RESUMEN
Biotinidase (BTD) is the only enzyme that can cleave biocytin, a product of the proteolytic digestion of holocarboxylases. Profound BTD deficiency (less than 10% mean normal activity in serum) is an autosomal recessive disorder that can result in neurological and cutaneous abnormalities. Both the cDNA and the genomic DNA of normal BTD gene have been isolated and characterized. The BTD gene is localized to chromosome 3p25. Thus far 61 mutations in three of the four exons of the BTD and one mutation in an intron gene that cause profound BTD deficiency have been reported. Mutations occur at different frequencies in symptomatic children than they do in children ascertained by newborn screening. Two mutations, 98-104del7ins3 and R538C, were present in 52% or 31 of 60 alleles found in symptomatic patients. Three other mutations, A755G, Q456H, and 511 G>A; 1330G>C (double mutation), accounted for 52% of the alleles detected by newborn screening in the United States. Two asymptomatic adults, parents of children with profound BTD deficiency detected by newborn screening, have been described. Additional different mutations have been found in Turkish, Saudi Arabian, and Japanese children with profound BTD deficiency. Partial BTD deficiency (10-30% of mean normal serum activity) is predominantly caused by the single 1330G>C mutation that results in D444H on one allele in combination with one of the mutations causing profound deficiency on the other allele. Four intragenic polymorphisms, three neutral and one amino acid change, have also been found. Although a preponderance of mutations causing the production of truncated BTD protein occurs in symptomatic children with profound deficiency, preliminary studies fail to demonstrate clear genotype-phenotype correlations.
Asunto(s)
Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Mutación/genética , Alelos , Biotinidasa , Cromosomas Humanos Par 3/genética , Exones/genética , Frecuencia de los Genes/genética , Pruebas Genéticas , Humanos , Intrones/genética , Polimorfismo Genético/genéticaRESUMEN
A search of protein databases revealed amino acid homologies among human biotinidase, bacterial aliphatic amidases, and bacterial and plant nitrilases. Amino acids YRK(210-212) of biotinidase are conserved among the enzyme families. This homology and naturally occurring mutations that cause biotinidase deficiency suggest that this region is essential for enzyme activity and is conserved from bacteria. Cys(245) is likely the cysteine in the active site of biotinidase.
Asunto(s)
Amidohidrolasas/genética , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/química , Secuencia de Aminoácidos , Biotinidasa , Dominio Catalítico , Secuencia Conservada , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Biotinidase deficiency is an autosomal recessive disorder of biotin recycling. Biotinidase cleaves the biotin from biocytin or short biotinyl-peptides to replenish the free biotin pool, or it can transfer the vitamin to specific proteins. The cDNA for human serum biotinidase has two in-frame start codons, potentially allowing for the synthesis of an enzyme with a signal peptide (SP) consisting of either 21 or 41 amino acids. In order to examine the requirements of the signal peptide region for the production and secretion of biotinidase, three different forms of the normal human serum biotinidase gene were constructed that encode either the 21-amino-acid SP (SP21-NL) or the 41-amino-acid SP (SP41-NL) or without a SP (NoSP-NL). These constructs were expressed in insect cells via a baculovirus expression system. Biotinidase from cells with SP41-NL and SP21-NL had immunoreactive and biotinyl-hydrolase-active enzyme in lysates and expression media. Cells with NoSP-NL had about 3% of the immunoreactive material and no enzyme activity in lysates and no immunoreactive protein or enzymatic activity in the expression medium. Lack of biotinidase from cells with NoSP-NL may be due to translation inefficiency or increased susceptibility of this species to protease degradation than the secreted forms. We have demonstrated that the 21-amino-acid signal peptide is sufficient to result in glycosylated, secreted biotinidase, but we cannot determine if the glycosylated biotinidase in the lysates or secreted in the medium of cells with SP41-NL use the first, second, or both ATGs in the SP region. Because this particular expression system has no mechanism for timing the movement of newly translated biotinidase protein, we cannot draw conclusions about the relative efficiency of SP41-NL versus SP21-NL, but it is possible that either is used in vivo depending on particular cellular conditions.