Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient

Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient Balachander Kannan, Vijayashree Priyadharsini Jayaseelan, Paramasivam Arumugam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4447507/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Aug, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 9 You are reading this latest preprint version Abstract Background Biotinidase deficiency (BD) is a rare, autosomal recessive metabolic disorder characterized by neurocutaneous symptoms. This study investigates a case of profound BD in an Indian patient and the underlying genetic basis. Methods A 10-month-old male presenting with seizures, hypotonia, ataxia, visual impairments, and developmental delay underwent biochemical and genetic analysis. Biotinidase activity was measured using an ELISA kit. Sanger sequencing of the BTD gene was performed to identify mutations. In silico analysis was employed to assess the potential impact of the identified variants. Results The patient exhibited profound biotinidase deficiency. Biallelic loss-of-function variations (c.903G > A and c.946C > T) in the BTD gene were identified, leading to premature stop codons and truncated, non-functional protein fragments. In silico analysis supported the functional significance of these variations, demonstrating their location within a critical domain essential for enzyme activity. Conclusion This case expands our knowledge of BD genetic diversity and underscores the critical role of early diagnosis and newborn screening programs in managing this treatable condition. Health Genetics Biotinidase deficiency BTD Mutation Neurology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Biotinidase deficiency (BD) is a rare, autosomal recessive metabolic disorder characterized by insufficient biotin utilization. The global incidence of BD exhibits regional variations, affecting approximately 1 in 60,000 newborns [ 1 – 3 ]. Biotin, a water-soluble B vitamin, functions as a crucial coenzyme for carboxylases. These enzymes participate in a multitude of metabolic pathways, including amino acid catabolism, fatty acid synthesis, and gluconeogenesis [ 4 , 5 ]. Biotinidase, an enzyme located within lysosomes and the endoplasmic reticulum, is responsible for liberating biotin from dietary sources and recycling endogenous biotin. Deficiency of biotinidase disrupts biotin homeostasis, consequently impacting the activity of biotin-dependent carboxylases [ 5 – 7 ]. Patients with BD primarily present with neurological and cutaneous manifestations. The phenotypic spectrum is diverse, suggesting multi-systemic involvement [ 2 , 8 ]. Clinical classification of BD is based on serum enzyme activity: profound deficiency (residual activity < 10%) and partial deficiency (residual activity 10–30%) [ 9 ]. Profound BD typically manifests with severe clinical symptoms, including alopecia, developmental delays, hearing loss, hypotonia, optic atrophy, and seizures. Sensorineural hearing loss, a frequent complication affecting up to 76% of symptomatic individuals with profound deficiency, is often irreversible [ 1 , 10 ]. Patients with partial deficiency exhibit milder clinical presentations and often experience symptoms during periods of stress. Fortunately, oral biotin supplementation offers a well-established and effective therapy for BD. However, certain complications, such as hearing loss, remain irreversible once established [ 11 , 12 ]. The human biotinidase ( BTD) gene, located on chromosome 3p25, encodes the biotinidase enzyme. Cole et al. first identified this gene in 1994 [ 13 ]. Mutations within the BTD gene are responsible for BD, with over 300 pathogenic variants reported worldwide [ 14 ]. A strong correlation often exists between specific mutations and the phenotypic severity observed in patients. The prevalence of specific BTD gene mutations can vary considerably across different ethnicities [ 2 , 15 – 18 ]. This knowledge can be instrumental in refining newborn screening programs and genetic counseling strategies for targeted populations at higher risk. This study delves into the case of an Indian patient diagnosed with profound BD. Our primary objective was to identify the underlying genetic cause by analyzing the BTD gene. By employing advanced genetic techniques, we successfully identified biallelic loss-of-function mutations in the BTD gene. This finding provides a definitive molecular basis for the observed enzymatic deficiency in this patient. Materials and Methods Patient and measurement of biotinidase activity This study recruited a ten-month-old male patient, who presented with seizures, hypotonia, ataxia, visual impairments, and developmental delay. Following ethical approval (Ref. No: 003/11/2023/IEC/SMCH) from the Institutional Human Ethics Committee (IHEC) Saveetha Institute of Medical and Technical Sciences, Chennai, written informed consent was obtained from the patient's parents. A commercially available ELISA kit for human biotinidase (BTD) was utilized to measure biotinidase activity. DNA sequencing DNA was extracted from blood using a Purelink DNA Mini Spin Kit (K182001, Invitrogen, MA, USA) according to the manufacturer's instructions. The extracted DNA's quality and quantity were evaluated using a Nanodrop One instrument (Thermo Fisher, USA) and 0.8% agarose gel electrophoresis. All exons and intron-exon boundaries of the BTD gene were amplified using primer sequences that were previously published [ 16 ]. A 50 µL PCR reaction mixture was prepared containing 25 µL of 2X Emerald green PCR master mix (Takara, Tokyo, Japan), 0.5 µM each of the forward and reverse primers, 5 µL of the isolated DNA, and nuclease-free water (DDH2O) to reach the final volume. The PCR amplification protocol consisted of initial denaturation: 