Successful outcomes of older adolescents and adults with profound biotinidase deficiency identified by newborn screening

PurposeWe began screening newborns for biotinidase deficiency disorder in 1984, and now all states in the United States and many countries perform this screening. The purpose of this study was to determine the outcomes of older adolescent and adult individuals with the disorder identified by newborn screening.Subjects and methodsWe located and surveyed, by questionnaire and telephone interviews, 44 individuals with profound biotinidase deficiency identified by newborn screening with a mean age of 23.1 years.ResultsAll individuals had successfully completed high school, and many were attending or had completed college or graduate school. Compliance in using biotin has been excellent. Several individuals developed a variety of symptoms when they discontinued biotin for days or weeks. These features readily resolved when biotin was resumed. In addition, five treated women had nine uneventful pregnancies and deliveries.ConclusionsNewborn screening for profound biotinidase deficiency and early treatment with biotin result in excellent outcomes for older adolescents and adults with the disorder. In addition, mothers with profound biotinidase deficiency who were treated with biotin had pregnancies with good outcomes. These outcome results indicate that newborn screening for biotinidase deficiency is one of the most successful newborn screening programs.Genet Med 19 4, 396–402.     

Analysis of mutations causing biotinidase deficiencya

Biotinidase deficiency is an inherited disorder in which the vitamin, biotin, is not recycled. Individuals with biotinidase deficiency can develop neurological and cutaneous symptoms if they are not treated with biotin. Biotinidase deficiency screening has been incorporated into essentially all newborn screening programs in the United States and in many countries. We now report 140 known mutations in the biotinidase gene (BTD) that cause biotinidase deficiency. All types of mutations have been found to cause biotinidase deficiency. Variants have been identified throughout the coding sequence. Essentially all the variants result in enzymatic activities with less than 10% of mean normal enzyme activity (profound biotinidase deficiency) with the exception of the c.1330G>C (p.D444H) mutation, which results in an enzyme having 50% of mean normal serum activity. The putative three‐dimensional structure of biotinidase has been predicted by homology to that of nitrilases/amidases. The effect of the various missense mutations can be predicted to affect various important sites within the structure of the enzyme. This compilation of variants causing biotinidase deficiency will be useful to clinical laboratories that are performing mutation analysis for confirmational testing when the enzymatic results are equivocal for children identified through newborn screening. Hum Mutat 31:983–991, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The neurology of biotinidase deficiency

Biotinidase deficiency is an autosomal recessively inherited metabolic disorder in which the enzyme, biotinidase, is defective and the vitamin, biotin, is not recycled. Individuals with biotinidase deficiency, if not treated with biotin, usually exhibit neurological and cutaneous abnormalities. Biotin treatment can ameliorate or prevent symptoms. Biotinidase deficiency meets the major criteria for inclusion in newborn screening programs. With the advent of universal newborn screening for the disorder, the "window-of-opportunity" to characterize the consequences of the untreated disease is essentially gone. To understand the neurology of biotinidase deficiency, we must depend on what is already known about symptomatic individuals with the disorder. Therefore, in this review, the neurological findings of symptomatic individuals with profound biotinidase deficiency have been compiled to catalog the characteristic features of the disorder and the consequences of biotin treatment on these findings. In addition, based on the available evidence, I have speculated on the cause of neurological problems associated with the disorder. Future studies in biotinidase-deficient animals should allow us to demonstrate more definitively if these speculations are correct.     

Double mutation (A171T) and (D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States

Biotinidase deficiency is inherited as an autosomal 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 alanine171 (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 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 biotinyl-hydrolase activity and no biotinyl-transferase activity. This double mutation allele (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States. Hum Mutat 11:410, 1998. © 1998 Wiley-Liss, Inc.     