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Baculoviridae/metabolismo , Expresión Génica , Biosíntesis de Proteínas/genética , Señales de Clasificación de Proteína/fisiología , Amidohidrolasas/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Transporte Biológico , Biotinidasa , Línea Celular , Codón Iniciador/genética , Medios de Cultivo Condicionados , Glicosilación , Humanos , Datos de Secuencia Molecular , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Spodoptera/virologíaRESUMEN
Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism that can lead to varying degrees of neurologic and cutaneous symptoms when untreated. Because this disorder meets the criteria for newborn screening, many states and countries perform this testing. Because newborn screening should result in complete ascertainment of mutations causing profound biotinidase deficiency (less than 10% of mean normal serum activity), we compared the mutations in a group of 59 children with profound biotinidase deficiency who were identified by newborn screening in the United States with 33 children ascertained by exhibiting symptoms. Of the 40 total mutations identified among the two populations, four mutations comprise 59% of the disease alleles studied. Two of these mutations occur in both populations, but in the symptomatic group at a significantly greater frequency. The other two common mutations occur only in the newborn screening group. Because two common mutations do not occur in the symptomatic population, it is possible that individuals with these mutations either develop mild or no symptoms if left untreated. However, inasmuch as biotin treatment is inexpensive and innocuous, it is still recommended that all children with profound biotinidase deficiency be treated.
Asunto(s)
Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Pruebas Genéticas , Deficiencia Múltiple de Carboxilasa/diagnóstico , Mutación , Tamizaje Neonatal , Sustitución de Aminoácidos , Biotinidasa , Mutación del Sistema de Lectura , Humanos , Recién Nacido , Deficiencia Múltiple de Carboxilasa/epidemiología , Deficiencia Múltiple de Carboxilasa/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa , Estados Unidos/epidemiologíaRESUMEN
For years, the major role of biotin has been as the coenzyme for four carboxylases in humans. Although there has been evidence that biotin might have other functions, none has been firmly established. The discovery that human serum biotinidase has biotinyl-transferase activity, in addition to biotinidase hydrolase activity, presents new possibilities for the role of biotinidase in biotin metabolism. Specific transfer of biotin to histones by biotinidase provides a possible explanation for why biotin is found in the nucleus and the nature of its role in the regulation of protein transcription. Future studies will help to determine the functions of biotinidase in biotin metabolism and in disease states.
Asunto(s)
Amidohidrolasas/metabolismo , Biotina/metabolismo , Amidohidrolasas/deficiencia , Transporte Biológico , Biotinidasa , Biotinilación , Coenzimas , Histonas/metabolismo , Histonas/fisiología , HumanosRESUMEN
The only known Japanese child with biotinidase deficiency was identified by newborn screening in Japan. He has 10.8% of mean normal serum biotinyl-hydrolase activity and trace biotinyl-transferase activity. The mutation results in 16% of normal cross-reacting material in serum with antibody to purified normal biotinidase. He is homozygous for a unique mutation, A1466 > C (Asn489Thr) in exon 4 of the biotinidase gene. The mutation appears to abolish a putative glycosylation site in a region in which other missense mutations have been identified, indicating that this region of the enzyme must be important for enzyme activity. This mutation may affect secretion or stability of the enzyme in serum. Interestingly, this child is now 8 years old, has not been on biotin supplementation for 3 years, and has remained asymptomatic.