95°C for 5 minutes, 35 cycles of denaturation: 95°C for 45 seconds, annealing: 55°C-60°C for 30 seconds, extension: 72°C for 1 minute, and final extension: 72°C for 5 minutes. The MiniAmpPlus thermal cycler (Applied Biosystems by Thermofisher, USA) was used for amplification. The quality of the PCR product was confirmed by running it on a 2% agarose gel. Bidirectional Sanger sequencing was then performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems by Thermofisher, USA) and analyzed on a 3730XL Genetic Analyzer (Applied Biosystems by Thermofisher, USA). The reference sequence for the BTD gene (NM_001281723.3) was retrieved from the National Center for Biotechnology Information (NCBI) and used for comparative analysis. In silico analysis Following the identification of variations in the BTD gene, a comprehensive in silico analysis was conducted to assess its potential pathogenicity and functional impact. Public databases and computational tools were used to collect relevant information and predict the potential impact of variations in the BTD gene. The Single Nucleotide Polymorphism Database (dbSNP) ( https://www.ncbi.nlm.nih.gov/SNP/ ) was queried to determine if the identified variations had been previously reported. The Genome Aggregation Database (gnomAD) browser ( https://gnomad.broadinstitute.org/ ) [ 20 ] was utilized to assess the polymorphic status of the variations within a large cohort of unrelated individuals (> 130,000), providing insights into its potential prevalence in the general population. Mutation Taster ( https://www.mutationtaster.org/ ) [ 20 ] was employed to predict the potential functional impact of variations on the encoded protein. This tool analyzes the variant's effect on amino acids and its potential for disrupting protein structure and function. Swiss-Model ( https://swissmodel.expasy.org/ ) [ 21 ] served as a valuable resource for locating the precise amino acid position affected by variations within the three-dimensional structure of the BTD protein. This information is crucial for understanding how the mutation might alter protein folding and activity. Finally, Meta Dome software ( https://stuart.radboudumc.nl/metadome/dashboard ) [ 22 ] was utilized to gain further insights into the mutation's tolerance. This tool integrates data from various sources and provides a more comprehensive evaluation of variants with unknown significance, aiding in a more informed interpretation of the identified variations. This comprehensive in silico approach aimed to elucidate the potential functional consequences of the BTD gene variations and its contribution to the observed clinical phenotype. Results This study involved a 10-month-old male infant with symptoms such as seizures, hypotonia, ataxia, visual impairments, and developmental delay. Due to the nature of these symptoms, we decided to prioritize screening the patient for biotinidase levels. Using an ELISA-based BTD deficiency screening kit, we discovered profound biotinidase deficiency in the patient (Fig. 1 A). Further analysis of the BTD gene identified two variations: c.903G > A and c.946C > T (Fig. 1 B). These variations introduce premature stop codons at positions p.Trp301Ter (p.W301X) and p.Gln316Ter (p.Q316X) in the protein sequence. We employed various bioinformatics tools to assess the functional significance of these variations, particularly their location within conserved regions. Data from the National Center for Biotechnology Information (NCBI) indicated that both p.W301X and p.Q316X reside within a crucial domain – the biotinidase-like and nitrolases superfamily (2A). Additionally, the presence of these amino acids in other species suggests a highly conserved region within the BTD gene (Fig. 2 B). Structural analysis of the BTD protein revealed that p.W301X and p.Q316X are situated in a critical location (Fig. 3 ). Introducing premature stop codons at these positions could potentially disrupt the protein structure and impair its function. Additionally, MutationTaster analysis predicted both variants as disease-causing due to nonsense-mediated mRNA decay. Moreover, MetaDome software predicted that p.W301X variation is intolerant (Fig. 4 ). Discussion This study presents a unique case of a patient in India diagnosed with profound BD caused by novel biallelic loss-of-function variations in the BTD gene. This report highlights the importance of early diagnosis and newborn screening programs for this treatable yet potentially devastating condition. We identified the compound heterozygous variations (c.903G > A and c.946C > T) in the BTD gene in an Indian patient with profound BD. These variations introduce premature stop codons, producing truncated, non-functional biotinidase protein fragments (p.W301X and p.Q316X). In silico analysis supported these findings, demonstrating that both variations reside within a critical domain essential for enzyme activity. This ultimately leads to a breakdown in biotin metabolism, causing a deficiency in this vital vitamin. Profound BD is a life-threatening condition if left untreated [ 22 ]. Biotin is a crucial vitamin involved in various metabolic pathways and acts as a coenzyme for four carboxylases in the human body. Insufficient biotin metabolism directly impacts the carboxylase cycle, resulting in a range of symptoms [ 23 ]. In patients with profound BD, the neurological system is often the most affected, with over 70% of children exhibiting seizures, hypotonia, skin rash, or alopecia. Partial BD typically results in milder symptoms, which are often exacerbated by stress, such as prolonged fasting or infection [ 14 , 23 , 24 ]. BD is genetic disorder, with the severity determined by the specific mutations in the BTD gene. The complete absence of enzyme activity is usually due to deletions, insertions, or nonsense