Sequence variants in the BTD underlying biotinidase deficiency in families of Pakistani origin.

BACKGROUND: Biotinidase deficiency (BTD) is a rare autosomal recessive metabolic disease, which develops neurological symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. The clinical features and mutation analysis of Pakistani children with BTD deficiency have rarely been described. Herein, for the first time, we report the clinical features, BTD gene mutations and biochemical analysis of seven symptomatic children with BTD deficiency from Pakistan. METHODS: Seven suspected BTD-deficient patients who presented abnormal organic acid profiles and clinical features were subjected to Sanger sequencing to identify pathogenic mutations in the BTD gene. The results were analyzed by Mutation Surveyor Software. RESULTS: All seven patients exhibited common biotinidase deficiency symptoms including hypotonia, developmental delay and seizures. Biochemical analysis shows marked excretion of 3-hydroxy isovalerate in all cases, followed by 3-hydroxy propionate and methyl citrate. Sanger sequencing revealed one frame-shift mutation, c.98_104delinsTCC (p.Cys33Phefs), and two missense mutations, c.1612C>A (p.Arg538Ser) and c.1330G>C (p.Asp444His). All mutations were in the homozygous state and classified as pathogenic in published studies and mutation databases. CONCLUSIONS: This study has validated the BTD variants as the underlying cause of biotinidase deficiency in which molecular testing of BTD is supported by urinary organic acid analysis and clinical diagnosis. Secondly, the strength of the local availability of this test in Pakistan will paved the way for the neonatal screening of biotinidase deficiency.

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.

Delayed Biotin Therapy in a Child with Atypical Profound Biotinidase Deficiency: Late Arrival of the Truth and a Lesson Worth Thinking.

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

Hepatic lipid metabolism disorders and immunotoxicity induced by cysteamine in early developmental stages of zebrafish.

Cysteamine, a sulfhydryl compound, is an intermediate in the metabolism of coenzyme A to taurine in living organisms. However, the potential side effects of cysteamine such as hepatotoxicity in pediatric patients have been reported in some studies. To evaluate the impact of cysteamine on infants and children, larval zebrafish (a vertebrate model) were exposed to 0.18, 0.36 and 0.54 mM cysteamine from 72 hpf to 144 hpf. Alterations in general and pathological evaluation, biochemical parameters, cell proliferation, lipid metabolism factors, inflammatory factors and Wnt signaling pathway levels were examined. Increased liver area and lipid accumulation were observed in liver morphology, staining and histopathology in a dose-dependent manner with cysteamine exposure. In addition, the experimental cysteamine group exhibited higher alanine aminotransferase, aspartate aminotransferase, total triglyceride and total cholesterol levels than the control group. Meanwhile, the levels of lipogenesis-related factors ascended whereas lipid transport-related factors descended. Oxidative stress indicators such as reactive oxygen species, MDA and SOD were upregulated after cysteamine exposure. Afterwards, transcription assays revealed that biotinidase and Wnt pathway-related genes were upregulated in the exposed group, and inhibition of Wnt signaling partially rescued the abnormal liver development. The current study found that cysteamine-induced hepatotoxicity in larval zebrafish is due to inflammation and abnormal lipid metabolism, which is mediated by biotinidase (a potential pantetheinase isoenzyme) and Wnt signaling. This provides a perspective on the safety of cysteamine administration in children and identifies potential targets for protection against adverse reactions.

Biotinidase deficiency: What have we learned in forty years?

