for the p.Asp444His mutation and a mutation that results in profound biotinidase deficiency are expected to have approximately 20%-25% of mean normal serum biotinidase enzyme activity or partial biotinidase deficiency [Swango et al 1998]. Individuals in this group are routinely treated with biotin [McVoy et al 1990].One BTD allele with both the p.Asp444His and p.Ala171Thr mutations in cis configuration, p.[Ala171Thr;Asp444His] (see Table 1), results in an allele causing profound biotinidase deficiency. An individual with an allele having these two mutations in cis configuration combined with another allele with a mutation for profound biotinidase deficiency has profound biotinidase deficiency and requires biotin therapy [Norrgard et al 1998].Individuals who are homozygous or have two alleles for the p.Asp444His mutation are expected to have approximately 45%-50% of mean normal serum biotinidase enzyme activity. This is similar to the activity of heterozygotes for profound biotinidase deficiency. Such individuals do not need biotin therapy.Several adults with profound biotinidase deficiency have never had symptoms and have never been treated [Wolf et al 1997] whereas some children with the same mutations have been symptomatic. Therefore, it has been speculated that some children with profound biotinidase deficiency may exhibit mild or no symptoms if left untreated. However, it is recommended that these children be treated nonetheless [Möslinger et al 2003].

Differential DiagnosisClinical features, such as vomiting, hypotonia, and seizures accompanied by metabolic ketolactic acidosis or mild hyperammonemia, are often observed in inherited metabolic diseases. Individuals with biotinidase deficiency may exhibit clinical features that are misdiagnosed as other disorders, such as isolated carboxylase deficiency, before they are correctly identified [Suormala et al 1985, Wolf 1992]. Other symptoms that are more characteristic of biotinidase deficiency (e.g., skin rash, alopecia) can also occur in children with nutritional biotin deficiency, holocarboxylase synthetase deficiency, zinc deficiency, or essential fatty acid deficiency.Biotin deficiency. Biotin deficiency can usually be diagnosed by dietary history. Individuals with biotin deficiency may have a diet containing raw eggs or protracted parenteral hyperalimentation without biotin supplementation.Low-serum biotin concentrations are useful in differentiating biotin and biotinidase deficiencies from holocarboxylase synthetase deficiency, but it is important to know the method used for determining the biotin concentration. Only methods that distinguish biotin from biocytin or bound biotin yield reliable estimates of free biotin concentrations.Isolated carboxylase deficiency. Urinary organic acid analysis is useful for differentiating isolated carboxylase deficiencies from the multiple carboxylase deficiencies that occur in biotinidase deficiency and holocarboxylase synthetase deficiency:Beta-hydroxyisovalerate is the most commonly elevated urinary metabolite in biotinidase deficiency, holocarboxylase synthetase deficiency, isolated beta-methylcrotonyl-CoA carboxylase deficiency, and acquired biotin deficiency.In addition to beta-hydroxyisovalerate, elevated concentrations of urinary lactate, methylcitrate, and beta-hydroxypropionate are indicative of the multiple carboxylase deficiencies.The multiple carboxylase deficiencies are biotin responsive, whereas the isolated carboxylase deficiencies are not. A trial of biotin can be useful for discriminating between the disorders.Isolated carboxylase deficiency can be diagnosed by demonstrating deficient enzyme activity of one of the three mitochondrial carboxylases in peripheral blood leukocytes (prior to biotin therapy) or in cultured fibroblasts grown in low biotin-containing medium and normal activity of the other two carboxylases.Holocarboxylase synthetase deficiency. Both biotinidase deficiency and holocarboxylase synthetase deficiency are multiple carboxylase deficiencies. Both are biotin responsive.The symptoms of biotinidase deficiency and holocarboxylase synthetase deficiency are similar, and clinical differentiation is often difficult.The age of onset of symptoms may be useful for distinguishing between holocarboxylase synthetase deficiency and biotinidase deficiency. Holocarboxylase synthetase deficiency usually presents with symptoms before age three months, whereas biotinidase deficiency usually presents after age three months; however, there are exceptions for both disorders.Organic acid abnormalities in