mutations, while missense mutations can have varied effects [ 14 , 24 , 25 ]. For instance, a study by Iqbal found the nonsense variant c.1275T > G in Austrian patients, leading to a premature stop codon and profound deficiency, although the affected infants did not exhibit symptoms initially [ 26 ]. Similarly, recent studies in Turkey identified nonsense variants such as c.171 T > G (p.Y57*) and compound heterozygous mutations c.499C > T (p.Pro167Ser) and c.572G > A (p.Arg191His), which severely impact BTD enzyme activity [ 27 , 28 ]. While several case reports in India have documented BD in various states, the focus has primarily been on enzymatic activity levels and treatment response [ 29 – 33 ]. Genetic analysis of BTD variants remains limited. This study adds to the growing body of Indian cases with a detailed analysis of novel biallelic variations (c.903G > A and c.946C > T) in the BTD gene. Our findings are similar to previous reports in India describing a case with a c.466-3T > G mutation causing profound BD and another with a c.133C > T (p.H447Y) mutation presenting as recurrent myelopathy [ 34 , 35 ]. These cases highlight the importance of BTD gene analysis alongside enzymatic activity assessment for a more comprehensive understanding of the genetic basis and potential for future genetic counseling to prevent recurrence in families. Bioinformatics tools play a crucial role in understanding the functional impact of genetic mutations. Studies by Carvalho et al. (2019), Wolf et al. (2005), and Swango et al. (2000) noted that BTD gene mutations resulting in truncated proteins due to nonsense mutations lead to profound BD [ 18 , 36 , 37 ]. The current study aligns with these findings, with the newly reported variations c.903G > A and c.946C > T causing premature stop codons and significantly impairing protein function. Fortunately, profound BD is a treatable condition. Children diagnosed with this form typically receive lifelong oral biotin supplementation. While biotin treatment is demonstrably effective and safe, there remains some uncertainty regarding the optimal dosage for patients with partial BD. Studies suggest that lower doses, ranging from 5 to 10 milligrams per day, might be sufficient for managing partial BD in children [ 12 , 38 ]. Early intervention is critical for achieving positive outcomes. Ideally, individuals with profound BD should be identified through newborn screening programs and promptly initiated on biotin therapy. This early intervention can significantly improve their chances of achieving normal physical and cognitive development [ 39 , 40 ]. Newborn screening programs play a vital role in the early detection and management of biotinidase deficiency. These programs enable prompt diagnosis and initiation of treatment, potentially preventing severe complications. While several developed countries have implemented comprehensive newborn screening (NBS) programs, many developing nations, including India, lack such widespread access. In these regions, NBS facilities are often limited to private healthcare settings or concentrated in urban areas [ 29 ]. These programs are crucial for early detection, allowing for timely intervention with biotin supplementation. This case underscores the urgent need to expand NBS programs, especially in regions with a high prevalence of consanguineous marriages, a known risk factor for BTD deficiency. Expanding newborn screening programs to encompass biotinidase deficiency, particularly in developing countries, is critical. Increased accessibility to these programs would ensure early detection and treatment for all infants, regardless of location or socioeconomic background. This expansion would significantly improve patient outcomes and quality of life for individuals with biotinidase deficiency. Conclusion The identification of novel variations in the BTD gene responsible for profound biotinidase deficiency in this Indian patient highlights the genetic heterogeneity of this metabolic disorder. Early diagnosis through newborn screening and prompt intervention with biotin supplementation are essential for preventing severe clinical manifestations. Expanding newborn screening programs to include biotinidase deficiency, especially in developing countries, is a crucial step towards improving patient outcomes and overall well-being. Declarations Acknowledgments The authors express their gratitude to the institution, staff, and technicians involved in this study. Special thanks are extended to the patients and their parents for their invaluable support. Funding This work was supported by the Science and Engineering Research Board (SERB), the Government of India (EMEQ/2019/000411). Conflicts of Interest The authors declare that they have no conflicts of interest. Authors' Contributions K. Balachander conducted the experiments, performed the analysis, and drafted the manuscript. Dr. Vijayashree Priyadharsini was responsible for formal analysis and proofreading the manuscript. Dr. A. Paramasivam contributed to the concept, formal analysis, result interpretation, and proofreading of the final manuscript. Dr. Hephzibah KN and Dr. Lal DV were involved in the clinical analysis of patients. All authors reviewed and approved the final version of the manuscript. Ethical Statement Ethical approval for this study was obtained from the Institutional Human Ethics Committee (IHEC) of Saveetha Institute of Medical and Technical Sciences, Chennai (Ref. No: 003/11/2023/IEC/SMCH). Written informed consent was obtained from the patient's parents. References Wolf B (2023) Biotinidase Deficiency. University of Washington, Seattle Canda E, Kalkan Uçar S, Çoker M (2020) Biotinidase Deficiency: Prevalence, Impact And Management Strategies. Pediatr Health Med Ther 11:127–133 Wolf B (1991) Worldwide survey of neonatal screening for biotinidase deficiency. J Inherit Metab Dis 14:923–927 Zempleni J, Kuroishi T (2012) Biotin Adv Nutr 3:213–214 Hymes J, Wolf B (1996) Biotinidase and its roles in biotin metabolism. Clin Chim Acta 255:1–11 Saleem H, Simpson B (2023) Biotinidase Deficiency. StatPearls Publishing Zempleni J, Hassan YI, Wijeratne SS (2008) Biotin and biotinidase deficiency. Expert Rev Endocrinol Metab 3:715–724 Tankeu AT, Van Winckel G, Elmers J et al (2023) Biotinidase deficiency: What have we learned in forty years? 