Asunto(s)
Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Amidohidrolasas/sangre , Sustitución de Aminoácidos , Asparagina/genética , Sitios de Unión/genética , Biotinidasa , Niño , Análisis Mutacional de ADN , Glicosilación , Homocigoto , Humanos , Japón , Masculino , Mutación/genética , Treonina/genéticaRESUMEN
Biotinidase deficiency is characterized by neurological and cutaneous abnormalities that can be prevented or ameliorated by oral biotin therapy. A child with biotinidase deficiency went undiagnosed for a long period and has irreversible neurological deficits despite biotin treatment. This child is homozygous for the most common mutation (G98:d7i3) found in symptomatic children with the disorder. The parents insisted on having prenatal diagnosis in a subsequent pregnancy to alleviate their anxiety about having another affected child. Mutation analysis of DNA obtained directly from amniotic fluid and from cultured amniocytes revealed that the fetus was heterozygous for the mutation. Maternal cell contamination of the amniocytes was excluded by genotype analysis. Biotinidase activity in extracts of cultured amniocytes revealed 40 per cent of mean normal activity. At birth, the infant was confirmed to be heterozygous by serum enzyme analysis. This is the first report of the use of molecular analysis for the prenatal diagnosis for biotinidase deficiency.
Asunto(s)
Amidohidrolasas/deficiencia , Tamización de Portadores Genéticos , Errores Innatos del Metabolismo/diagnóstico , Diagnóstico Prenatal , Amidohidrolasas/sangre , Amidohidrolasas/genética , Líquido Amniótico/química , Líquido Amniótico/citología , Biotinidasa , Células Cultivadas , Análisis Mutacional de ADN , Femenino , Genotipo , Homocigoto , Humanos , Lactante , Masculino , Mutación , EmbarazoRESUMEN
Children with biotinidase deficiency usually exhibit symptoms at several months to years of age. We describe four children who had symptoms later in childhood or during adolescence; they had motor limb weakness, spastic paresis, and eye problems, such as loss of visual acuity and scotomata, rather than the more characteristic symptoms observed in young untreated children with the disorder. These older children each have different mutations, but they are the same as those of children who have exhibited symptoms at an early age. Biotinidase deficiency should be considered in older children who suddenly experience limb weakness and/or spastic paresis and eye symptoms.
Asunto(s)
Aciltransferasas/deficiencia , Amidohidrolasas/deficiencia , Errores Innatos del Metabolismo , Aciltransferasas/genética , Adolescente , Edad de Inicio , Amidohidrolasas/genética , Biotinidasa , Niño , Femenino , Humanos , Masculino , Errores Innatos del Metabolismo/genética , MutaciónRESUMEN
Biotinidase deficiency is inherited as an antosomal recessive trait that, unless treated with pharmacologic doses of biotin, can result in neurologic and cutaneous symptoms. We have identified two new mutations in exon D of the biotinidase gene of children with profound biotinidase deficiency ascertained by newborn screening. Transition 511G->A near the 5' end of exon D results in a substitution of threonine for alanine 171 (A171T) and transversion 1330G->C occurs close to the 3' end of exon D causing a substitution of histidine for aspartic acid 444 (D444H). The D444H mutation was detected in four individuals from our normal population whose mean serum biotinidase activity is 5.25 nmol/min/ml, which is significantly lower than the mean normal activity (7.1 nmol/min/ml). We calculated that this mutation causes a 52% loss of activity in the aberrant enzyme. Twenty-three individuals with the D444H mutation were found by allele specific oligonucleotide analysis of DNA from 296 randomly-selected, anonymous dried-blood spots. We estimate the frequency of this allele in the general population to be 0.039. In contrast, no individuals in 376 have the A171T mutation. Fourteen children (eleven probands and three siblings) out of the 31 enzyme-deficient children have both the A171T and D444H mutations. Both mutations are inherited from a single parent as a double mutation allele. The nine families in which this allele was identified are of mostly European ancestry, although the mutation cannot be attributed to a specific nationality or ethnic group. The serum of a child who is homozygous for the double mutation allele has very little CRM and the aberrant enzyme has very low biotinylhydrolase activity and no botinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficience in children ascertained by newborn screening in the United States.
