Biotinidase-deficient Patients Research Articles

Comprehensive analysis of genotypic and phenotypic characteristics of biotinidase deficiency patients in the eastern region of Türkiye.

Biotin is a water-soluble vitamin that plays a key role in carboxylation. The formation of free biotin is impaired in biotinidase deficiency (BD), resulting in impaired biotin-dependent carboxylase functions. Based on the percentage of residual serum enzyme activity, BD is classified as partial and profound. Retrospective data including gender, age, parental consanguinity, family history, biotinidase activity analyses, type of deficiency (partial-profound), physical examination, treatment, and genotypes were evaluated in patients diagnosed with biotinidase deficiency in a single center in the eastern region of Türkiye. Patients whose biotinidase enzyme activity was below 30% with biallelic variants in the BTD gene were diagnosed as BD. A total of 302 patients were included in the study. Parental consanguinity was present in 135 (44.7%) of them. Two hundred eighty-six (94.7%) were diagnosed by neonatal screening, 14 (4.6%) by family screening and two (0.06%) by clinical symptoms. Ninety-two (30.5%) of the patients were followed-up with profound deficiency and 210 (69.5%) with partial deficiency. A total of 306 variants were detected. Twenty different variants (3 novel - 3 rare) and 31 different genotypes were detected. The 3 most frequently detected variants were c.410G>A (p.Arg137His; 47.3%), c.1270G>C (p.Asp424His; 29.7%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36; 15.3%). The 3 most frequently identified genotypes were c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) compound heterozygous (32.4%), c.410G>A (p.Arg137His) homozygous (24.8%), and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.1270G>C (p.Asp424His) compound heterozygous (12.2%). Patients with c.410G>A (p.Arg137His) homozygous variant, c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) homozygous variant and c.38_44delGCGGCTGinsTCC (p.Cys13Phefs*36) / c.410G>A (p.Arg137His) compound heterozygous variant were statistically significantly associated with profound deficiency. Compound heterozygosity of c.410G>A (p.Arg137His) / c.1270G>C (p.Asp424His) variants were significantly associated with partial deficiency. The association between the BTD genotype and biochemical phenotype is not always consistent. Our study provides valuable data by adding variants with genotype-phenotype correlations to the literature and three novel variants, which can provide significant guidance in clinical follow-up.

Identification of the mutations in BTD gene in Iranian patients with biotinidase deficiency and evaluating their genotype-phenotype correlations

BackgroundBiotinidase (BTD, encoded by the BTD gene) deficiency is an autosomal recessive neurometabolic disease caused by abnormal BTD activity in the biotin cycle. The clinical symptoms of patients, which are mainly neurocutaneous, range from mild to severe based on the enzyme activity level. This study aimed to identify BTD gene mutations in suspected BTD deficiency patients for the first time in the southwest of Iran and evaluate their genotype-phenotype correlations. Methods11 clinically and biochemically suspected patients from nine unrelated families and their available family members were subjected to Sanger sequencing. Segregation analysis was performed for novel mutation. The effect of each mutation on protein stability and hydropathicity, as well as the pathogenicity prediction of all detected mutations were assessed using various in silico analysis tools. ResultsSix mutations including a novel and five previously reported mutations were identified in patients with different ethnicities. Three out of five known mutations were reported for the first time in Iran. Various common clinical manifestations and a rarely reported coexistence of celiac disease in biotinidase deficiency patients were observed. The novel missense variant c.787G > A (p.Glu263Lys) was detected in exon 4 of the BTD gene, within the biotinidase-like domain of the BTD protein. This variation was found in two cousins of a family, both developed the same initial clinical presentation. In silico analyses revealed that this missense substitution decreased protein stability and increased protein hydrophilicity. Additionally, the known frameshift mutation c.1264delG (p.val422serfster59), was the most frequent allele in the studied population. We also predicted and visualized the effects of the novel and frameshift mutations on protein structure. ConclusionOur findings expanded the mutational spectrum of the BTD gene and provided valuable data on genotype-phenotype correlations, which helps in genetic counseling. Furthermore, the necessity of performing molecular analysis along with enzymatic analysis was highlighted by this study.