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Cite Share Download PDF Status: Published Journal Publication published 09 Aug, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 19 Jun, 2024 Reviews received at journal 16 Jun, 2024 Reviews received at journal 12 Jun, 2024 Reviewers agreed at journal 07 Jun, 2024 Reviewers agreed at journal 07 Jun, 2024 Reviewers invited by journal 07 Jun, 2024 Editor assigned by journal 21 May, 2024 Submission checks completed at journal 20 May, 2024 First submitted to journal 20 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4447507","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308248907,"identity":"250d06d0-19d5-4134-9b69-bb8c6aad19b4","order_by":0,"name":"Balachander Kannan","email":"","orcid":"","institution":"Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University","correspondingAuthor":false,"prefix":"","firstName":"Balachander","middleName":"","lastName":"Kannan","suffix":""},{"id":308248908,"identity":"72da70e0-1cf9-4ba0-9ef8-bfc481db4bc3","order_by":1,"name":"Vijayashree Priyadharsini Jayaseelan","email":"","orcid":"","institution":"Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University","correspondingAuthor":false,"prefix":"","firstName":"Vijayashree","middleName":"Priyadharsini","lastName":"Jayaseelan","suffix":""},{"id":308248909,"identity":"279cde51-365c-446d-bd1a-19cf905babd5","order_by":2,"name":"Paramasivam Arumugam","email":"data:image/png;base64,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","orcid":"","institution":"Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University","correspondingAuthor":true,"prefix":"","firstName":"Paramasivam","middleName":"","lastName":"Arumugam","suffix":""},{"id":308248910,"identity":"c1af6021-79e5-4e23-b7c0-4e9b4bce2203","order_by":3,"name":"Hephzibah Kirubamani Navamani","email":"","orcid":"","institution":"Saveetha Medical College and Hospital, Saveetha University","correspondingAuthor":false,"prefix":"","firstName":"Hephzibah","middleName":"Kirubamani","lastName":"Navamani","suffix":""},{"id":308248911,"identity":"b06a405b-f8e7-4f00-a5f1-ed7bfd1cd786","order_by":4,"name":"Lal DV","email":"","orcid":"","institution":"Saveetha Medical College and Hospital, Saveetha University","correspondingAuthor":false,"prefix":"","firstName":"Lal","middleName":"","lastName":"DV","suffix":""}],"badges":[],"createdAt":"2024-05-20 07:54:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4447507/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4447507/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-024-09827-5","type":"published","date":"2024-08-09T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57864879,"identity":"59dae775-e812-4434-826f-d05db8e3197e","added_by":"auto","created_at":"2024-06-06 15:34:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiotinidase Activity and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBTD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Gene Mutations in the Patient\u003c/strong\u003e. A) Pedigree: Graph depicting 10 moth old male affected profound deficiency. B) \u003cem\u003eBTD\u003c/em\u003e Gene Variations: Sequencing chromatograms showcasing the identified variations in the \u003cem\u003eBTD\u003c/em\u003egene: reference control sequence (upper panel), c.903G\u0026gt;A and c.946C\u0026gt;T variations (lower panel).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4447507/v1/0c4bd8ead80978405282121d.png"},{"id":57864880,"identity":"e9469b21-9644-45f9-8d46-dc15253cae77","added_by":"auto","created_at":"2024-06-06 15:34:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":380221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization of Variations within the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBTD\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGene\u003c/strong\u003e A) Schematic Representation of the BTD Protein: A diagram illustrating the functional domains of the BTD protein. The location of variations identified in this study (p.W301X and p.Q316X) is highlighted within a critical domain. B) Protein Sequence Alignment: A multiple sequence alignment demonstrating the conservation of the amino acids affected by variations (p.W301 and p.Q316) across various species. This signifies a highly conserved region within the \u003cem\u003eBTD\u003c/em\u003e gene.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4447507/v1/661f64d36ac1b4dd2c8ebfd5.png"},{"id":57864882,"identity":"a04dc028-b36d-4382-94cd-19daecbf880e","added_by":"auto","created_at":"2024-06-06 15:34:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":429880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural Impact of Variations on the BTD Protein\u003c/strong\u003e. (A-C) Three-dimensional structure of the BTD protein with the locations of the identified variations (p.W301X and p.Q316X) marked. The potential impact of these variations on the protein structure and function is highlighted.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4447507/v1/a3fc7031aba6a08529503817.png"},{"id":57864881,"identity":"ced763cb-9ad0-4c26-bfcf-5047d2ef1b53","added_by":"auto","created_at":"2024-06-06 15:34:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":219499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn Silico\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ePrediction of Mutation Significance\u003c/strong\u003e. Peak graphs depicting the results of \u003cem\u003ein silico\u003c/em\u003e analysis for the identified mutations aminoacids effects (p.W301X and p.Q316X) using software MetaDome. MetaDome shows p.W301X intolerance level (A) and p.Q316X are highly tolerance (B) location in the BTD protein.