Abstract
Background:
Biotinidase is an enzyme recycling endogenous biotin by hydrolyzing ε-N-biotinyl lysine (biocytin) to lysine and biotin. Biotin acts as a coenzyme in various carboxylation reactions. Biotinidase deficiency is rare, with an incidence of 1 per 112,271 individuals.
Objectives:
Genetic characterization of a 4-month-old boy born out of a consanguineous marriage presented with cutaneous manifestations, alopecia, developmental delay, hypotonia with absent neck holding, respiratory problems, and biotinidase deficiency.
Methods:
Biochemical characterization was performed using tandem mass spectrometry and immunofluorescence enzyme assays. Pathogenic genetic variant was identified by Sanger sequencing of the BTD (biotinidase) gene in a patient and his parents.
Results:
Biochemical characterization indicated elevated methylmalonylcarnitine/hydroxyisovalerylcarnitine and decreased biotinidase, citrulline, glycine, and ornithine levels, suggesting a profound biotinidase deficiency. Sequencing of BTD gene indicates the presence of a 7-bp deletion and a 3-bp insertion (98-104del7ins3) in a homozygous state, resulting in a change of amino acid from cystine to phenylalanine at the 33rd position, causing a frameshift and premature amino acid termination at the 68th position.
Conclusions:
A 7-bp deletion and a 3-bp insertion (98-104del7ins3) homozygous variation in the BTD gene segregating in a homozygous recessive manner in the family, causing truncated, nonfunctional biotinidase deficiency in the proband. This is a recurrent mutation and has the potential to be used as a biomarker for screening patients with biotinidase deficiency.
Introduction
Biotin, a water-soluble essential vitamin, acts as a coenzyme for four carboxylase enzymes having essential roles in gluconeogenesis, catabolism of branched-chain amino acids, and fatty acid synthesis (Bonjour, 1977; Hymes and Wolf, 1996). Enzyme biotin-amide amidohydrolase (biotinidase, EC 3.5.1.12) hydrolyzes biocytin (ε-N-biotinyl lysine) to lysine and the vitamin biotin (Wolf et al., 1990). Endogenous biotin is recycled and possibly liberated from protein-bound sources with the help of the enzyme biotinidase (Wolf et al., 1984). Seizures, hypotonia, ataxia, skin rash, baldness, hearing loss, optic atrophy, and sepsis-like metabolic crisis are the chief neurocutaneous symptoms of biotinidase deficiency (Wolf, 2015). The incidence of profound biotinidase deficiency is estimated to be 1 per 112,271 (Wolf, 1991). The biotinidase deficiency is an autosomal recessive inherited metabolic disorder caused by pathogenic variations in the BTD gene, resulting in reduced or absent biotinidase activity (Cole et al., 1994b). Here, we present the possible genetic mechanism based on the analysis of the biotinidase gene in a 4-month-old patient and his parents, who experienced a severe metabolic crisis associated with biotinidase enzyme deficiency.
Materials and Methods
Isolation of genomic DNA from blood samples
About 3 mL of the venous blood was collected through venipuncture from the patient and his parents. Genomic DNA was extracted using FlexiGene® DNA kit (QIAGEN, Germany). DNA integrity and purity were verified by 0.8% agarose gel electrophoresis/ethidium bromide staining and optical density 260/280 absorbance ratio of 1.8.
Evaluation of biochemical parameters
Biochemical parameters, protein levels, and glucose levels were measured in serum samples. Protein levels were measured in cerebrospinal fluid (CSF). Routine urine examination was carried out to detect the presence of abnormal constituents. Inborn errors of metabolism, resulting from deficient enzymes of metabolic pathways, were evaluated by tandem mass spectrometry and gas chromatography–mass spectrometry (GCMS) analysis by outsourcing the blood and urine samples to LifeCell Diagnostics, Chennai.
Genetic evaluation and direct Sanger sequencing of the BTD gene
The BTD gene has four exons, with a total length of 1629 bp, and is located on chromosome 3p25 (Cole et al., 1994a; Knight et al., 1998). The exon 1 is 79 bp long, exon 2 is 265 bp, exon 3 is 150 bp, and exon 4 is 1502 bp (Knight et al., 1998). The primers for BTD gene exons 1–4 were designed using messenger RNA Ref-Seq NM_001407374.1 as a reference sequence, and Exon Primer software was used to design intronic primers for the PCR amplification of exons. The primer sequences are available on request. The illustra™ GFX PCR DNA and Gel Band purification kit, Cat. No. 28-9034-70 from GE Healthcare was used to purify the PCR amplicons. Using dye terminator chemistry (BigDye® Terminator v3.1 Cycle Sequencing Kit) and an automated capillary sequencer (ABI 3500 Genetic Analyzer, Applied Biosystems®, Foster City, CA), both forward and backward purified PCR amplicons were sequenced for genetic evaluation. Chromas v2.5.1 (Technelysium Pty Ltd, South Brisbane, Australia) and Sequence Scanner Software v2.0 (Applied Biosystems) were used to analyze the sequence electropherograms.
