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

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 infant 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 biotinidase (BTD) gene was performed to identify genetic variations. In silico analysis was employed to assess the potential impact of the identified variants. RESULTS: The infant biotinidase activity was undetectable and its suggest profound biotinidase deficiency. Novel biallelic loss-of-function variations (c.903G > A and c.946 C > T) in the BTD gene were identified, leading to premature stop codons and truncated, non-functional protein fragments. 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.

Oxford nanopore sequencing-based assay for BTD gene screening: Design, clinical validation, and variant frequency assessment in the Turkish population.

Biotinidase deficiency (BTD) is an autosomal recessive disorder characterized by impaired recycling of the water-soluble vitamin biotin which leads to a spectrum of clinical manifestations ranging from mild to severe, including mainly neurological and cutaneous symptoms. Biotin supplementation is a cornerstone of treatment, but diagnosis often relies on measuring serum enzyme activity, which needs to be confirmed by genetic analysis. Thus, molecular methods become necessary in the differential diagnosis of BTD. Accordingly, countries with a high-incidence have implemented next-generation sequencing (NGS) techniques to newborn screening programs for BT. Nevertheless, NGS platforms, while well-established, present challenges in cost, labor, accessibility, and duration for newborn screening programs targeting BTD, therefore these limitations necessitate the exploration of alternative systems to ensure efficient and widespread screening. Here, third-generation sequencing platforms, notably Oxford Nanopore Technology (ONT), present promising solutions to the associated challenges. Hence, in the present study, we aimed to develop an ONT-based assay for the screening of BTD gene. After designing and optimizing primers for long-PCR using reference DNA, we assessed the performance of the ONT assay in BTD patients previously diagnosed by enzyme assay and confirmed using Illumina-based sequencing. The results demonstrate a strong correlation between the two methods, indicating the reliability of the ONT-based assay. Moreover, this first in-house single gene testing specifically tailored for BTD successfully detected previously known genetic variants with high sequencing depths, affirming the effectiveness of ONT-based sequencing in human genetics.

Biotin Homeostasis and Human Disorders: Recent Findings and Perspectives.

Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/"pharmacological" doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin-thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed.

Biotinidase biochemical and molecular analyses: Experience at a large reference laboratory.

BACKGROUND: Biotinidase deficiency is caused by absent activity of the biotinidase, encoded by the biotinidase gene (BTD). Affected individuals cannot recycle the biotin, leading to heterogeneous symptoms that are primarily neurological and cutaneous. Early treatment with biotin supplementation can prevent irreversible neurological damage and is recommended for patients with profound deficiency, defined as enzyme activity <10% mean normal (MN). Molecular testing has been utilized along with biochemical analysis for diagnosis and management. In this study, our objective was to correlate biochemical phenotype/enzyme activity to BTD genotype in patients for whom both enzyme and molecular testing were performed at our lab, and to review how the correlations inform on variant severity. METHODS: We analyzed results of biotinidase enzyme analysis and BTD gene sequencing in 407 patients where samples were submitted to our laboratory from 2008 to 2020. RESULTS: We identified 84 BTD variants; the most common was c.1330G>C, and 19/84 were novel BTD variants. A total of 36 patients had enzyme activity <10% of MN and the most common variant found in this group was c.528G>T. No variant was reported in one patient in the profound deficiency group. The most common variant found in patients with enzyme activity more than 10% MN was c.1330G>C. CONCLUSIONS: Although enzyme activity alone may be adequate for diagnosing profound biotinidase deficiency, molecular testing is necessary for accurate carrier screening and in cases where the enzyme activity falls in the range where partial deficiency and carrier status cannot be discriminated.

Ophthalmic manifestations of biotinidase deficiency: report of a case and review of literature.