Asunto(s)
Aciltransferasas/deficiencia , Mutación/genética , Tamizaje Neonatal , Aciltransferasas/genética , Alanina/genética , Ácido Aspártico/genética , Histidina/genética , Humanos , Recién Nacido , Treonina/genéticaRESUMEN
Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin biotin. The disorder can cause neurologic and cutaneous abnormalities that can be treated effectively with pharmacologic doses of biotin. We identified 21 mutations that cause profound biotinidase deficiency in 37 symptomatic children (30 different probands and 7 siblings), as well as provide relevant biochemical and clinical information for each child. The two most common mutations (G98:d7i3 and R538C) were found in 31 of 60 alleles (52%), whereas the remainder of the alleles are accounted for by the 19 other unique mutations. Serum samples were available from 18 children, of these 11 had no detectable cross-reacting material (CRM) to antibody prepared against normal human serum biotinidase, three had reduced quantities of CRM and four had normal quantities of CRM in serum. All of these mutations result in complete absence of biotinyl-transferase activity in serum. Two polymorphisms were also identified in normal individuals. It is apparent that a child who inherits any of these mutations, either in the homozygous state or in combination, can develop the clinical features of the disorder if untreated. There are, however, no clear genotype/phenotype correlations that would allow for the prediction of the type, severity, or age of onset of symptoms.
Asunto(s)
Aciltransferasas/genética , Amidohidrolasas/genética , Aciltransferasas/sangre , Aciltransferasas/deficiencia , Amidohidrolasas/sangre , Amidohidrolasas/deficiencia , Biotinidasa , Niño , Pruebas Genéticas , Genotipo , Humanos , Mutación , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Biotinidase deficiency is an autosomal recessive disorder that can result in neurologic and cutaneous symptoms if not treated with biotin supplementation. We have identified the most common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. 1368A-->C results in a substitution of histidine for glutamine 456 (Q456H) in exon D of the biotinidase gene. This mutation was found in at least one allele in 14 unrelated children from 27 different families or 15 of 54 alleles studied (28%). This mutation was not identified in 41 normal adults using SSCA, nor was it found in 296 normal newborns using allele-specific oligonucleotide analysis, suggesting that this change is not a polymorphism. In addition, biochemical data from a child homozygous for Q456H suggest that the aberrant enzyme has very low biotinyl-hydrolase activity, lacks biotinyl-transferase activity, and is not recognized by antibody prepared to purified, normal human biotinidase. The ethnic backgrounds of the parents contributing the Q456H allele are varied but are generally northern European.
Asunto(s)
Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Tamizaje Neonatal , Mutación Puntual/genética , Aciltransferasas/metabolismo , Adulto , Amidohidrolasas/inmunología , Biotinidasa , Pruebas Genéticas , Humanos , Recién Nacido , Estados UnidosRESUMEN
Biotinidase deficiency is an autosomal recessively inherited disorder in the recycling of the vitamin biotin. The most common mutation that causes profound biotinidase deficiency in symptomatic individuals is a deletion/insertion (G98:d7i3) that occurs in exon B of the biotinidase gene. We now report the second most common mutation, a C-to-T substitution (position 1612) in a CpG dinucleotide in exon D of the biotinidase gene. This mutation results in the substitution of a cysteine for arginine538 (designated R538C) and was found in 10 of 30 symptomatic children with profound biotinidase deficiency, 5 of whom also have the G98:d7i3 mutation. This mutation was not found in DNA samples from 32 individuals with normal biotinidase activity, but was found in one individual with enzyme activity in the heterozygous range. This mutation was not detected in 371 randomly selected, normal individuals using allele-specific oligonucleotide hybridization analysis. Aberrant biotinidase protein was not detectable in extracts of fibroblasts from a child who is homozygous for the R538C mutation, but was present in less than normal concentration in identical extracts treated with beta-mercaptoethanol. Because there is no detectable biotinidase protein in sera of children who are homozygous for the R538C mutation and in combination with the deletion/insertion mutation, the R538C mutation likely results in inappropriate intra- or intermolecular disulfide bond formation, more rapid degradation of the aberrant enzyme, and failure to secrete the residual aberrant enzyme from the cells into blood.