In silico analyses
The Nucleotide Basic Local Alignment Tool was used to align the sequences. The Mutation Taster24 (http://mutationtaster.org/), PolyPhen-225 (http://genetics.bwh.harvard.edu/pph2/), and SIFT26 (http://sift.jcvi.org/) were used to evaluate the potential pathogenic nature of the observed BTD variant. The nucleotide sequences of both wild-type (obtained using NCBI Nucleotide) and mutated (containing an indel and obtained through Sanger sequencing) BTD genes were converted into the amino acid sequence using the Expasy Translate tool (Swiss Institute of Bioinformatics). The Alphafold-3 server was used to generate the protein structures of the protein sequence.
Results
Case description
A 4-month-old male infant born out of a consanguineous marriage was a lethargic child with a dull and expressionless face, hypotonia, and absent neck holding (developmental delay). He had positive cutaneous manifestations, such as a macular skin rash and dry skin. Symptoms of alopecia were also present. The patient also had respiratory problems.
Recruitment of the subjects
After the clinical examination, the patient and both his unaffected parents were recruited for the genetic screening, noting the familial pedigree. Unfortunately, the proband deceased while the study was still being carried out. The family was included in the study after obtaining informed consent from the participants/their parents according to the Declaration of Helsinki. The study was approved (Approval No. RA/24/640) by the Institutional Ethical Committee at the University of Jammu, Jammu.
Clinical diagnosis of the disorder
The patient was diagnosed with biotinidase deficiency on the basis of clinical and biochemical evaluation. The analyses of the pedigree indicated consanguinity in two generations of the family and an autosomal recessive mode of inheritance of the disease. It has been documented that BTD gene variations cause enzyme deficiencies (Canda et al., 2020; Hymes et al., 2001).
Results of biochemical evaluation
CSF examination revealed elevated protein levels. Further investigations included estimation of serum ammonia, urine profile for ketone, and reducing substances. Among these three, serum ammonia was elevated (240 µg/dL), and urine was positive for reducing substances. Tandem mass spectrometry and GCMS analysis indicated increased levels of methylmalonylcarnitine/hydroxyisovalerylcarnitine, 3 µmol/L (range 0.00–1.00 µmol/L). Immunofluorescence enzyme assay showed absence of biotinidase enzyme associated with decreased levels of citrulline, 2.39 µmol/L (3.86–62.11 µmol/L), glycine 116 µmol/L (117.35–1270.0 µmol/L), and ornithine 22.1 µmol/L (22.75–330.0 µmol/L). These biochemical observations indicated profound biotinidase enzyme deficiency.
Confirmation of the presence of indel
Analysis of DNA sequences of the PCR-amplified exons of the BTD gene revealed a 7-bp deletion and a 3-bp insertion (c.38_44delinsTCC), immediately 3-prime to a 12-bp polypyrimidine sequence in exon 2, segregating in the family in an autosomal recessive manner. The patient was found to be homozygous for the variation. Both parents were found to be asymptomatic carriers of the variant allele (heterozygous). The electropherograms indicating the nucleotide position of the observed BTD exon 2 (rs80338684) variant are represented along with the pedigree in Figure 1. Raw sequencing data are available upon request.
Pedigree of the family representing the status of the observed BTD exon 2 (rs 80338684) variant proband and his family, along with their genotypes. Males are represented in squares, while females are represented in circles (affected with solid representations). The proband has been marked with a black arrow.
In silico analyses of c.98_104delinsTCC variation
The 7-bp deletion and 3-bp insertion (c.98_104delinsTCC) were found on chr3:15676984_15676990delinsTCC. The deleted 7-nucleotide sequence (GCGGCTG) present on exon 2 encoding amino acids cystine-glycine-cystine in biotinidase enzyme is highly conserved throughout most of the mammalian species (Fig. 2). In silico analysis by MutationTaster and SIFT predicted the pathogenic nature of this variation, resulting in a change at the 33rd amino acid position from cystine to phenylalanine and further frameshift in amino acid sequence (HGMD: CD166227) and premature termination at amino acid 68 (Fig. 3), resulting in a considerable truncation of the biotinidase enzyme (Figs. 4, 5).
Screenshot from UCSC Genome Browser representing conservation of the variant nucleotide (NM_001407374.1: c.38_44delinsTCC) of the BTD exon 2 across 100 vertebrates.
The normal translated protein sequence shown above and the truncated protein resulting from Indel. The change in the 33rd amino acid Cystine to Phenylalanine (C>F) is highlighted in yellow colour. The frameshift of the following amino acids and premature termination at amino acid 68 is highlighted in red. The reduction in size of the Biotinidase protein from 543 amino acid protein to a 68 amino acid peptide with altered sequence can be seen.
The normal translated functional Biotinidase protein structure containing 543-amino-acids drawn using AlphaFold-3 server.