Outcomes of individuals with profound and partial biotinidase deficiency ascertained by newborn screening in Michigan over 25 years

PurposeBiotinidase deficiency, if untreated, usually results in neurological and cutaneous symptoms. Biotin supplementation markedly improves and likely prevents symptoms in those treated early. All states in the United States and many countries perform newborn screening for biotinidase deficiency. However, there are few studies about the outcomes of the individuals identified by newborn screening.MethodsWe report the outcomes of 142 children with biotinidase deficiency identified by newborn screening in Michigan over a 25-year period and followed in our clinic; 22 had profound deficiency and 120 had partial deficiency.ResultsIndividuals with profound biotinidase and partial deficiency identified by newborn screening were started on biotin therapy soon after birth. With good compliance, these children appeared to have normal physical and cognitive development. Although some children exhibited mild clinical problems, these are unlikely attributable to the disorder. Biotin therapy appears to prevent the development of neurological and cutaneous problems in our population.ConclusionIndividuals with biotinidase deficiency ascertained by newborn screening and treated since birth appeared to exhibit normal physical and cognitive development. If an individual does develop symptoms, after compliance and dosage issues are excluded, then other causes must be considered.     

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.     

Real time PCR assays to detect common mutations in the biotinidase gene and application of mutational analysis to newborn screening for biotinidase deficiency

Biotinidase deficiency is an autosomal recessive disorder of biotin metabolism caused by defects in the biotinidase gene. Symptoms of biotinidase deficiency are resolved or prevented with oral biotin supplementation and as such newborn screening is performed to prospectively identify affected individuals prior to the onset of symptoms. Biotinidase deficiency is detected by determining the activity of the biotinidase enzyme utilizing the newborn dried blood spot and colorimetric end point analysis. While newborn screening by enzyme analysis is effective, external factors may compromise results of the enzyme analysis and difficulty is encountered in distinguishing between complete and partial enzyme deficiencies. In the United States, the four mutations most commonly associated with complete biotinidase deficiency are c98:d7i3, Q456H, R538C, and the double mutation D444H:A171T. Partial biotinidase deficiency is almost universally attributed to the D444H mutation. To more effectively distinguish between profound and partial biotinidase deficiency, a panel of assays utilizing real time PCR and melting curve analysis using Light Cycler technology was developed. Employing DNA extracted from the original dried blood specimens from newborns identified through prospective newborn screening as presumptive positive for biotinidase deficiency, the specimens were analyzed for the presence of the five common mutations. Using this approach it was possible to separate newborns with partial and complete deficiency from each other as well as from many of those with false positive results. In most cases it was also possible to correlate the genotype with the degree of residual enzyme activity present. In newborn screening for biotinidase deficiency, we have shown that the analysis of common mutations is useful in distinguishing between partial and complete enzyme deficiency as well as improving specificity. Combining biotinidase enzyme analysis with genotypic data also increases the sensitivity of screening for biotinidase deficiency and provides information useful to clinicians earlier than would otherwise be possible.     

Combined pedigree and twin family study to determine the sources of variation in serum biotinidase activity: The usefulness of multiple study designs

Biotinidase, the enzyme responsible for recycling the vitamin biotin, is deficient in most individuals with late-onset multiple carboxylase deficiency. Based on clinical criteria, biotinidase deficiency appears to be inherited as an autosomal recessive trait; however, the inheritance of biotinidase serum activity as a quantitative trait has not been studied previously. In this study, both segregation analysis of proband families and the analysis of twin family data were used to determine the relative contributions of a major gene, polygenes and environment to the variation in serum biotinidase activity. Segregation analysis of 24 families of biotinidase-deficient individuals indicated that serum biotinidase activity is determined by the segregation of a single codominant major gene with the variability about the mean of each major genotype attributable to environmental effects. Significant polygenic effects could not be detected by this analysis. Variance component analysis of 128 twin families, which included the twins, their spouses, and their offspring, indicated that 70% of total variance in biotinidase activity is attributable to additive genetic effects, 22% to individual environmental effects, and 8% to shared environmental effects. The model also included an age effect for females. A portion (27%) of the estimated additive variance may be attributed to the segregation of the major gene. This study emphasizes the usefulness of studying multiple data sets representing different types of family relationships. © 1993 Wiley-Liss, Inc.     