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4447507/v1/5845d82e6d4ef6b9da930461.png"},{"id":62298261,"identity":"fff72168-428c-4dae-b294-d1a2a3b11bc4","added_by":"auto","created_at":"2024-08-12 16:11:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1622956,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4447507/v1/c76c2563-9339-46e0-bd31-72065cac3752.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiotinidase deficiency (BD) is a rare, autosomal recessive metabolic disorder characterized by insufficient biotin utilization. The global incidence of BD exhibits regional variations, affecting approximately 1 in 60,000 newborns [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Biotin, a water-soluble B vitamin, functions as a crucial coenzyme for carboxylases. These enzymes participate in a multitude of metabolic pathways, including amino acid catabolism, fatty acid synthesis, and gluconeogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Biotinidase, an enzyme located within lysosomes and the endoplasmic reticulum, is responsible for liberating biotin from dietary sources and recycling endogenous biotin. Deficiency of biotinidase disrupts biotin homeostasis, consequently impacting the activity of biotin-dependent carboxylases [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePatients with BD primarily present with neurological and cutaneous manifestations. The phenotypic spectrum is diverse, suggesting multi-systemic involvement [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clinical classification of BD is based on serum enzyme activity: profound deficiency (residual activity\u0026thinsp;\u0026lt;\u0026thinsp;10%) and partial deficiency (residual activity 10\u0026ndash;30%) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Profound BD typically manifests with severe clinical symptoms, including alopecia, developmental delays, hearing loss, hypotonia, optic atrophy, and seizures. Sensorineural hearing loss, a frequent complication affecting up to 76% of symptomatic individuals with profound deficiency, is often irreversible [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Patients with partial deficiency exhibit milder clinical presentations and often experience symptoms during periods of stress. Fortunately, oral biotin supplementation offers a well-established and effective therapy for BD. However, certain complications, such as hearing loss, remain irreversible once established [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe human biotinidase (\u003cem\u003eBTD)\u003c/em\u003e gene, located on chromosome 3p25, encodes the biotinidase enzyme. Cole et al. first identified this gene in 1994 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mutations within the \u003cem\u003eBTD\u003c/em\u003e gene are responsible for BD, with over 300 pathogenic variants reported worldwide [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A strong correlation often exists between specific mutations and the phenotypic severity observed in patients. The prevalence of specific \u003cem\u003eBTD\u003c/em\u003e gene mutations can vary considerably across different ethnicities [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This knowledge can be instrumental in refining newborn screening programs and genetic counseling strategies for targeted populations at higher risk. This study delves into the case of an Indian patient diagnosed with profound BD. Our primary objective was to identify the underlying genetic cause by analyzing the \u003cem\u003eBTD\u003c/em\u003e gene. By employing advanced genetic techniques, we successfully identified biallelic loss-of-function mutations in the \u003cem\u003eBTD\u003c/em\u003e gene. This finding provides a definitive molecular basis for the observed enzymatic deficiency in this patient.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient and measurement of biotinidase activity\u003c/h2\u003e \u003cp\u003eThis study recruited a ten-month-old male patient, who presented with seizures, hypotonia, ataxia, visual impairments, and developmental delay. Following ethical approval (Ref. No: 003/11/2023/IEC/SMCH) from the Institutional Human Ethics Committee (IHEC) Saveetha Institute of Medical and Technical Sciences, Chennai, written informed consent was obtained from the patient's parents. A commercially available ELISA kit for human biotinidase (BTD) was utilized to measure biotinidase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA sequencing\u003c/h2\u003e \u003cp\u003eDNA was extracted from blood using a Purelink DNA Mini Spin Kit (K182001, Invitrogen, MA, USA) according to the manufacturer's instructions. The extracted DNA's quality and quantity were evaluated using a Nanodrop One instrument (Thermo Fisher, USA) and 0.8% agarose gel electrophoresis. All exons and intron-exon boundaries of the \u003cem\u003eBTD\u003c/em\u003e gene were amplified using primer sequences that were previously published [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A 50 \u0026micro;L PCR reaction mixture was prepared containing 25 \u0026micro;L of 2X Emerald green PCR master mix (Takara, Tokyo, Japan), 0.5 \u0026micro;M each of the forward and reverse primers, 5 \u0026micro;L of the isolated DNA, and nuclease-free water (DDH2O) to reach the final volume. The PCR amplification protocol consisted of initial denaturation: 95\u0026deg;C for 5 minutes, 35 cycles of denaturation: 95\u0026deg;C for 45 seconds, annealing: 