biotinidase deficiency and holocarboxylase synthetase deficiency are similar and may be reported as consistent with multiple carboxylase deficiency. However, the tandem mass spectroscopic methodology that is being incorporated into many newborn screening programs should identify metabolites that are consistent with multiple carboxylase deficiency. Because most children with holocarboxylase synthetase deficiency excrete these metabolites in the newborn period, the disorder should be identifiable using this technology.Definitive enzyme determinations are required to distinguish between the two disorders. Biotinidase activity is normal in serum of individuals with holocarboxylase synthetase deficiency; therefore, the enzymatic assay of biotinidase activity used in newborn screening is specific for biotinidase deficiency and does not identify children with holocarboxylase synthetase deficiency.Both biotinidase deficiency and holocarboxylase synthetase deficiency are characterized by deficient activities of the three mitochondrial carboxylases in peripheral blood leukocytes prior to biotin treatment. In both disorders, these activities increase to near-normal or normal after biotin treatment.Individuals with holocarboxylase synthetase deficiency have deficient activities of the three mitochondrial carboxylases in extracts of fibroblasts that are incubated in medium containing only the biotin contributed by fetal calf serum (low biotin), whereas individuals with biotinidase deficiency have normal carboxylase activities in fibroblasts. The activities of the carboxylases in fibroblasts of individuals with holocarboxylase synthetase deficiency become near-normal to normal when cultured in medium supplemented with biotin (high biotin).Sensorineural hearing loss (see Deafness and Hereditary Hearing Loss Overview). Sensorineural hearing loss has many causes. Biotinidase deficiency can be excluded as a cause by determining biotinidase enzyme activity in serum. This test should be performed specifically on children with hearing loss who are exhibiting other clinical features consistent with biotinidase deficiency.Ataxia (see Hereditary Ataxia Overview). Ataxia has multiple causes. Biotinidase deficiency can be excluded as a cause by determining biotinidase enzyme activity in serum. The test should be performed especially on children with ataxia who are exhibiting other clinical features consistent with biotinidase deficiency.Note to clinicians: For a patient-specific ‘simultaneous consult’ related to this disorder, go to , an interactive diagnostic decision support software tool that provides differential diagnoses based on patient findings (registration or institutional access required).Profound biotinidase deficiencyPartial biotinidase deficiency

ManagementEvaluations Following Initial DiagnosisTo establish the extent of disease in an individual diagnosed with biotinidase deficiency, the following evaluations are recommended:History of seizures, balance problems, feeding problems, breathing problems, loss of hair, fungal infections, skin rash, conjunctivitisPhysical examination for hypotonia, ataxia, eye findings such as optic atrophy, eczematous skin rash, alopecia, conjunctivitis, breathing abnormalities such as stridor, thrush, and/or candidiasisEvaluation for sensorineural hearing loss and psychomotor deficitsIdentification of biochemical abnormalities such as metabolic ketolactic acidosis, hyperammonemia, and organic aciduriaIdentification of cellular immunologic abnormalitiesQuantitative determination of biotinidase enzyme activity in serum/plasmaTreatment of ManifestationsCompliance with biotin therapy improves symptoms in symptomatic individuals.Some features such as optic atrophy, hearing loss, or developmental delay may not be reversible; they should be addressed with ophthalmologic evaluations and intervention, hearing aids or cochlear implants, and appropriate interventions for developmental deficits.Prevention of Primary ManifestationsAll individuals with profound biotinidase deficiency, even those who have some residual biotinidase enzyme activity, should be treated with biotin independent of their genotype [Wolf 2003].Biotinidase deficiency is treated by supplementation with oral biotin in free form as opposed to the bound form. Children with biotinidase deficiency identified by newborn screening will remain asymptomatic with compliance to biotin therapy.All symptomatic children with biotinidase deficiency have improved after treatment with 5-10 mg oral biotin per day.The biochemical abnormalities and seizures