Asunto(s)
Aciltransferasas/genética , Amidohidrolasas/genética , Fosfatos de Dinucleósidos/genética , Mutación Puntual , Aciltransferasas/deficiencia , Aciltransferasas/metabolismo , Alelos , Amidohidrolasas/deficiencia , Amidohidrolasas/inmunología , Amidohidrolasas/metabolismo , Anticuerpos/inmunología , Arginina , Automatización , Biotina , Biotinidasa , Células Cultivadas , Niño , Cisteína , ADN , Exones , Femenino , Fibroblastos/citología , Humanos , Masculino , Linaje , Análisis de Secuencia de ADNRESUMEN
Biotinidase deficiency is an autosomal-recessive disorder of biotin recycling. Children with profound biotinidase deficiency usually have neurological and cutaneous symptoms in early childhood, but they may not develop symptoms until adolescence. We now report on a man and a woman with profound biotinidase deficiency who are asymptomatic and who were diagnosed only because their biotinidase-deficient children were identified by newborn screening. These adults have never exhibited symptoms of the disorder and are homozygous for two different mutations resulting in different aberrant enzymes. There is no evidence of an increased dietary intake of biotin to explain why they have remained asymptomatic. Although these adults may still be at risk for developing symptoms, they could represent a small group of individuals with profound biotinidase deficiency who will never develop clinical problems. Their lack of symptoms suggests that there are probably epigenetic factors that protect some enzyme-deficient individuals from developing symptoms. These individuals broaden the spectrum of expression of biotinidase deficiency.
Asunto(s)
Amidohidrolasas/deficiencia , Adulto , Biotina/sangre , Biotina/orina , Biotinidasa , Femenino , Humanos , Lisina/análogos & derivados , Lisina/sangre , Lisina/orina , MasculinoRESUMEN
Biotinidase is the enzyme responsible for the recycling of the vitamin biotin. Biotinidase acts as a hydrolase by cleaving biocytin and biotinyl-peptides, thereby liberating biotin for reutilization. Biotinidase is also important for making biotin bioavailable from bound dietary sources. The interest in this enzyme has been increased by the discovery of biotinidase deficiency, an inherited biotin-responsive disorder that can result in neurological and cutaneous abnormalities, but can be treated effectively with biotin supplementation. Biotinidase has recently been shown to be biotinylated in the presence of biocytin, but not biotin, at neutral and alkaline pH. This raises the possibility that biotinidase acts as a biotin-binding or biotin-carrier protein. Biotinidase has also been shown to have biotinyl-transferase activity resulting in the transfer of biotin from biocytin to nucleophilic acceptors, such as histones. Transferase activity occurs at physiological pH and at physiological concentrations of biocytin and, therefore, may be the main function of the enzyme in serum and other tissues. These novel functions of the enzyme may indicate that biotinidase plays a critical role in the metabolism of biotin in nuclei, particularly of neuronal cells. The role of biotinidase in biotin metabolism may be a paradigm for better understanding the metabolism of other vitamins.
Asunto(s)
Aciltransferasas/metabolismo , Amidohidrolasas/metabolismo , Biotina/metabolismo , Amidohidrolasas/deficiencia , Animales , Biotinidasa , Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Histonas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lisina/análogos & derivados , Lisina/metabolismo , Neuronas/metabolismoRESUMEN
Experimental studies indicate that Angll is involved in the process of tissue destruction in chronic renal diseases. This notion has been verified in a number of small-to large-scale clinical studies using angiotensin (ANG) I converting enzyme inhibitors (ACEI). Although the repertoire of the pathophysiologic cascade underlying the progressive destruction of renal tissue has continued to expand over recent years, from proteinuria and physical forces to growth factors and metalloproteinase disregulations, studies now suggest that Angll is involved in many, if not all, of these processes. Because these expanded pathophysiologic potentials of Angll are based primarily on observations in vitro, their significance in vivo, and in humans in particular, needs to be established. Recent studies in animals and humans indicate that the role of Angll in renal tissue destruction is subject to the modulation of multiple environmental and genetic factors, such as dietary habit and ACE genotype. Further delineation of the role of Angll in this respect for specific renal diseases and patients will enable us to design an efficient therapeutic intervention for this otherwise most complex problem of today's nephrology.