BACKGROUND: Biotinidase deficiency (BD) is an autosomal recessively inherited disorder that was first described in 1982. Forty years after its first description, we compiled available clinical data on BD with the aim of generating a more comprehensive picture of this condition. METHODS: A systematic search strategy was performed in relevant databases without limits for publication date or languages. We screened 3966 records and included 144 articles reporting individuals with BD and their clinical presentation as well as the outcomes, when available. RESULTS: This study included 1113 individuals with BD. More than half (51.5%) of these individuals were diagnosed by newborn screening, 43.3% in presence of clinical symptoms and 5.2% due to family screening. We grouped symptomatic individuals into four main clinical presentations: neonatal-onset (<1 month; 7.9%), early childhood-onset (<2 years; 59.2%), juvenile-onset (2-16 years; 25.1%) and adult-onset (>16 years; 7.7%). BD affected five main organ systems: nervous system (67.2%), skin (53.7%), eye (34.4%), auditory (26.9%) and respiratory system (17.8%). Involvement was mainly multisystemic (82.2%) of individuals, whereas isolated system presentation was seen in only 17.2% of individuals. When reported, metabolic acidosis was present in 42.4% of symptomatic individuals and characteristic abnormal organic acid metabolites were found in 57.1%. Biotin treatment led to clinical stability or improvement in 89.2% of individuals. 1.6% of reported individuals with BD died due to non-availability of treatment or late diagnosis. CONCLUSION: Newborn screening has had a major positive impact on the outcome of many individuals with BD. However, undiagnosed and non-treated BD remains a health concern. Given the risk of mortality or complications associated with late or missed diagnosis if newborn screening is not available, a trial of biotin should be considered in undiagnosed infants and adults exhibiting suspected clinical signs. Enzymatic activity and/or analysis of genetic variants can readily confirm the diagnosis of BD.

Neuroimaging Features of Biotinidase Deficiency.

Biotinidase deficiency is an autosomal recessive condition caused by pathogenic variants in the BTD gene. Resultant deficiency of free biotin leads to impaired activity of the enzyme carboxylase and related neurologic, dermatologic, and ocular symptoms. Many of these are reversible on treatment, but early recognition and commencement of biotin supplementation are critical. This practice is especially important in countries where routine neonatal screening for biotinidase deficiency is not performed. In this report comprising 14 patients from multiple centers, we demonstrate the MR imaging patterns of this disorder at various age groups. Knowledge of these patterns in the appropriate clinical context will help guide early diagnosis of this treatable metabolic disorder.

Recovery of enzyme activity in biotinidase deficient individuals during early childhood.

Deficiency of the biotinidase (BTD) enzyme is an inborn error of biotin metabolism caused by biallelic pathogenic variants in the BTD gene. There are two forms, partial and profound BTD deficiency, which both can be successfully treated with pharmacological doses of biotin, justifying the inclusion of this disorder in the newborn screening in numerous countries. We investigated the BTD deficiency cohort (N = 87) in our metabolic center, as it was detected upon newborn screening since 2005, and aimed to better understand the long-term course of BTD enzyme activity and how it may relate to the patients' genetic background. We observed that individuals with partial BTD deficiency display an elevation of BTD enzyme activity with increasing age in 48% of cases-a recovery which allowed adjustment or stop of biotin supplementation in 20% of all individuals. In addition, we were able to recruit 56 patients (64%) for genetic testing, revealing 19 different variants (2 novel), and constituting 22 different genotypes. Genotype-phenotype correlations revealed that the most abundant allele in our cohort p.(Asp444His) was also the most common variant in patients displaying recovery of BTD enzyme activity. Based on our results, we recommend to retest all patients with partial BTD deficiency at the age of 5 years, as this may result in an impact on therapy. Moreover, genetic testing of BTD deficient individuals can allow prediction of the severity of BTD deficiency and of the likelihood of BTD enzyme activity recovery with age.

Two novel BTD mutations causing profound biotinidase deficiency in a Chinese patient.

BACKGROUND: Biotinidase deficiency (OMIM 253260) is an autosomal recessively inherited disorder affecting about 1/60,000 people worldwide. The absence or deficiency of biotinidase impairs free biotin recycling and affects biotin-dependent carboxylase functions. METHODS: A Chinese patient with spontaneous recurrent epilepsy, an eczema-like rash, hair loss, hypotonia, and hearing loss began at three months of age. Her biotinidase activity was 1.0 nmol/ml/min, 9.5% of the mean control activity, which confirmed profound biotinidase deficiency. RESULTS: Compound heterozygous for c.250-1G > C and c.878dupT variants in the BTD gene were identified in this patient. These two variants were novel and absent in the population matched controls and any databases. CONCLUSIONS: This study expanded the mutation spectrum of alterations of the BTD gene. Our patient also emphasized the critical role of biotinidase activity measurement combined with mutation analysis in early diagnosis of biotinidase deficiency.