Molecular characterisation of 34 patients with biotinidase deficiency ascertained by newborn screening and family investigation

This study characterises the spectrum of biotinidase mutations in 21 patients (17 families) with profound biotinidase deficiency (BD) and 13 unrelated patients with partial BD using a denaturing gradient gel electrophoretic mutation screening and selective sequencing approach. In 29 from 30 unrelated families we found biallelic mutations including four common mutations, D444H (frequency 23.3%), G98:d7i3(20.0%), Q456H(20.0%), T532M (15.0%) and nine rare mutations (V62M, R157H, A171T+D444H, C423W, D543H, L279W, N172S, V109G, 12236G-A) with frequencies less than 5.0%. Only three profound BD patients with G98:d7i3/G98:d7i3 and Q456H/Q456H genotypes and residual biotinidase activities of 0.0%, and 0.9% of normal activity developed clinical symptoms before biotin supplementation at 8 weeks of age. All other patients remained asymptomatic within the first months of life or even longer without treatment. Two patients homozygous for the frameshift mutation G98:d7i3 had no measurable residual enzyme activity. Twelve patients with partial BD had the D444H mutation in at least one allele. We conclude that, based on mutation analysis and biochemical examinations of the enzyme, it is currently not clearly predictable whether an untreated patient will develop symptoms or not, although it seems that patients with activities lower than 1% are at a high risk for developing symptoms of the disease early in life.     

Novel mutations causing biotinidase deficiency in individuals identified by the newborn screening program in Minas Gerais,Brazil

Biotinidase deficiency is an autosomal recessive inherited metabolic disorder caused by mutations in the BTD gene. Clinical manifestations can be treated and effectively prevented with pharmacological doses of biotin. Nine novel mutations in BTD are reported in 14 children diagnosed by the newborn screening program in Minas Gerais, Brazil, from June 2013 to December 2017. Serum BTD enzyme activity was determined for all cases and some parents. Two of the mutations are deletions and seven missense mutations located in the exonic region of the BTD gene, mostly in exon 4. Two newborns were profoundly biotinidase‐deficient (one homozygous p.A534V [c.1601C > T] and another, double heterozygous for a novel mutation p.R211S [c.631C > A] co‐inherited with an already described mutation p.T532 M [c.1595C > T]). Two mutations were associated with a partial deficiency of biotinidase (p.F361 V [c.1081 T > G] in two homozygous children, and p.S311 T [c.932G > C] in a compound heterozygous child who co‐inherited a known severe mutation p.Y438X [c.1314 T > A]). The remaining five mutations were found in compound heterozygous children. Hence, a definitive conclusion about the degree of biotinidase deficiency is not possible yet. These results emphasize the importance of sequencing the BTD gene as an important tool to gain a better understanding of the correlation between biochemical phenotype and genotype.     

Mutations in BTD causing biotinidase deficiency

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. Hum Mutat 18:375–381, 2001. © 2001 Wiley‐Liss, Inc.     

Technical standards and guidelines for the diagnosis of biotinidase deficiency

Biotinidase deficiency is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences if untreated. Fortunately, the clinical features of the disorder can be ameliorated or prevented by administering pharmacological doses of the vitamin biotin. Newborn screening and confirmatory diagnosis of biotinidase deficiency encompasses both enzymatic and molecular testing approaches. These guidelines were developed to define and standardize laboratory procedures for enzymatic biotinidase testing, to delineate situations for which follow-up molecular testing is warranted, and to characterize variables that can influence test performance and interpretation of results.     

Double mutation (A171T and D444H) is a common cause of profound biotinidase deficiency in children ascertained by newborn screening the the United States. Mutations in brief no. 128. Online

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.     

Application of diagnostic and treatment criteria for common variable immunodeficiency disorder

Common variable immunodeficiency disorder (CVID) is the most frequent symptomatic primary immune deficiency disorder in adults. It probably comprises a spectrum of polygenic disorders, with hypogammaglobulinemia being the overarching feature. While the majority of patients with CVID can be identified with relative ease, a significant proportion can present with minimal symptoms in spite of profound laboratory abnormalities. Here we discuss three patients who were presented to the Auckland Hospital immunoglobulin treatment committee to determine if they qualified for immunoglobulin replacement. Two were asymptomatic with profound laboratory abnormalities while the third patient was severely ill with extensive bronchiectasis. The third patient had less severe laboratory abnormalities compared with the two asymptomatic patients. We have applied four sets of published diagnostic and treatment criteria to these patients to compare their clinical utility. We have chosen these patients from the broad phenotypic spectrum of CVID, as this often illustrates differences in diagnostic and treatment criteria.     