INTRODUCTION: Biotinidase deficiency (BD) is an inherited autosomal recessive metabolic disorder. BD has been associated with optic nerve atrophy, eye infections, and retinopathy. The most prevalent ophthalmic manifestation of BD is optic atrophy, which might be misdiagnosed as multiple sclerosis or neuromyelitis optica, especially in late-onset BD cases. METHODS: In this article, we report a 9-year-old boy with gradual vision loss. Ophthalmologic examination, Brain MRI, and several laboratory tests such as Aquaporin-4 IgG level and biotinidase level were done on the patient. RESULTS: Bilateral optic atrophy and impaired visual acuity were detected on examination. The patient had a biotin level of 1.25 U/min/ml (normal range 3-9 U/min/ml), favoring the BD. CONCLUSION: In this study, we report a 9-year-old boy with vision loss diagnosed with BD. We also reviewed the literature to highlight the ophthalmic manifestations of BD. Ophthalmologists must consider BD in children with unexplained ophthalmologic complaints, especially when other characteristic signs of BD (e.g., developmental delay, seizure) are present. Also, patients with BD should undergo regular annual ophthalmologic examinations to be checked for any signs of eye involvement.

Evaluation of clinical, laboratory, and molecular genetic features of patients with biotinidase deficiency.

Biotinidase deficiency (BD) is an autosomal recessive inherited metabolic disorder which results from the inability of biotin-dependent carboxylase enzymes to function due to the release and absorption of biotin, leading to neurological and cutaneous findings. In the present study, evaluation of demographic characteristics, clinical findings, laboratory results, molecular genetic characteristics, and genotype-phenotype correlations of cases with BD. Two hundred forty-seven cases were included in the study who were admitted to the Department of Pediatric Metabolism of Ankara Bilkent City Hospital after being identified with potential BD through the Newborn Screening Program (NBS), during family screening or based on suspicious clinical findings, or following the detection of a pathogenic variant in a BTD genetic analysis during the period of October 2020 and February 2022. The medical files of the cases were reviewed retrospectively. An analysis of the admission routes of all cases to our clinic revealed 89.5% NBS, 5.7% family screening, and 4.9% suspicious clinical findings suggestive of BD. Complete enzyme deficiency was identified in 19.8%, partial enzyme deficiency in 55.1%, and heterogenous enzyme deficiency in 9.7%. The most common pathogenic variants were c.1270G > C (p.Asp424His), c.410G > A (p.Arg137His), and c.38_44delGCGCTGinsTCC (p.Cys13Phefs*36) in BTD gene. The c.1270G > C variant was most common in patients with cutaneous symptoms. The c.410G > A and c.38_44delGCGCTGinsTCC variants were more common in the patients with neurological symptoms. The mean activity level in patients with the c.1270G > C homozygous variant was statistically significantly higher than the mean activity level in the c.1270G > C compound heterozygous patients and the activity level of patients without the c.1270G > C variant. The mean activity level in c.410G > A homozygous patients was statistically significantly lower than the mean activity level of the c.410G > A compound heterozygous patients and the activity level of patients without the c.410G > A variant. In the course of our study, four new pathogenic variants were detected, namely: c.190G > A (p.Glu64Lys), c.249 + 5G > T, c.228delA (p.Val77*), and c.682A > G (p.Ile228Val).     Conclusions: The present study has determined the clinical and genetic spectrum of a large group of patients with BD in a single center. The frequent mutations in our study were similar to those reported in literature, and four novel variants were also described. What is Known: • Biotinidase deficiency is an autosomal recessive, treatable inborn error of metabolism. Two hundred ninety-four pathogenic variants in the BTD gene have been identified and the c.1270G > C variant is the most frequent BTD gene mutation in both Turkey and around the world. What is New: • Four new pathogenic variants (c.190G > A, p.Glu64Lys; c.249 + 5G > T; c.228delA, p.Val77*; and c.682A > G, p.Ile228Val) have been identified. It is believed that the c.38_44delGCGGCTGinsTCC variant is more commonly seen in individuals with ocular issues; however, further genotype-phenotype correlations are needed.

Resultados del diagnóstico neonatal del déficit de Biotinidasa en la provincia de Guantánamo 2015-2019 / Results of neonatal diagnosis of Biotinidase deficiency in the province of Guantánamo 2015 - 2019