Biotinidase deficiency presenting as Neuromyelitis Optica Spectrum Disorder.

Biotinidase deficiency disorder is a rare inherited metabolic disorder with typical neurological manifestations of hypotonia, developmental delay, rashes, seizures, hearing and vision impairment. We present two cases with different and unusual clinical profiles, whose neuroimaging resembled Neuromyelitis Optica Spectrum Disorder. Case 1 was initially treated with immunomodulation with steroids and intravenous immunoglobulins, with partial improvement. However reinvestigation for worsening of symptoms showed more extensive changes on spine magnetic resonance imaging. Raised lactate and alanine levels on repeat cerebrospinal fluid testing resulted in further investigations that revealed a biotinidase deficiency. Case 2 presented mainly with respiratory symptoms: a barium swallow suggested bulbar dysfunction. Neuroimaging of brain and spine was similar to that in case 1 and the child was promptly investigated for and confirmed to have biotinidase deficiency. Both cases responded to biotin supplementation. It is important to be cognisant of atypical neurological presentations of biotinidase deficiency including those that mimic immune mediated neurodemyelination disorders, as biotinidase deficiency is potentially treatable.

The novel homozygous p.Asn197_Ser201del mutation in BTD gene is associated with profound biotinidase deficiency in an Iranian consanguineous family.

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

Biotin: overview of the treatment of diseases of cutaneous appendages and of hyperseborrhea.

One of the most common micronutrient deficiencies with cutaneous findings is the vitamin B, also known as biotin, deficiency. Biotin deficiency may be due to congenital lack of biotinidase, or acquired following some conditions that interfere with its absorption, such as inflammatory bowel disorders, a diet too rich in avidin, magnesium deficiency, smoking habit and treatment with broad-spectrum antibiotics, anticonvulsants and sulfonamides. This review highlights the role of biotin in the most common skin disorders such associated with biotin deficiency and an approach to their treatment. Biotin administration may improve the treatment of hair loss when deficiency is detected on the basis of a careful patient history, clinical examination and the determination of serum biotin levels. The use of biotin is rationale in seborrheic dermatitis as the vitamin intercepts the main metabolic pathways underlying the pathogenesis of the disease. Treatment with biotin could also be useful in comedonal acne characterized by a high rate of seborrhea, and may be helpful for acne treated with topical retinoids, contributing to the control of flaking and irritation. The tolerability of biotin is excellent and there is no risk of hypervitaminosis even in the case of high doses. It is important that administration is controlled by physicians and follows a medical diagnosis and prescription. Correct doses used in dermatological conditions are safe and are not at risk of interference with laboratory tests.

Effect of BTD gene variants on in vitro biotinidase activity.

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

Biotin in metabolism, gene expression, and human disease.

Biotin is a water-soluble vitamin that belongs to the vitamin B complex and which is an essential nutrient of all living organisms from bacteria to man. In eukaryotic cells biotin functions as a prosthetic group of enzymes, collectively known as biotin-dependent carboxylases that catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Enzyme-bound biotin acts as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In recent years, evidence has mounted that biotin also regulates gene expression through a mechanism beyond its role as a prosthetic group of carboxylases. These activities may offer a mechanistic background to a developing literature on the action of biotin in neurological disorders. This review summarizes the role of biotin in activating carboxylases and proposed mechanisms associated with a role in gene expression and in ameliorating neurological disease.

Clinical features, BTD gene mutations, and their functional studies of eight symptomatic patients with biotinidase deficiency from Southern China.