rapidly resolve after biotin treatment, followed by improvement of the cutaneous abnormalities. Hair growth returns over a period of weeks to months in children who have alopecia. Optic atrophy and hearing loss may be resistant to therapy, especially if a long period has elapsed between their onset and the initiation of treatment. Some treated children have rapidly achieved developmental milestones, whereas others have continued to show delays.Only a few anecdotal reports exist regarding symptoms in children with partial biotinidase deficiency who were not treated with biotin. Because there is no known toxicity for biotin, children with partial deficiency are usually treated with 1-10 mg oral biotin per day.Biotin therapy is lifelong.More data are required to determine the dosage of biotin that is necessary for older children with either profound or partial biotinidase deficiency, but essentially all children have tolerated 10 mg/day of oral biotin with no side effects. Anecdotally, two girls with profound biotinidase deficiency developed hair loss during adolescence that resolved following increase of their biotin dosages from 10 mg per day to 15 or 20 mg per day. A protein-restricted diet is not necessary.SurveillanceFor all children with biotinidase deficiency:Yearly ophthalmologic examination and auditory testingRegularly scheduled appointments with primary care physicians or as neededYearly evaluation by a medical geneticist or metabolic specialistSymptomatic children with residual clinical problems should be seen as directed by the appropriate sub-specialists:Evaluation of urinary organic acids if return of symptoms with biotin therapy (most commonly the result of non-compliance)
Note: Measurement of biotin concentrations in blood or urine is not useful except to determine compliance with therapy.Agents/Circumstances to AvoidRaw eggs should be avoided because they contain avidin, an egg-white protein that binds biotin, thus decreasing its bioavailability. (Thoroughly cooked eggs present no problem because heating inactivates avidin, rendering it incapable of binding biotin.)Evaluation of Relatives at RiskSibs who have never been tested, even if asymptomatic, should have biotinidase enzyme testing.Any relative with symptoms consistent with biotinidase deficiency should have biotinidase enzyme testing.See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.Pregnancy ManagementThe only special pregnancy management considerations for a woman who is carrying a baby with biotinidase deficiency or is at risk of having a baby with biotinidase deficiency is consideration of biotin supplementation of the mother. Therapies Under InvestigationSearch ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions. Note: There may not be clinical trials for this disorder.

Molecular GeneticsInformation in the Molecular Genetics and OMIM tables may differ from that elsewhere in the GeneReview: tables may contain more recent information. —ED.Table A. Biotinidase Deficiency: Genes and DatabasesView in own windowGene SymbolChromosomal LocusProtein NameLocus SpecificHGMDBTD3p25​.1BiotinidaseBiotinidase Deficiency (BTD)
BTD @ LOVDBTDData are compiled from the following standard references: gene symbol from HGNC; chromosomal locus, locus name, critical region, complementation group from OMIM; protein name from UniProt. For a description of databases (Locus Specific, HGMD) to which links are provided, click here.Table B. OMIM Entries for Biotinidase Deficiency (View All in OMIM) View in own window 253260BIOTINIDASE DEFICIENCY 609019BIOTINIDASE; BTDNormal allelic variants. The human gene encoding biotinidase consists of four exons, designated 1-4, with sizes of 79 bp, 265 bp, 150 bp, and 1502 bp, respectively [Knight et al 1998]. Intron 1, separating exons 1 and 2, is at least 12.5 kb; intron 2 is 6.2 kb, and intron 3 is 0.7 kb. Two putative translation initiation codons exist in the gene; the first is encoded within exon 1 and the other within exon 2, which contains the N-terminal methionine of the mature enzyme. The presence of an intron between the two possible initiation codons could allow for alternative splicing, which could produce transcripts encoding a protein with a 41- or a 21-residue signal peptide.The nucleotide sequence upstream of exons 1 and 2 has been examined for putative promoter elements. Promoter features identified from -600 to +400 are consistent with the ubiquitous expression of biotinidase with characteristics of a CpG island, lack of a TATA element, six consensus methylation sites, and three initiator (Inr) sequences, which are thought to be important in transcription initiation of TATA-less promoters. A consensus sequence for the liver-specific transcription factor HNF-5 is present at -352. The nucleotide sequence 5' of exon 2, which contains the second putative ATG initiation codon, has features associated with housekeeping genes but does contain a consensus sequence for the liver-specific transcription factor C/EBP within 300 bp of the 5' end of exon 2.Normal allelic variants have been found among individuals with normal biotinidase activity.Pathologic allelic variants. Pindolia et al [2010] have compiled mutations causing biotinidase deficiency. A new continually updated database of current mutations has been established. See www.arup.utah.edu.Approximately 100 mutations have been described in symptomatic children with profound biotinidase deficiency, including the following [Pomponio et al 1997a, Muhl et al 2001, Wolf et al 2002a]:A seven-base deletion/three-base insertion (p.Cys33Phefs*36 ) that occurs in at least one allele of the biotinidase gene in approximately 50% of symptomatic children [Pomponio et al 1995]A missense mutation, p.Arg538Cys, the second most common cause of profound biotinidase deficiency in symptomatic children [Pomponio et al 1997b]Multiple mutations have been reported in children identified by newborn screening who had profound biotinidase deficiency [Norrgard et al 1999]. Of this group, two mutations occurred most commonly:A p.Gln456His missense mutationp.[Ala171Thr;Asp444His] [Norrgard et al 1997, Norrgard et al 1998]: p.[Ala171Thr;Asp444His] (p.Asp444His in cis configuration with the p.Ala171Thr mutation) results in a profound biotinidase deficiency allele.An allele with the double mutation combined with a second allele for profound biotinidase deficiency causes profound biotinidase deficiency.Individuals who are compound heterozygous for the p.Asp444His mutation and a mutation that results in profound biotinidase deficiency are expected to have approximately 20%-25% of mean normal serum biotinidase activity (i.e., partial biotinidase deficiency) [Swango et al 1998].Individuals who are homozygous for the p.Asp444His mutation are expected to have approximately 50% of mean normal serum biotinidase deficiency. This is similar to the activity of heterozygotes for profound biotinidase deficiency.Several of these pathologic allelic variants are included in OMIM 253260 (see Table B).Table 2. Selected BTD Pathologic Allelic Variants View in own windowDNA Nucleotide Change
(Alias ¹)Protein Amino Acid ChangeReference Sequencesc.98_104delinsTCC
(G98del3ins)p.Cys33Phefs*36NM_000060​.2
NP_000051​.1c.511G>Ap.Ala171Thrc.1330G>Cp.Asp444Hisc.1368A>Cp.Gln456Hisc.1612C>Tp.Arg538CysSee Quick Reference for an explanation of nomenclature. GeneReviews follows the standard naming conventions of the Human Genome Variation Society (www​.hgvs.org).1. Variant designation that does not conform to current naming conventionsNormal gene product. Biotinidase is essential for the recycling of the vitamin biotin [Wolf et al 1985a]. Biotinidase has been shown to have biotinyl-hydrolase and biotinyl-transferase activities (see Abnormal gene product) [Hymes & Wolf 1996].The cDNA for human biotinidase from a human cDNA hepatic library has two possible ATG initiation codons and an open reading frame of 1629 bp, relative to the first ATG codon [Cole et al 1994]. The cDNA encodes for a mature protein of 543 amino acids with a molecular mass of 56,771 d. The amino terminus of the mature serum biotinidase is in the same reading frame with both of the ATG codons, consistent with the two putative signal peptides.Northern blot analysis, using a 2000-bp probe consisting of the cDNA sequence, revealed that the biotinidase message is present in human lung, liver, skeletal muscle, kidney, pancreas, heart, brain, and placenta under the hybridization conditions used.The author and several other investigators have purified human biotinidase to homogeneity from plasma [Craft et al 1985, Chauhan & Dakshinamurti 1986, Wolf et al 1987]. The enzyme is a monomeric sialylated glycoprotein with a molecular weight of 76-77 kd. Normal serum or plasma biotinidase has at least nine isoforms (four major and five minor isoforms) between pH 4.15 and 4.35 observed by isoelectric focusing [Hart et al 1991].There are six potential N-linked glycosylation sites (N-X-T/S) in the deduced amino acid sequence. Glycosylation of the protein could increase its mass by 13 to 19 kd; the molecular mass of the glycosylated enzyme is thus estimated at between 70 