Asunto(s)
Angiotensina II/fisiología , Enfermedades Renales/fisiopatología , Modelos Biológicos , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Progresión de la Enfermedad , Ambiente , Humanos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/genéticaRESUMEN
Serum biotinidase has biotinyl-transferase activity in addition to biocytin hydrolase activity. A sensitive assay for biotinyl-transferase activity was developed based on the transfer of biotin from biocytin to histones. Biotinidase biotinyl-transferase occurs at physiological and alkaline pHs, whereas hydrolysis of biocytin occurs optimally at pH 4.5 to 6.0. Measurement of hydrolysis requires micromolar concentrations of biocytin, whereas biotinylation of histones can be detected readily at 1.5 nM biocytin. Because polylysine is readily biotinylated by biotinidase in the presence of biocytin, whereas polyarginine is not, the enzyme likely transfers biotin to the epsilon-amino group of lysyl residues. To determine if patients who are deficient in biocytin hydrolase activity are also deficient in biotinyl-transferase activity, serum from 103 children (25 identified by exhibiting clinical symptoms and 78 detected by newborn screening) with profound biotinidase deficiency (less than 10% of mean normal biotinyl-p-aminobenzoate hydrolyzing activity) were assessed for biotinyl-transferase activity and for the presence of cross-reacting material (CRM) to antibodies prepared against purified serum biotinidase. Sera from all symptomatic patients, both CRM-negative and CRM-positive, had no biotinyl-transferase activity. Sera that was CRM-negative from children ascertained by newborn screening also had no biotinyl-transferase activity, whereas sera from 67% of the CRM-positive children identified by newborn screening had varying degrees of biotinyl-transferase activity. These results indicate that there is a large group of enzyme-deficient children detected by newborn screening who are different biochemically from those who are symptomatic. The clinical relevance of having some degree of biotinyl-transferase activity for individuals with biotinidase deficiency remains to be determined. In addition, it is important to determine if biotinyl-transferase activity, especially biotinylation of histones, is a physiological function of biotinidase.
Asunto(s)
Amidohidrolasas/sangre , Amidohidrolasas/deficiencia , Biotina/metabolismo , Histonas/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Animales , Biotinidasa , Western Blotting , Bovinos , Niño , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Peso MolecularRESUMEN
Human serum biotinidase, purified to homogeneity (1920 units/mg protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5-9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors.
Asunto(s)
Amidohidrolasas/sangre , Biotina/metabolismo , Lisina/análogos & derivados , Amidohidrolasas/aislamiento & purificación , Anticuerpos Monoclonales , Avidina/metabolismo , Biotinidasa , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Immunoblotting , Cinética , Lisina/metabolismoRESUMEN
Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnormalities, and can be successfully treated with biotin supplementation. Sequences of tryptic peptides of the purified human serum enzyme were used to design oligonucleotide primers for polymerase chain reaction amplification from human hepatic total RNA to generate putative biotinidase cDNA fragments. Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe revealed an open reading frame of 1629 bases encoding a protein of 543 amino acid residues, including 41 amino acids of a potential signal peptide. Comparison of the open reading frame with the known biotinidase tryptic peptides and recognition of the expressed protein encoded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA. Southern analyses suggested that biotinidase is a single copy gene and revealed that human cDNA probes hybridized to genomic DNA from mammals, but not from chicken or yeast. Northern analysis indicated the presence of biotinidase mRNA in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, and pancreas.