55\u0026deg;C-60\u0026deg;C for 30 seconds, extension: 72\u0026deg;C for 1 minute, and final extension: 72\u0026deg;C for 5 minutes. The MiniAmpPlus thermal cycler (Applied Biosystems by Thermofisher, USA) was used for amplification. The quality of the PCR product was confirmed by running it on a 2% agarose gel. Bidirectional Sanger sequencing was then performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems by Thermofisher, USA) and analyzed on a 3730XL Genetic Analyzer (Applied Biosystems by Thermofisher, USA). The reference sequence for the \u003cem\u003eBTD\u003c/em\u003e gene (NM_001281723.3) was retrieved from the National Center for Biotechnology Information (NCBI) and used for comparative analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eanalysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the identification of variations in the \u003cem\u003eBTD\u003c/em\u003e gene, a comprehensive in silico analysis was conducted to assess its potential pathogenicity and functional impact. Public databases and computational tools were used to collect relevant information and predict the potential impact of variations in the BTD gene. The Single Nucleotide Polymorphism Database (dbSNP) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/SNP/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/SNP/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was queried to determine if the identified variations had been previously reported. The Genome Aggregation Database (gnomAD) browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gnomad.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was utilized to assess the polymorphic status of the variations within a large cohort of unrelated individuals (\u0026gt;\u0026thinsp;130,000), providing insights into its potential prevalence in the general population. Mutation Taster (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mutationtaster.org/\u003c/span\u003e\u003cspan address=\"https://www.mutationtaster.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was employed to predict the potential functional impact of variations on the encoded protein. This tool analyzes the variant's effect on amino acids and its potential for disrupting protein structure and function. Swiss-Model (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] served as a valuable resource for locating the precise amino acid position affected by variations within the three-dimensional structure of the BTD protein. This information is crucial for understanding how the mutation might alter protein folding and activity. Finally, Meta Dome software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://stuart.radboudumc.nl/metadome/dashboard\u003c/span\u003e\u003cspan address=\"https://stuart.radboudumc.nl/metadome/dashboard\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was utilized to gain further insights into the mutation's tolerance. This tool integrates data from various sources and provides a more comprehensive evaluation of variants with unknown significance, aiding in a more informed interpretation of the identified variations. This comprehensive in silico approach aimed to elucidate the potential functional consequences of the BTD gene variations and its contribution to the observed clinical phenotype.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThis study involved a 10-month-old male infant with symptoms such as seizures, hypotonia, ataxia, visual impairments, and developmental delay. Due to the nature of these symptoms, we decided to prioritize screening the patient for biotinidase levels. Using an ELISA-based BTD deficiency screening kit, we discovered profound biotinidase deficiency in the patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of the \u003cem\u003eBTD\u003c/em\u003e gene identified two variations: c.903G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.946C\u0026thinsp;\u0026gt;\u0026thinsp;T (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These variations introduce premature stop codons at positions p.Trp301Ter (p.W301X) and p.Gln316Ter (p.Q316X) in the protein sequence. We employed various bioinformatics tools to assess the functional significance of these variations, particularly their location within conserved regions. Data from the National Center for Biotechnology Information (NCBI) indicated that both p.W301X and p.Q316X reside within a crucial domain \u0026ndash; the biotinidase-like and nitrolases superfamily (2A). Additionally, the presence of these amino acids in other species suggests a highly conserved region within the \u003cem\u003eBTD\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStructural analysis of the BTD protein revealed that p.W301X and p.Q316X are situated in a critical location (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Introducing premature stop codons at these positions could potentially disrupt the protein structure and impair its function. Additionally, MutationTaster analysis predicted both variants as disease-causing due to nonsense-mediated mRNA decay. Moreover, MetaDome software predicted that p.W301X variation is intolerant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study presents a unique case of a patient in India diagnosed with profound BD caused by novel biallelic loss-of-function variations in the \u003cem\u003eBTD\u003c/em\u003e gene. This report highlights the importance of early diagnosis and newborn screening programs for this treatable yet potentially devastating condition.