Alterations in optical coherence tomography and optical coherence tomography angiography findings in children with partial biotinidase deficiency.

The aim of the study was to investigate whether retinal neurovascular structural impairment in children with partial biotinidase deficiency (BD) could be detected early via optical coherence tomography (OCT) and OCT angiography (OCTA). Eighty patients with partial BD and 80 control cases without any known systemic or ocular diseases were enrolled in this prospective study. Retinal neurovascular structures in the macular and optic disc regions were examined in using OCT and OCTA. Data from subjects with partial BD were compared with data from healthy controls. A statistically significant decrease in the vessel density (VD) was observed in both superior and inferior parts of the superficial capillary plexus (SCP) and the deep capillary plexus (DCP) in the partial BD patient group compared with the control group (p<0.05). A significant decrease in the VD was also observed in the superior and inferior peripapillary SCP-DCP regions of the optic disc area in the patients with partial BD (p<0.05). Both the superficial and deep foveal avascular zone (FAZ) areas were significantly enlarged in the partial BD group with respect to the control group (p=0.026, p=0.021 respectively). In the partial BD patients, the vascular density in the macula and optic disc region is decreased in childhood, when compared to the control group. In patients with partial BD, it would be very useful to detect the early neurovascular changes that may occur during the course of the disease by means of a noninvasive technique such as OCTA.

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.

Biotinidase deficiency and its impact on the auditory system in Iranian children

Background and Aim: Biotinidase deficiency (BTD) is a rare autosomal recessive abnormality of biotin metabolism. If left untreated, it may lead to auditory symptoms. In this study, we examined the possible relationship between BTD and hearing impairment among Iranian children. Methods: This descriptive cross-sectional study was performed on 9 children (8 boys, 1 girl) with BTD, who referred to Imam Hossein Hos­pital in Isfahan City, Iran, in 2018. After coll­ecting their demographic data, including age, gender, weight, height, and history of diseases, we performed routine otolaryngologic and neu­rologic examination, audiological examinations, including otoscopic, acoustic immittance mea­surements, and auditory brainstem response (ABR). We recorded cochlear microphonic res­ults in most cases, too. Results: The subjects’ mean ± SD age of BTD diagnosis was 4.33 ± 5.36 months. Of all par­ticipants, 11.1% had a positive family history of the disease, and 66.7% of families had the first-degree consanguineous marriage. About 44.5% of participants had a normal hearing; 22.2% had moderate sensorineural hearing loss, and 33.3% showed no response to ABR test. All subjects showed normal acoustic immittance results. However, children with profound hearing loss showed bilateral absence of acoustic reflexes. Conclusion: BTD has a high impact on a chi­ld’s hearing system. The high prevalence of hearing loss among BTD patients suggests that parents of BTD children (diagnosed at birth) should pay special attention to auditory scree­ning and follow-up programs, as early diagnosis is important for preventing hearing loss. Also, families with first-degree of consanguineous marriages should consider genetic counseling before having children. Keywords: Biotinidase deficiency; hearing impairment; children

Clinical, Biochemical and Genetic Analysis of Biotinidase Deficiency in Iranian Population.

