OBJECTIVE: In the present study, we aimed to evaluate the genotype-phenotype relation in patients with biotinidase enzyme deficiency based on repeated biotinidase enzyme measurements.
METHODS: The hospital file information of patients with biotinidase, enzyme deficiency was assessed retrospectively, and the relationship between the BTD gene mutations analysis results and biotinidase enzyme activity following the first and repeated enzyme activity assessments was analyzed.
RESULTS: One-hundred-ten patients were included. In the first enzyme evaluation, profound biotinidase enzyme deficiency was identified in 15 (13.6 %), partial biotinidase enzyme deficiency in 63 (57.3 %), and heterozygous biotinidase enzyme deficiency in 32 (29.1 %) of the patients. The BTD genetic analysis revealed 42 (38.2 %) homozygous, 42 (38.2 %) heterozygous, and 26 (23.6 %) compound heterozygous variants. The most common homozygous variant, p.Asp444His, was evaluated with 130 repeated enzyme measurements and was consistent with a partial biotinidase enzyme deficiency in 55.4 % of cases, heterozygous biotinidase enzyme deficiency in 43.8 % of cases, and profound biotinidase enzyme deficiency in one (0.8 %) case. Clinical symptoms developed in 17 patients during follow-up, of which 70.6 % were related to neurodevelopment. The most common variant was homozygous p.Asp444His (29.4 %) among the patients who developed symptoms.
CONCLUSIONS: This is the first study to date to evaluate the genotype-phenotype relationship in patients with biotinidase deficiency through repeated measurements of biotinidase enzyme activity. The study reveals that biotinidase enzyme activity alone is inadequate for diagnosing biotinidase enzyme deficiency or evaluating disease severity, as genetic investigations are also required for a definitive diagnosis of biotinidase enzyme deficiency.

Biotinidase (BTD) deficiency (OMIM 253260) is an autosomal recessively inherited metabolic disorder resulting from deficient activity of the BTD enzyme, which can cleave and release biotin from a variety of biotin-dependent carboxylases, and is therefore recognized as a tool to recycle biotin. Being a condition caused by variations on BTD gene with a consequence of free biotin shortage, BTD deficiency may impair the activity of biotin-dependent carboxylases, and thus bring about a buildup of potentially toxic compounds in the body, primarily 3-hydroxyisovaleryl-carnitine in plasma as well as 3-hydroxyisovaleric acid in urine. The phenotype of BTD deficiency may vary dramatically, from asymptomatic adults to severe neurological anomalies, even death in infancy. In the present study, we reported on a 5-month-old boy, whose parents sought for medical consultation in our clinic for their son due to his loss of consciousness, repeated tetany, and motor retardation. Detailed clinical features included severe psychomotor retardation, hypotonia, as well as failure to thrive. The brain MRI at 12 months showed cerebellar hypoplasia and multiple foci of leukodystrophy. The result of antiepileptic therapy was not satisfying. During hospitalization, BTD deficiency was suggested by elevated concentration of 3-hydroxyisovaleryl-carnitine in the blood spots and 3-hydroxyisovaleric acid in the urine. The child was then diagnosed with profound BTD deficiency based on the above findings and low BTD enzyme activity. Subsequent mutational analysis revealed a novel homozygous variant, c.637_637delC (p.H213Tfs*51) in exon 4 of BTD gene in the proband, which was recognized as a further support to the diagnosis. Therefore, biotin treatment was started immediately, eventually with satisfactory outcomes achieved in terms of prevention of epileptic seizure, performance in deep tendon reflexes, and improvement of muscular hypotonia, but unfortunately, the therapy failed to show any evident effects on poor feeding and intellectual disability. This painful lesson suggests that newborn screening for inherited metabolic diseases is essential for early identification and treatment, which should have been performed in this case to avoid this tragedy.

The biotinidase (BTD) enzyme is essential for recycling biotin, a water-soluble B-complex vitamin that is the coenzyme of four carboxylases involved in fatty acid synthesis, amino acid catabolism and gluconeogenesis. If untreated, total or partial BTD deficiencies lead to an autosomal recessive inherited organic aciduria whose clinical features, mainly presenting in the first years of life, include, seizures, skin rash, and alopecia. Based on residual BTD enzyme activity it is possible to identify partial or total biotinidase deficiency. The incidence of profound and partial biotinidase deficiency worldwide is estimated to be about 1 in 60.000. We report twelve years of experience in the newborn screening of biotinidase deficiency on 466.182 neonates. When a positive screening result occurred, a clinical evaluation was made of the patient and genetic counselling was offered to the family. Molecular analysis the BTD gene was carried out in all recalled neonates. Newborn screening lead to the identification of 75 BTD deficiencies with an incidence of about 1:6.300 births, ten times higher than the reported worldwide incidence. BTD deficiency was confirmed at a genomic level in all patients, demonstrating a high frequency of the p.(Asp444His) amino acid substitution and the complex allele p.(Ala171Thr)/p.(Asp444His) in the analyzed Italian newborns. Four new mutations (two small deletions, one stop mutation and one missense mutation) and a new combined allelic alteration were identified. Our data suggests that there is a high incidence of the biotinidase defect in the Italian population, most likely due to the high frequency of certain mutations.