\u003c/p\u003e \u003cp\u003eWe identified the compound heterozygous variations (c.903G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.946C\u0026thinsp;\u0026gt;\u0026thinsp;T) in the \u003cem\u003eBTD\u003c/em\u003e gene in an Indian patient with profound BD. These variations introduce premature stop codons, producing truncated, non-functional biotinidase protein fragments (p.W301X and p.Q316X). \u003cem\u003eIn silico\u003c/em\u003e analysis supported these findings, demonstrating that both variations reside within a critical domain essential for enzyme activity. This ultimately leads to a breakdown in biotin metabolism, causing a deficiency in this vital vitamin.\u003c/p\u003e \u003cp\u003eProfound BD is a life-threatening condition if left untreated [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Biotin is a crucial vitamin involved in various metabolic pathways and acts as a coenzyme for four carboxylases in the human body. Insufficient biotin metabolism directly impacts the carboxylase cycle, resulting in a range of symptoms [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In patients with profound BD, the neurological system is often the most affected, with over 70% of children exhibiting seizures, hypotonia, skin rash, or alopecia. Partial BD typically results in milder symptoms, which are often exacerbated by stress, such as prolonged fasting or infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBD is genetic disorder, with the severity determined by the specific mutations in the \u003cem\u003eBTD\u003c/em\u003e gene. The complete absence of enzyme activity is usually due to deletions, insertions, or nonsense mutations, while missense mutations can have varied effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For instance, a study by Iqbal found the nonsense variant c.1275T\u0026thinsp;\u0026gt;\u0026thinsp;G in Austrian patients, leading to a premature stop codon and profound deficiency, although the affected infants did not exhibit symptoms initially [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similarly, recent studies in Turkey identified nonsense variants such as c.171 T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.Y57*) and compound heterozygous mutations c.499C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Pro167Ser) and c.572G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Arg191His), which severely impact BTD enzyme activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile several case reports in India have documented BD in various states, the focus has primarily been on enzymatic activity levels and treatment response [\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Genetic analysis of \u003cem\u003eBTD\u003c/em\u003e variants remains limited. This study adds to the growing body of Indian cases with a detailed analysis of novel biallelic variations (c.903G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.946C\u0026thinsp;\u0026gt;\u0026thinsp;T) in the \u003cem\u003eBTD\u003c/em\u003e gene. Our findings are similar to previous reports in India describing a case with a c.466-3T\u0026thinsp;\u0026gt;\u0026thinsp;G mutation causing profound BD and another with a c.133C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.H447Y) mutation presenting as recurrent myelopathy [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These cases highlight the importance of \u003cem\u003eBTD\u003c/em\u003e gene analysis alongside enzymatic activity assessment for a more comprehensive understanding of the genetic basis and potential for future genetic counseling to prevent recurrence in families.\u003c/p\u003e \u003cp\u003eBioinformatics tools play a crucial role in understanding the functional impact of genetic mutations. Studies by Carvalho et al. (2019), Wolf et al. (2005), and Swango et al. (2000) noted that \u003cem\u003eBTD\u003c/em\u003e gene mutations resulting in truncated proteins due to nonsense mutations lead to profound BD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The current study aligns with these findings, with the newly reported variations c.903G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.946C\u0026thinsp;\u0026gt;\u0026thinsp;T causing premature stop codons and significantly impairing protein function.\u003c/p\u003e \u003cp\u003eFortunately, profound BD is a treatable condition. Children diagnosed with this form typically receive lifelong oral biotin supplementation. While biotin treatment is demonstrably effective and safe, there remains some uncertainty regarding the optimal dosage for patients with partial BD. Studies suggest that lower doses, ranging from 5 to 10 milligrams per day, might be sufficient for managing partial BD in children [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Early intervention is critical for achieving positive outcomes. Ideally, individuals with profound BD should be identified through newborn screening programs and promptly initiated on biotin therapy. This early intervention can significantly improve their chances of achieving normal physical and cognitive development [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNewborn screening programs play a vital role in the early detection and management of biotinidase deficiency. These programs enable prompt diagnosis and initiation of treatment, potentially preventing severe complications. While several developed countries have implemented comprehensive newborn screening (NBS) programs, many developing nations, including India, lack such widespread access. In these regions, NBS facilities are often limited to private healthcare settings or concentrated in urban areas [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These programs are crucial for early detection, allowing for timely intervention with biotin supplementation. This case underscores the urgent need to expand NBS programs, especially in regions with a high prevalence of consanguineous marriages, a known risk factor for BTD deficiency.