Three dimensional structure of human biotinidase: computer modeling and functional correlations

Untreated individuals with deficient activity of biotinidase, the enzyme responsible for recycling the vitamin biotin, usually exhibit neurological and cutaneous findings. To better understand the variability in expression of the disorder it is important to understand the structure of the enzyme and the putative effects of various mutations on its activity. Past attempts to express and purify sufficient quantities of the enzyme by us and others have failed. Therefore, we have resorted to computer modeling using homologous related, crystallized nitrilases/amidases to predict the 3-dimensional structure of biotinidase. The resultant structure is a two domain protein with the catalytic triad consisting of glutamate, lysine and cysteine, within the larger domain. The model predicts multiple glycosylation sites at the surface of the enzyme and multiple disulfide bonds. The precise location of the biotin-binding site could not be determined. Characteristics of 45 missense mutations known to cause profound and partial biotinidase deficiency were examined, including their location, their distance from the catalytic triad, and their potential effect on the structure of the enzyme. Although there are obviously short-comings in predicting the 3-dimensional structure of a protein without crystallographic data, because of the marked homology between biotinidase and specific crystallized amidases/nitrilases, the predicted 3-dimensional structure of biotinidase is probable and should be useful providing clues to structure-function relationships and ultimately the effect of mutations on altering the enzyme's hydrolase and transferase activities.     

Biotinidase deficiency: "if you have to have an inherited metabolic disease, this is the one to have"

Biotinidase recycles the vitamin biotin. Biotinidase deficiency is an autosomal recessively inherited neurocutaneous disorder. The symptoms of the disorder can be successfully treated or prevented by administering pharmacological doses of biotin. The biotinidase gene (BTD) has been cloned and sequenced; its genomic organization has been determined and more than 150 mutations have been identified. The disorder meets the major criteria for newborn screening and is being universally adopted in the United States and in many countries around the world. Newborn screening will limit our understanding about the natural history of the disorder. Regardless, the disorder is an ideal example of an inherited metabolic disorder that if untreated can result in major disabilities, but if identified early can be readily treated by the oral administration of a vitamin.     

Examination of the signal peptide region of human biotinidase using a baculovirus expression system

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.     

Holocarboxylase synthetase deficiency: novel clinical and molecular findings

Tammachote R, Janklat S, Tongkobpetch S, Suphapeetiporn K and Shotelersuk V. Holocarboxylase synthetase deficiency: novel clinical and molecular findings. Multiple carboxylase deficiency (MCD) is an autosomal recessive metabolic disorder caused by defective activity of biotinidase or holocarboxylase synthetase (HLCS) in the biotin cycle. Clinical symptoms include skin lesions and severe metabolic acidosis. Here, we reported four unrelated Thai patients with MCD, diagnosed by urine organic acid analysis. Unlike Caucasians, which biotinidase deficiency has been found to be more common, all of our four Thai patients were affected by HLCS deficiency. Instead of the generally recommended high dose of biotin, our patients were given biotin at 1.2 mg/day. This low‐dose biotin significantly improved their clinical symptoms and stabilized the metabolic state on long‐term follow‐up. Mutation analysis by polymerase chain reaction‐sequencing of the entire coding region of the HLCS gene revealed the c.1522C>T (p.R508W) mutation in six of the eight mutant alleles. This suggests it as the most common mutation in the Thai population, which paves the way for a rapid and unsophisticated diagnostic method for the ethnic Thai. Haplotype analysis revealed that the c.1522C>T was on three different haplotypes suggesting that it was recurrent, not caused by a founder effect. In addition, a novel mutation, c.1513G>C (p.G505R), was identified, expanding the mutational spectrum of this gene.