Biotinidase deficiency (BTD) is an autosomal recessive disorder of biotin metabolism. Biotin is a coenzyme that enhances the action of the four enzymes that play an important role in carbohydrates, amino acid, and fatty acid metabolism. Defects in these pathways cause severe metabolic disorder in the body. In general, biotinidase deficiency can be classified into two levels: partial and profound. The incidence of BTD is 1:40,000 to 1:60,000 births in the world, even though no convincing statistical data on the prevalence of this disorder exist in Iran. In this study, we aimed to set up a test for determining biotinidase activity among the Iranian population and report BTD mutations. The quantitative method for the determination of biotinidase activity was set up in the National Biochemistry Reference Laboratory (NBRL) of Pasteur Institute of Iran in Tehran. To detect mutations in BTD, polymerase chain reaction (PCR) was performed followed by DNA sequencing. The biotinidase activity range values were 3.81 - 8.25 nmol/min/mL. We identified 8 BTD patients out of 47 cases with neurologic signs. We detected two mutations, c.98-104del7ins3 and p.Arg79Cys, in 5 patients with profound BTD, and one p.Asp444His mutation in 3 patients with partial BTD. Infants suffering from BTD seem healthy during their first months of life. At present, the screening program for metabolic disorders such as BTD is in progress. The patients that are BTD deficient benefit from the availability of the tests, and consequently receive the Biotin supplements before being clinically affected.

Clinical, Biochemical and Outcome Profile of Biotinidase Deficient Patients from Tertiary Centre in Northern India.

Biotinidase deficiency is an inherited metabolic disorder with estimated birth incidence of 1 in 61,000 for profound and partial deficiency. Estimated incidence of profound and partial biotinidase deficiency is 1 in 1, 37,000 and 1 in 1, 10,000 respectively. The carrier frequency in general population is 1 in 120. We attempt to study clinical, biochemical and outcome from 10 Biotinidase deficient patients. A retrospective case record study was conducted to record Clinical, biochemical and outcome profile from genetic records. Biotinidase level was measured using spectrophotometric method. Study group comprised of 8 males and 2 females with median age of presentation 6 (2-45.75) months. Median (interquartile range) Biotinidase level in study group 0.3 (0.08-1.5) nmol/ml/min. Study group was further divided in to early onset group (< 12 months, n-6) and late onset group (> 12 months, n-4). Seizure, alopecia and hearing loss were predominant phenotypes in study group. The other rare presentations were: hypotonia, ataxia, skin rash, seborrhoea. The most common seizure type was focal seizure. Control of seizure activity was important immediate outcome measured in study group. Median duration (interquartile range) of seizure control in early onset group was 3 (2-4)days against 13.5 (12.25-14.75) days in late onset group. This study highlights the need of early diagnosis for favourable outcome for a potentially treatable inherited metabolic disorder.

Gene mutation analyses in Chinese children with multiple carboxylase deficiency

To confirm the diagnosis of multiple carboxylase deficiency (MCD) on the gene level and explore the mutations in Chinese children with MCD. 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. 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. 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.

Impaired Biotinidase Activity Disrupts Holocarboxylase Synthetase Expression in Late Onset Multiple Carboxylase Deficiency

Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5'-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation.

Determination of biotin (vitamin H) by the high-performance affinity chromatography with a trypsin-treated avidin-bound column

A method for measuring biotin by affinity-chromatography was developed using a trypsin-treated avidin silica gel column. Elution was by a linear gradient of propan-2-ol in an acidic phosphate buffer system containing 0.7 M NaCl (pH 2.4). Biotin was derivatized with 9-anthryldiazomethane (ADAM) to the fluorescent biotin-ADAM ester and a linear calibration line was obtained from 0 to 1.39 pmol with a detection limit of 69.5 fmol. Biotin was measured after hydrolysis in 2.25 M sulphuric acid for 1 h at 120 °C and the recovery for biocytin was 65.7 ± 2.53%, and hence a correction factor of 1.52 was used for the total biotin analysis. The recovery of added biotin from the serum was more than 98% using this correction factor of 1.52. Biotin was also measured in nutritional supplemental foods and foodstuffs, and we found that chicken egg yolk, “natto”, rice bran, royal jelly, and dried yeast contained highest levels of biotin. Biotin was also found in ferments by Bacillus natto, yeast, and some acetic acid bacterium. Storage foods such as beans, nuts and eggs also contained abundant biotin. Biotin was also determined and replacement monitored in the serum of suspected biotinidase deficiency patients. This affinity-chromatographic method for biotin determination was shown to be a robust and reliable and is well suited for biochemical and nutritional research.