Biotinidase (BTD) deficiency is a rare autosomal recessive metabolic disease, which develops neurological and cutaneous symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. Clinical features and mutation analysis of Chinese children with BTD deficiency were rarely described. Herein, for the first time, we reported the clinical features, BTD gene mutations and their functional studies of eight symptomatic children with BTD deficiency from southern China. Fatigue, hypotonia, proximal muscular weakness, hearing deficits, rash and respiratory problems are common clinical phenotype of our patients. Seizures are observed only in patients with profound BTD deficiency. Five novel mutations were detected, among which c.637delC (H213TfsTer51) was found in 50% of our patients and might be considered as a common mutation. In vitro studies confirmed three mild mutations c.1368A>C (Q456H), c.1613G>A (R538H), and c.644T>A (L215H) which retained 10-30% of wild type enzyme activity, and six severe mutations c.235C>T (R79C), c.1271G>C (C424S), c.1412G>A (C471Y), c.637delC (H213TfsTer51), c.395T>G (M132W), c.464T>C (L155P), and c.1493dupT (L498FfsTer13) which retained <10% of wild type enzyme activity. c.1330G>C (D444H) decreased the protein expression but not activity of BTD enzyme, and H213TfsTer51 was structurally damaging while L498FfsTer13 was functionally damaging. These results will be helpful in establishing the definitive diagnosis of BTD deficiency at the gene level, offering appropriate genetic counseling, and providing clues to structure/function relationships of the enzyme.

Laboratory diagnosis of biotinidase deficiency, 2017 update: a technical standard and guideline of the American College of Medical Genetics and Genomics.

Disclaimer: These ACMG Standards and Guidelines are intended as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these Standards and Guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of others that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, clinical laboratory geneticists should apply their professional judgment to the specific circumstances presented by the patient or specimen. Clinical laboratory scientists and geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Biotinidase deficiency is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences if untreated. Fortunately, the clinical features of the disorder can be ameliorated or prevented by administering pharmacological doses of the vitamin biotin. Newborn screening and confirmatory diagnosis of biotinidase deficiency encompasses both enzymatic and molecular testing approaches. These guidelines were developed to define and standardize laboratory procedures for enzymatic biotinidase testing, to delineate situations for which follow-up molecular testing is warranted, and to characterize variables that can influence test performance and interpretation of results.

Developmental window of sensorineural deafness in biotinidase-deficient mice.

Biotinidase deficiency is an autosomal recessively inherited disorder that results in the inability to recycle the vitamin, biotin. If untreated, the disorder can result in a range of neurological and cutaneous symptoms, including sensorineural deficits and deafness. To understand early mechanistic abnormalities that may precede more generalized and nonspecific effects of metabolic deficits such as weight loss and acidosis, we have analyzed auditory brainstem responses (ABRs) in biotinidase-deficient knockout (Btd -/- ) mice in the periweaning period with or without dietary biotin supplementation. We find significant increases in the latency of wave V of the ABR elicited by pure tone stimuli at one octave intervals, which precede substantial increases in ABR thresholds. Finer interpeak latency analyses of these changes indicate they are confined to the latter ABR waves associated with the CNS and likely reflect slowed brainstem transmission time. In contrast, peripheral nervous system conduction velocity appears normal. Further, we find that biotin-supplementation after the onset of symptoms reverses the latency shifts, which has significant relevance for early treatment in patients. Finally, ABR latencies in Btd -/- mice fed a biotin-supplemented diet for the first month of life appear refractory to transmission time slowing during a subsequent bout of biotin deficiency. These data suggest a transient vulnerability window for biotin deficiency in the auditory brainstem. Finally, we also observe a developmental vulnerability window involving follicular melanosome production or melanocyte survival. Sensorineural deafness precedes peripheral hearing loss in developmental biotinidase deficiency and is transient if rescued by dietary biotin within a short developmental window.

Biotinidase deficiency: Genotype-biochemical phenotype association in Brazilian patients.