and 76 kd, which is consistent with that of the glycosylated serum enzyme reported by the author's laboratory and other investigators [Craft et al 1985, Chauhan & Dakshinamurti 1986, Wolf et al 1987, Oizumi et al 1989]. Most of the microheterogeneity observed on isoelectric focusing results from differences in the degree of sialylation.Biotinidase is a thiol enzyme that migrates to the α1-region on agarose electrophoresis. The serum enzyme has a pH optimum of 5-6 when biocytin or biotinyl-p-aminobenzoate (artificial substrate) is the substrate [Pispa 1965, Craft et al 1985, Chauhan & Dakshinamurti 1986]. Biocytin is cleaved more readily than larger biotinyl-peptides [Craft et al 1985]. Biotinidase apparently does not cleave biotin from intact holocarboxylases at acid pH. The biotinyl-binding site of biotinidase is specific for the ureido group of the biotinyl moiety of various substrates [Knappe et al 1963, Chauhan & Dakshinamurti 1986]. Biotinidase plays a role in the processing of dietary protein-bound biotin [Heard et al 1984, Wolf et al 1985a] and has recently been shown to transfer biotin from biocytin to nucleophiles, such as histones [Hymes et al 1995]; the physiologic significance of the latter activity is not known.Abnormal gene product. Biotinidase is essential for the recycling of the vitamin biotin [Wolf et al 1985a]. Biotinidase has been shown to have biotinyl-hydrolase and biotinyl-transferase activities [Hymes & Wolf 1996]:Biotinyl-hydrolase activity. Hymes and Wolf [1996] have determined that both the polyclonal and monoclonal antibodies react on immunoblot with biotinidase in extracts of normal fibroblasts and lymphoblasts. These antibodies react with normal serum biotinidase that has been sialylated by treatment with neuraminidase. Individuals with profound biotinidase deficiency can be classified into at least nine distinct biochemical phenotypes on the basis of the presence or absence of cross-reacting material (CRM) to biotinidase, the number of isoforms, and the distribution frequency of the isoforms. All CRM-positive individuals had normal-size serum biotinidase on SDS-immunoblots. None of the individuals with CRM had an abnormal K_m of the substrate for the enzyme. No relationship exists between the age of onset or severity of symptoms and the isoform patterns or CRM status of the symptomatic children. The isoform patterns of children identified by newborn screening are not different from those of symptomatic children.
Hart et al [1992b] have performed biochemical and immunologic characterization of biotinidase in sera from children with partial biotinidase deficiency. All individuals had CRM in their sera. Individuals with partial biotinidase deficiency can be classified into six distinct biochemical phenotypes on the basis of the number of isoforms and the distribution frequency of the isoforms. Kinetic studies were performed on samples from these individuals and were found to be normal in all cases. The isoform patterns observed in the individuals with partial biotinidase deficiency were not different from those of individuals with profound biotinidase deficiency who had CRM.Biotinyl-transferase activity. More than 100 children with profound biotinidase deficiency were assessed for biotinyl-transferase activity and the presence of CRM to antibodies prepared against purified serum biotinidase [Hymes et al 1995]. Sera from all of the symptomatic individuals studied (both CRM-negative and CRM-positive) had no biotinyl-transferase activity. Sera from children detected by newborn screening who were CRM-negative had no biotinyl-transferase activity, whereas a large group of the CRM-positive children had varying degrees of transferase activity. Statistically, a significant difference in biotinyl-transferase activity exists between the population of symptomatic enzyme-deficient children and the population of children who were identified by newborn screening. Hart et al [1992a] have previously shown a difference in the biotinyl-hydrolase activity between the symptomatic and newborn screening group. The significance of these differences is not yet known. These differences may indicate variations in the domains of the enzyme resulting from different mutations. The authors do not know if all children with profound biotinidase deficiency who are detected by newborn screening will become symptomatic. Transfer of biotin to histones, which may represent a physiologic function, may ultimately be a criterion for determining which children with profound enzyme deficiency are likely to become symptomatic.