Asymptomatic adults and older siblings with biotinidase deficiency ascertained by family studies of index cases

We report 32 biotinidase-deficient patients detected by family studies in the index cases. The study group consisted of 10 mothers, 4 fathers and 18 siblings. There were 17 individuals (3 mothers, 4 fathers and 10 siblings) with profound biotinidase deficiency (BD) (< 10% of mean normal activity) and 15 (7 mothers and 8 siblings) with partial BD (10-30% of mean normal activity). In the profound BD group, only three siblings were symptomatic. Dermatitis, microcephaly, developmental delay and convulsions were observed. The patients with partial BD did not have any clinical symptoms except one sibling with borderline IQ score. None of the parents was symptomatic. Family investigation of patients with BD is very important for the detection of asymptomatic patients who are at risk of exhibiting symptoms at any age. Careful evaluation of these untreated individuals with BD is important to obtain additional information about the natural history of this disorder and may provide clues to phenotype-genotype relationships and treatment regimes.

Molecular characterisation of 34 patients with biotinidase deficiency ascertained by newborn screening and family investigation.

This study characterises the spectrum of biotinidase mutations in 21 patients (17 families) with profound biotinidase deficiency (BD) and 13 unrelated patients with partial BD using a denaturing gradient gel electrophoretic mutation screening and selective sequencing approach. In 29 from 30 unrelated families we found biallelic mutations including four common mutations, D444H (frequency 23.3%), G98:d7i3(20.0%), Q456H(20.0%), T532M (15.0%) and nine rare mutations (V62M, R157H, A171T+D444H, C423W, D543H, L279W, N172S, V109G, 12236G-A) with frequencies less than 5.0%. Only three profound BD patients with G98:d7i3/G98:d7i3 and Q456H/Q456H genotypes and residual biotinidase activities of 0.0%, and 0.9% of normal activity developed clinical symptoms before biotin supplementation at 8 weeks of age. All other patients remained asymptomatic within the first months of life or even longer without treatment. Two patients homozygous for the frameshift mutation G98:d7i3 had no measurable residual enzyme activity. Twelve patients with partial BD had the D444H mutation in at least one allele. We conclude that, based on mutation analysis and biochemical examinations of the enzyme, it is currently not clearly predictable whether an untreated patient will develop symptoms or not, although it seems that patients with activities lower than 1% are at a high risk for developing symptoms of the disease early in life.

Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood-brain barrier.

The uptake of biotin and the closely related biocytin was characterized in primary cultures of calf brain microvessel endothelial (CBME) cells. Biotin uptake was found to be Na(+)-gradient dependent and independent of changes in the membrane potential. Concentration dependence revealed a single saturation mechanism with a K(m) of 47 microM and a V(max) of 101 pmol/min/mg. Inhibition studies demonstrated dependence on metabolic energy and the necessity for a free carboxyl group for transport activity. The anticonvulsants primidone and carbamazepine had no inhibitory effect. Biotin uptake into CBME cells is a secondary active, electroneutral, saturable and specific process. Biocytin which accumulates in biotinidase deficiency, a human congenital disorder, did not inhibit biotin uptake and was not transported into these cells. The presence of human serum with normal biotinidase activity significantly reduced biotin uptake by about 50%. Further, added biocytin was hydrolyzed to biotin, which accumulated intracellularly but to a lesser extent than added free biotin. Biotin uptake after addition of plasma of biotinidase-deficient patients was not different from that in the presence of normal serum. These results indicate that the absence of biotinidase activity in serum does not reduce blood-brain barrier transport of biotin.

N-d-Biotinyl-7-amino-4-methylcoumarin as a Novel Fluorigenic Substrate for the Determination of Biotinidase Activity

Abstract A new fluorigenic substrate for the determination of biotinidase, N-d-biotinyl-7-amino-4-methylcoumarin has been synthesized by using BOP reagent. This substrate was used for measuring the enzyme activity in plasma of normal and biotinidase deficient patients. The methodology used here should be applicable to other biological fluids and homogenates to detect biotinidase deficiency.