INTRODUCTION: The association between the BTD genotype and biochemical phenotype [profound biotinidase deficiency (BD), partial BD or heterozygous activity] is not always consistent. This study aimed to investigate the genotype-biochemical phenotype association in patients with low biotinidase activity. METHODS: All exons, the 5'UTR and the promoter of the BTD gene were sequenced in 72 Brazilian individuals who exhibited low biotinidase activity. For each patient, the expected biochemical phenotype based on the known genotype was compared with the observed biochemical phenotype. Additional non-genetic factors that could affect the biotinidase activity were also analysed. RESULTS: Most individuals were identified by neonatal screening (n = 66/72). When consecutive results for the same patient were compared, age, prematurity and neonatal jaundice appeared to affect the level of biotinidase activity. The biochemical phenotype at the time of the second blood collection changed in 11/22 patients compared to results from the first sample. Three novel variants were found: c.1337T>C (p.L446P), c.1466A>G (p.N489S) and c.962G>A (p.W321*). Some patients with the same genotype presented different biochemical phenotypes. The expected and observed biochemical phenotypes agreed in 68.5% of cases (concordant patients). The non-coding variants c.-183G>A, c.-315A>G and c.-514C>T were present in heterozygosis in 5/17 discordant patients. In addition, c.-183G>A and c.-514C>T were also present in 10/37 concordant patients. CONCLUSIONS: The variants found in the promoter region do not appear to have a strong impact on biotinidase activity. Since there is a disparity between the BTD genotype and biochemical phenotype, and biotinidase activity may be affected by both genetic and non-genetic factors, we suggest that the diagnosis of BD should be based on more than one measurement of plasma biotinidase activity. DNA analysis can be of additional relevance to differentiate between partial BD and heterozygosity.

Biotinidase Resistant 68Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation.

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly 68Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled with 68Ga(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC50 of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. µPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

Effects of Biotin Deficiency on Biotinylated Proteins and Biotin-Related Genes in the Rat Brain.

Biotin is a water-soluble vitamin that functions as a cofactor for biotin-dependent carboxylases. The biochemical and physiological roles of biotin in brain regions have not yet been investigated sufficiently in vivo. Thus, in order to clarify the function of biotin in the brain, we herein examined biotin contents, biotinylated protein expression (e.g. holocarboxylases), and biotin-related gene expression in the brain of biotin-deficient rats. Three-week-old male Wistar rats were divided into a control group, biotin-deficient group, and pair-fed group. Rats were fed experimental diets from 3 wk old for 8 wk, and the cortex, hippocampus, striatum, hypothalamus, and cerebellum were then collected. In the biotin-deficient group, the maintenance of total biotin and holocarboxylases, increases in the bound form of biotin and biotinidase activity, and the expression of an unknown biotinylated protein were observed in the cortex. In other regions, total and free biotin contents decreased, holocarboxylase expression was maintained, and bound biotin and biotinidase activity remained unchanged. Biotin-related gene (pyruvate carboxylase, sodium-dependent multivitamin transporter, holocarboxylase synthetase, and biotinidase) expression in the cortex and hippocampus also remained unchanged among the dietary groups. These results suggest that biotin may be related to cortex functions by binding protein, and the effects of a biotin deficiency and the importance of biotin differ among the different brain regions.

Repression of Biotin-Related Proteins by Benzo[a]Pyrene-Induced Epigenetic Modifications in Human Bronchial Epithelial Cells.

Benzo[a]pyrene (B[a]P) exposure has been associated with the alteration in epigenetic marks that are involved in cancer development. Biotinidase (BTD) and holocarboxylase synthetase (HCS) are 2 major enzymes involved in maintaining the homeostasis of biotinylation, and the deregulation of this pathway has been associated with a number of cancers. However, the link between B[a]P exposure and the dysregulation of BTD/HCS in B[a]P-associated tumorigenesis is unknown. Here we showed that the expression of both BTD and HCS was significantly decreased upon B[a]P treatment in human bronchial epithelial (16HBE) cells. Benzo[a]pyrene exposure led to the global loss of DNA methylation by immunofluorescence, which coincided with the reduction in acetylation levels on histones H3 and H4 in 16HBE cells. Consistent with decreased histone acetylation, histone deacetylases (HDACs) HDAC2 and HDAC3 were significantly upregulated in a dosage-dependent manner. When DNA methylation or HDAC activity was inhibited, we found that the reduction in BTD and HCS was separately regulated through distinct epigenetic mechanisms. Together, our results suggested the potential link between B[a]P toxicity and deregulation of biotin homeostasis pathway in B[a]P-associated cancer development.