Introducción: La deficiencia de biotinidasa es un desorden autosómico recesivo. Se clasifica según la actividad de la enzima en: deficiencia parcial y profunda.Objetivo: Describir los resultados del Programa Nacional de Diagnóstico para Deficiencia de Biotinidasa en Guantánamo durante el período 2015-2019.Métodos: Se realizó estudio observacional descriptivo transversal de un universo de 32 606 pacientes nacidos en Guantánamo en el período 2015-2019. Se analizaron 48 casos que previamente resultaron positivos en el Programa de Pesquisa Neonatal de Biotinidasa. Se utilizó el método espectrofotométrico descrito por Wolf para la confirmación de la enfermedad. Resultados: Durante el quinquenio estudiado fueron diagnosticados tres niños con deficiencia total y dos con deficiencia parcial de biotinidasa con una incidencia de 0,9:10000 y 0,6:10000 respectivamente.Conclusiones: Con el Programa Nacional de Diagnóstico para Deficiencia de Biotinidasa en el período del 2015-2019 se identificaron 5 casos en la provincia de Guantánamo, lo que permitió el asesoramiento a la familia sobre la naturaleza genética de la entidad, recurrencia del trastorno en otros miembros, el riesgo para próximos embarazos, así como las pautas para el seguimiento del individuo afectado...(AU)

Auditory Neuropathy/Dyssynchrony in Biotinidase Deficiency

Biotinidase deficiency is a disorder inherited autosomal recessively showing evidence of hearing loss and optic atrophy in addition to seizures, hypotonia, and ataxia. In the present study, a 2-year-old boy with Biotinidase deficiency is presented in which clinical symptoms have been reported with auditory neuropathy/auditory dyssynchrony (AN/AD). In this case, transient-evoked otoacoustic emissions showed bilaterally normal responses representing normal function of outer hair cells. In contrast, acoustic reflex test showed absent reflexes bilaterally, and visual reinforcement audiometry and auditory brainstem responses indicated severe to profound hearing loss in both ears. These results suggest AN/AD in patients with Biotinidase deficiency.

Gene mutation analyses in Chinese children with multiple carboxylase deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD.</p><p><b>METHODS</b>Biotinidase (BT) and holocarboxylase synthetase (HLCS) genes were analyzed by PCR and direct sequencing for the 4 BT deficiency patients and 8 HLCS deficiency patients, respectively. The identified mutations in the parents of the patients and 50 normal controls were screened by PCR-restriction fragment length polymorphism and direct DNA sequencing.</p><p><b>RESULTS</b>Total detection rate of gene mutation is 100% in the 12 children with MCD. Six mutations were detected in the 4 children with BT deficiency, they were c. 98-104del7ins3, c. 1369G>A (V457M), c. 1157G>A(W386X), c. 1284C>A(Y428X), c. 1384delA and c. 1493_1494insT. The last four were novel mutations. Four mutations were found in the 8 children with HLCS deficiency. They were c. 126G>T (E42D), c. 1994G>C (R665P), c. 1088T>A (V363D) and c. 1522C>T (R508W). The last two were hot-spot mutations [75%(12/16)], and c. 1994G>C (R665P) was a novel mutation.</p><p><b>CONCLUSION</b>This study confirmed the diagnosis of 12 patients with MCD on the gene level. Six mutations were found in the BT gene and 4 in the HLCS gene, including 5 novel mutations. Two mutations of the HLCS gene are probably hot-spot mutations in Chinese children with HLCS deficiency.</p>

Diagnosis, treatment and gene mutation analysis in children with holocarboxylase synthetas deficiency / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease.</p><p><b>METHODS</b>Eleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed.</p><p><b>RESULTS</b>All 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment.</p><p><b>CONCLUSIONS</b>HCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.</p>

Cribado neonatal de las enfermedades congénitas / Neonatal screening for congenital diseases

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Gene mutation analysis in four Chinese patients with multiple carboxylase deficiency / 中华儿科杂志