BACKGROUND: Biotinidase deficiency is an autosomal recessive inherited inborn error of biotin metabolism. Biotin as a water-soluble vitamin is the prosthetic group of biotin-dependent carboxylase enzymes, and by enhancing their function plays a key role in amino acid catabolism, fatty acid synthesis, and gluconeogenesis. Beyond its prosthetic group role, it has been recognized that biotin regulates the level of gene transcription in the eukaryotic cells, therefore any defect in these pathways causes a multisystem metabolic disorder characterized by neurological and cutaneous symptoms.
RESULTS: We report the identification of a novel pathogenic variant in the BTD gene, c.528_542del15 (p.Asn197_Ser201del, UniProt P43251-1) in an Iranian consanguineous family with a severe form of the disease. The segregation analysis in the family was consistent with phenotype and the identified variant was predicated as a pathogenic mutation by the in-silico prediction tools. Computer structural modeling suggests the deleted amino acid residues are located near the biotinidase active site and disrupt the special conformations which are critical for the enzyme activity, and also N-glycosylation.
CONCLUSIONS: This study further expands the mutation spectrum of the BTD gene underlying cause of profound biotinidase deficiency.

Biotinidase deficiency (BD), an autosomal recessive disease, is classified into profound (activity <10%) or partial BD (activity 10-30%). The most frequent variant in patients worldwide is c.1330G > C (p.Asp444His), which is associated with partial BD. In vivo studies indicate that this variant reduces the biotinidase activity by 50%. The objective of this study was to evaluate the in vitro effect of p.Asp444His and of five novel variants identified among Brazilian individuals showing low activity of biotinidase in serum.
The variants c.119 T > C (p.Leu40Pro), c.479G > A (p.Cys160Tyr), c.664G > A (p.Asp222Asn), c.1330G > C (p.Asp444His), c.1337 T > C (p.Leu446Pro), c.1466A > G (p.Asn489Ser) and the wild type (wt) BTD gene were expressed in HEK 293 cells. Biotinidase activity was quantified by colorimetric method in cells homogenates and culture medium. The wtBTD activity was considered 100%.
The p.Leu40Pro, p.Cys160Tyr and p.Leu446Pro variants were associated to impaired biotinidase activity (activity in cells: 33%, 14%, 0%, respectively; activity in medium: 7%, 0.3%, 2%, respectively) and undetectable amount of protein in intra and extracellular space. The p.Asn489Ser variant had these effects restricted to the extracellular space (activity in medium: 43%), and the p.Asp222Asn variant showed normal activity. The expression of p.Asp444His variant resulted in detectable protein and slightly reduced activity only in cells (activity in cells: 46%; activity in medium: 115%).
Our findings suggest that the p.Leu40Pro, p.Cys160Tyr and p.Leu446Pro variants are deleterious; the p.Asn489Ser is probably related to a mild biochemical phenotype; and p.Asp222Asn variant is probably not deleterious. The p.Asp444His variant seems to code for a protein with variable activity.

The BTD gene codes for production of biotinidase, the enzyme responsible for helping the body reuse and recycle the biotin found in foods. Biotinidase deficiency is an autosomal recessively inherited disorder resulting in the inability to recycle the vitamin biotin and affects approximately 1 in 60,000 newborns. If untreated, the depletion of intracellular biotin leads to impaired activities of the biotin-dependent carboxylases and can result in cutaneous and neurological abnormalities in individuals with the disorder. Mutations in the biotinidase gene (BTD) alter enzymatic function. To date, more than 165 mutations in BTD have been reported. Our group has developed a database that characterizes the known mutations and sequence variants in BTD (http://arup.utah.edu/database/BTD/BTD_welcome.php). All sequence variants have been verified for their positions within the BTD gene and designated according to standard nomenclature suggested by Human Genome Variation Society (HGVS). In addition, we describe the change in the protein, indicate whether the variant is a known or likely mutation vs. a benign polymorphism, and include the reference that first described the alteration. We also indicate whether the alteration is known to be clinically pathological based on an observation of a known symptomatic individual or predicted to be pathological based on enzymatic activity or putative disruption of the protein structure. We incorporated the published phenotype to help establish genotype-phenotype correlations and facilitate this process for those performing mutation analysis and/or interpreting results. Other features of this database include disease information, relevant links about biotinidase deficiency, reference sequences, ability to query by various criteria, and the process for submitting novel variations. This database is free to the public and will be updated quarterly. This database is a paradigm for formulating databases for other inherited metabolic disorders.