Na(+)-dependent biotin transport into brush-border membrane vesicles from rat kidney

The mechanisms of biotin reabsorption in rat kidney cortex were investigated using isolated brush-border membrane vesicles. An inwardly directed Na+ gradient specifically stimulated a transient biotin overshoot. Biotin transport was not affected by a valinomycin-induced K(+)-diffusion potential, and biotin(-)-Na+ stoichiometry was found to be 1:1. As a function of concentration, the uptake showed saturation in the presence of a Na+ gradient with an apparent Michaelis constant (Km) of 55 microM and Vmax of 217 pmol.mg protein-1.25 s-1. Desthiobiotin, 250 microM, norbiotin, bisnorbiotin, thioctic acid, valeric acid, probenecid, and nonanoic acid inhibited the transport of 30 microM biotin, whereas other biotin derivatives, as well as biocytin and organic acids found in the urine of biotinidase-deficient patients, did not. Preloading of the vesicles with biotin, desthiobiotin, norbiotin, and thioctic acid in the presence of Na+ increased initial uptake of biotin from the incubation medium (trans-stimulation). Our results indicate that biotin absorption in rat kidney fulfills the criteria for a specific carrier-mediated and electroneutral Na(+)-biotin- cotransport in a 1:1 ratio. The results are discussed in context with congenital biotinidase deficiency in humans.

Comparison of patients with complete and partial biotinidase deficiency: Biochemical studies

Seventeen partially biotinidase-deficient patients detected by neonatal screening or family studies were compared with four patients with classical biotinidase deficiency. Using a sensitive HPLC method for biotinidase in plasma (substrate: biocytin) the patients could be divided into two groups: one with residual biotinidase activity, and the second with undetectable biotinidase activity (0-activity). Biocytin excretion, characteristically elevated in 0-activity patients, decreased rapidly with increasing residual biotinidase activity and was almost normal when residual activity exceeded 2-3% of mean normal. In one patient with classical disease (0-activity) biotin deficiency, typical organic aciduria and multiple carboxylase deficiency were found as early as at the second week of life. In contrast, 13 infants with residual activities from 1.2% to 23% had no remarkable clinical or biochemical abnormalities. However, in three 5-, 14- and 15-year-old healthy siblings with residual biotinidase activities between 2.3% and 4.2%, biotin deficiency was proven by decreased activities of the mitochondrial carboxylases in lymphocytes (30-57% of mean normal) and, in the older siblings, also by subnormal plasma biotin concentrations. In biotinidase deficiency, biotin depletion presumably occurs earlier in the brain than in other tissues and may thus first affect the central nervous system. For this reason and because of discrete biochemical abnormalities found in a patient with residual biotinidase activity of 8%, we suggest that at least all patients with residual activities below 10% should be treated with biotin.

Quantitative determination of biocytin in urine of patients with biotinidase deficiency using high-performance liquid chromatography (HPLC)

A specific method for the quantitative determination of biocytin from urine of biotinidase deficient patients is described using HPLC-separation and quantitative determination by an avidin binding method. Partial purification of biocytin from urine was achieved with an anion exchange resin and concentration of the eluate by lyophilisation. The recovery of biocytin from urines was 95.3 ± 5.9 (mean ± sd). The precision of biocytin estimation in patients urines including the HPLC-sample preparation procedure varied between 5.9% and 10.5% (CV). Biocytin concentrations were measured in urine samples of 5 patients obtained during and/or before biotin therapy. Before treatment biocytin excretion ranged from 6.2–28.8 nmol/mmol creatinine. During therapy biocytin excretion increased to the 1.3 to 4-fold level in 3 out of 4 patients. However, there was no dose-related increase of biocytin excretion when pharmacological doses were administered. Apart from biocytin and biotin, patients excrete additional biotin derivatives. Some of these have been preliminary identified as bisnorbiotin and oxidation products of bisnorbiotin, biocytin and biotin.