<p><b>OBJECTIVE</b>Multiple carboxylase deficiency (MCD) is an autosomal recessive disorder. MCD is characterized by skin rash, metabolic acidosis, vomiting and psychomotor retardation. Depending on deficiency of the enzyme, MCD includes two different forms, biotinidase deficiency (BTD, OMIM 253260) and holocarboxylase synthetase deficiency (HLCSD, OMIM 253270). In this study, we analyzed gene mutations of four Chinese MCD patients and to explore the mutation spectrum and possibility of a molecular diagnosis.</p><p><b>METHODS</b>All exons and their flanking introns of biotinidase gene and HLCS gene were screened by polymerase chain reaction combined with DNA direct sequencing in four Chinese MCD patients. Genomic DNA was extracted using a kit from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 2% agarose gel electrophoresis and were subsequently sequenced with both the forward and reverse primers.</p><p><b>RESULTS</b>All patients showed mutations in HLCS gene, whereas no mutation was found in biotinidase gene, proving that all the four patients had HLCS deficiency. Four previously reported mutations in HLCS gene were detected (Y456C, R508W, D634N and 780delG). A missense mutation of 1522C > T in exon 11 of HLCS gene, which was a homozygotic mutation, was identified in patient 1; a mutation of 1522C > T in exon 11 combined with a mutation of 1367A > G in exon 9, which was a compound heterozygotic mutation, was identified in patient 2; a mutation of 1522C > T in exon 11 combined with a mutation of 1900G > A in exon 13, which was a compound heterozygotic mutation, was identified in patient 3; a mutation of 1522C > T in exon 11 combined with a mutation of 780delG in exon 7, which was a compound heterozygotic mutation, was identified in patient 4. All the parents were carriers of mutations. No additional carrier of this four mutations was identified from 50 samples of Chinese controls.</p><p><b>CONCLUSION</b>The 1522C > T (R508W) mutation probably represents a mutational hot-spot in Chinese HLCS deficiency patients while the 780delG mutation which was reported only in Japanese patients was found firstly in Chinese patients.</p>

Cribado de hipotiroidismo congénito, fenilcetonuria, galactosemia y deficiencia de biotinidasa en una muestra de retrasados mentales de Ciudad de La Habana / Screening for congenital hypothyroidism, phenylketonuria, galactosemia and biotinidase deficiency in a sample of mentaly retarded patients in the city of Havana

Introducción.El hipotiroidismo congénito (HC), la fenilcetonuria (FCU), la galactosemia (GAL) y la deficiencia de biotinidasa (DB) son errores innatos del metabolismo que tienen como característica común el retraso mental (RM) de diferentes gradaciones. Objetivo. Realizar el cribado de HC, FCU, GAL y DB en individuos con RM de etiología desconocida. Pacientes y métodos. Se recolectaron muestras de sangre venosa en tarjetas de papel de filtro S&S 903 de 55 individuos con RM sin etiología específica, nacidos en el período de 1977-1997. El diagnóstico de HC se realizó mediante las determinaciones de tiroxina total (T4) y hormona estimulante del tiroides (TSH), con los estuches de reactivos UMELISA T4 y TSH neonatal, respectivamente, y la detección de FCU, GAL y DB mediante las determinaciones de fenilalanina (Phe), galactosa total (Gal) y la actividad de la enzima biotinidasa (Biot), con los UMTEST PKU, GAL y BIOTINIDASA. Resultados. Los valores medios obtenidos para los análitos evaluados fueron: 0,8 mUI de TSH/L de sangre total (EEM: ñ0,2), 113,1 nmol de T4/L de suero (EEM: ñ5,4), 67,7 µmol de Phe/L de sangre total (EEM: ñ0,1), 0,1 mmol de Gal/L de sangre total (EEM:ñ0,01) y la actividad de Biot fue normal en todos los casos. Conclusiones. Este estudio permitió conocer los niveles de T4, TSH, Phe y Gal en una muestra de población cubana con RM de etiología desconocida. Además, se encontraron niveles ligeramente elevados de T4 en niños que tenían hipercinesia (AU)

Biotinidase deficiency: a treatable genetic disorder in the Saudi population

Biotinidase deficiency is an autosomal recessive genetic disorder which is not uncommon in the Saudi population. Biotinidase is responsible for biotin recycling and biotin is an essential cofactor for activation of the carboxylase enzymes. Absence of biotinidase leads to infantile or early childhood encephalopathy, seizure disorder, dermatitis, alopecia, neural deafness and optic atrophy. The disease can be diagnosed by simple fluorometric enzyme assay. Treatment with biotin is both cheap and simple, resulting in rewarding clinical recovery and normalization of the biochemical, neuroradiological and neurophysiological parameters. If neglected, however, a patient may die of acute metabolic acidosis or may suffer from permanent neural deafness and optic atrophy, with mental and motor handicap. We describe the detection and treatment of 20 cases of biotinidase deficiency in our hospital and recommend the introduction of a neonatal screening programme for this disorder