\u003c/p\u003e \u003cp\u003eExpanding newborn screening programs to encompass biotinidase deficiency, particularly in developing countries, is critical. Increased accessibility to these programs would ensure early detection and treatment for all infants, regardless of location or socioeconomic background. This expansion would significantly improve patient outcomes and quality of life for individuals with biotinidase deficiency.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe identification of novel variations in the \u003cem\u003eBTD\u003c/em\u003e gene responsible for profound biotinidase deficiency in this Indian patient highlights the genetic heterogeneity of this metabolic disorder. Early diagnosis through newborn screening and prompt intervention with biotin supplementation are essential for preventing severe clinical manifestations. Expanding newborn screening programs to include biotinidase deficiency, especially in developing countries, is a crucial step towards improving patient outcomes and overall well-being.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors express their gratitude to the institution, staff, and technicians involved in this study. Special thanks are extended to the patients and their parents for their invaluable support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThis work was supported by the Science and Engineering Research Board (SERB), the Government of India (EMEQ/2019/000411).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. Balachander conducted the experiments, performed the analysis, and drafted the manuscript. Dr. Vijayashree Priyadharsini was responsible for formal analysis and proofreading the manuscript. Dr. A. Paramasivam contributed to the concept, formal analysis, result interpretation, and proofreading of the final manuscript. Dr. Hephzibah KN and Dr. Lal DV were involved in the clinical analysis of patients. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Institutional Human Ethics Committee (IHEC) of Saveetha Institute of Medical and Technical Sciences, Chennai (Ref. No: 003/11/2023/IEC/SMCH). Written informed consent was obtained from the patient\u0026apos;s parents.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWolf B (2023) Biotinidase Deficiency. University of Washington, Seattle\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanda E, Kalkan U\u0026ccedil;ar S, \u0026Ccedil;oker M (2020) Biotinidase Deficiency: Prevalence, Impact And Management Strategies. 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Eur J Hum Genet 24:1424\u0026ndash;1429\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Health, Genetics, Biotinidase deficiency, BTD, Mutation, Neurology","lastPublishedDoi":"10.21203/rs.3.rs-4447507/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4447507/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBiotinidase deficiency (BD) is a rare, autosomal recessive metabolic disorder characterized by neurocutaneous symptoms. This study investigates a case of profound BD in an Indian patient and the underlying genetic basis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA 10-month-old male presenting with seizures, hypotonia, ataxia, visual impairments, and developmental delay underwent biochemical and genetic analysis. Biotinidase activity was measured using an ELISA kit. Sanger sequencing of the \u003cem\u003eBTD\u003c/em\u003e gene was performed to identify mutations. \u003cem\u003eIn silico\u003c/em\u003e analysis was employed to assess the potential impact of the identified variants.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe patient exhibited profound biotinidase deficiency. Biallelic loss-of-function variations (c.903G\u0026thinsp;\u0026gt;\u0026thinsp;A and c.946C\u0026thinsp;\u0026gt;\u0026thinsp;T) in the \u003cem\u003eBTD\u003c/em\u003e gene were identified, leading to premature stop codons and truncated, non-functional protein fragments. \u003cem\u003eIn silico\u003c/em\u003e analysis supported the functional significance of these variations, demonstrating their location within a critical domain essential for enzyme activity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis case expands our knowledge of BD genetic diversity and underscores the critical role of early diagnosis and newborn screening programs in managing this treatable condition.\u003c/p\u003e","manuscriptTitle":"Biallelic loss-of-function variations in BTD cause profound biotinidase deficiency in an Indian patient","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-06 15:34:43","doi":"10.21203/rs.3.rs-4447507/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-19T14:11:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-16T16:21:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-12T23:50:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136459683569503896249291374720706667579","date":"2024-06-07T21:56:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75582277475069351637780361695635196251","date":"2024-06-07T14:04:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-07T13:13:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T13:52:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-21T01:09:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-05-20T07:53:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"167e8a4b-4dc7-491a-8a46-a1660ccc6c18","owner":[],"postedDate":"June 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:00:18+00:00","versionOfRecord":{"articleIdentity":"rs-4447507","link":"https://doi.org/10.1007/s11033-024-09827-5","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-08-09 15:57:09","publishedOnDateReadable":"August 9th, 2024"},"versionCreatedAt":"2024-06-06 15:34:43","video":"","vorDoi":"10.1007/s11033-024-09827-5","vorDoiUrl":"https://doi.org/10.1007/s11033-024-09827-5","workflowStages":[]},"version":"v1","identity":"rs-4447507","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4447507","identity":"rs-4447507","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}