Structure of non-functional, truncated biotinidase protein resulting from indel (c.98_104delinsTCC) causing a change in the 33rd amino acid from Cystine to Phenylalanine (C>F) and premature termination at 68th position drawn using the AlphaFold-3 server.
Discussion
Biotinidase deficiency is a metabolic disorder caused by a defect in the recycling of the vitamin biotin (Muhl et al., 2001; Pispa, 1965). With a mean age of 3.5 months, the majority of BTD deficiency symptoms manifest between the ages of 1 week and 10 years (Wolf et al., 1985a). Biotin supplementation can markedly improve the neurological and cutaneous symptoms of affected children and prevent symptoms in children (Wolf, 2010; Wolf et al., 1985b). There are about 300 known harmful variations in the BTD gene that can result in biotinidase deficiency (Alyasi et al., 2023). In this study, we present a familial case of biotinidase deficiency from the Union territory of Jammu & Kashmir, India. A 7-bp insertion and a 3-bp deletion (rs80338684) result in a change in the 33rd amino acid of the biotinidase enzyme from cystine to phenylalanine. Furthermore, there was a frameshift in the next 34 amino acids before premature truncation of the protein at the 68th amino acid instead of 543rd amino acid position. This premature termination of the protein chain results in a nonfunctional, truncated biotinidase enzyme that is unable to recycle endogenous biotin from biocytin. Deficient biotinidase activity impairs biotin recycling and reduces the availability of free biotin, thereby compromising biotin-dependent carboxylation reactions. Without biotin supplementation, the resulting deficiency of multiple carboxylase activities leads to the characteristic metabolic and clinical manifestations of biotinidase deficiency. The clinical phenotype of the patient was found to be concordant with a classic case of biotinidase deficiency. 98G:del7ins3 variation (rs80338684) resulting in biotinidase deficiency has been reported in the population of United States (Pomponio et al., 1997). Two cases reported from Spain, a 1.5-month-old baby with neurological crisis and a 2.5-month-old baby with progressive encephalopathy, hypotonia, and seizures, were carrying similar indel (98G:1del7ins3) in a homozygous state (Iqbal et al., 2010). At least two cases have been reported from India: a 2-year-old baby with clinical symptoms of seizures without skin or hair changes and another 1.5-year-old baby with developmental delay, alopecia, and seizures. Similarly, an 8-day-old baby with no clinical symptoms from Austria and a 3-month-old baby with moderate dermatitis from Morocco were found to have a similar homozygous indel (Iqbal et al., 2010). In a study conducted in 31 Turkish children with profound biotinidase deficiency, who were symptomatic or ascertained by newborn screening, seven children in the age group of 10 days to 4 months were found to be homozygous for the 98G:del7ins3 variant (Pomponio et al., 2000a). Similarly, a 24-month-old patient in Saudi Arabia presenting with clinical features including mental retardation and hearing impairment was diagnosed with a homozygous 98 G:del7ins3 variation (Pomponio et al., 2000b). Since this variant has been reported in the Indian population, future cases from this region with biotinidase deficiency-like symptoms should immediately be screened for this indel (98G:1del7ins3) for early, cost-effective detection, so that early treatment can be started and precious lives be saved.
Conclusion
The biotinidase deficiency resulting from a nonfunctional truncated biotinidase enzyme caused by a homozygous indel (98-104del7ins3) has been reported, to the best of our knowledge, for the first time from Jammu and Kashmir, India. 98G:del7ins3 (rs80338684) variation is a recurrent variation observed in different parts of the world and has been reported in a North Indian patient. This variant can be used as a biomarker for genetic screening of patients before sequencing the whole gene, immediately after establishing a diagnosis based on clinical examination.
Authors’ Contributions
R.S.: Experimental work, collection of blood samples, data analysis, and article writing—original draft. A.M.: Article writing, data analysis, and conceptualization. N.K., S.K., and S.B.: Article writing—review and editing. A.B.: Clinical characterization of the patient and family and conceptualization. D.S.: Clinical characterization of the patient and family. M.K.D.: Article writing—review and editing and conceptualization.
Ethics Approval and Consent of the Participants
The study was approved (Approval No. RA/24/640) by the Institutional Ethical Committee at the University of Jammu, Jammu. The participants were included in the study after obtaining informed consent from parents according to the Declaration of Helsinki.
Consent for Publication
All authors approved the publication of the final version of the article.
Footnotes
Data Availability
Acknowledgments
The authors thank the Director, School of Biotechnology, University of Jammu, Jammu, for providing laboratory and library facilities. The authors also gratefully acknowledge Dr. Gaurav Bhardwaj, Research Assistant Professor, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA, for reviewing the article and helping improve its readability and grammatical correctness.
Author Disclosure Statement
No conflicts of interest, financial or otherwise, are declared by the authors.
Funding Information
A.M. acknowledges a Seed Grant from the University of Jammu (reference no. Fin./2022-23/3246-52).