IntroductionThe 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.MethodsAll 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.ResultsMost 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.ConclusionsThe 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.

BackgroundBiotinidase deficiency (BD) is an inborn error of metabolism in which some genetic variants correlate with the level of enzyme activity. Biotinidase activity, however, may be artifactually low due to enzyme lability, premature birth, and jaundice; this hinders both phenotypic classification and the decision to implement therapy. This study sought to characterize the clinical and genetic profile of a sample of Brazilian patients exhibiting reduced biotinidase activity.MethodsThis observational, multicenter study used a convenience sampling strategy, with sequencing of exons 2, 3, and 4 of the BTD gene.ResultsThe sample comprised 38 individuals with biochemical phenotypes defined a priori on the basis of biotinidase activity in serum/plasma (2 with profound deficiency, 9 with partial deficiency, 15 heterozygous, 1 borderline between partial deficiency and heterozygosity, 2 borderline between heterozygous and normal) or dried blood spot sample (n = 9, all with unspecified deficiency). Most patients were from Southern Brazil (n = 29/38) and were identified by neonatal screening (n = 33/38). Parental consanguinity was reported in two cases. The most commonly found genetic variants were c.1330G > C (p.D444H), c.755A > G (p.D252G), and c.[511G > A;1330G > C] (p.[A171T;D444H]), with allele frequencies of 50%, 9.4%, and 5.4% respectively. Three novel pathogenic variants were identified (c.119 T > C or p.L40P, c.479G > A or p.C160Y, and c.664G > A or p.D222N). Twenty-nine patients had two pathogenic variants detected (with cis/trans status ascertained in 26/29), six had only one variant, and three had no pathogenic variants detected. Genotyping confirmed the original phenotypic classification based on enzyme activity in 16/26 cases. Three polymorphic variants were identified in control individuals, of which two were nonpathogenic (c.1171C > T or p.P391S and c.1413 T > C or p.C471C, with a frequency of 1.5% and 5.5% respectively) and one pathogenic (c.1330G > C, frequency 4%).ConclusionsOur findings suggest that partial BD is the most common form of BD in Brazil, and expand current knowledge on the allelic heterogeneity of this condition.

BackgroundClassical homocystinuria (HCU) is a monogenic disease caused by the deficient activity of cystathionine β‐synthase (CβS). The objective of this study was to identify the CBS mutations in Brazilian patients with HCU.Methods gDNA samples were obtained for 35 patients (30 families) with biochemically confirmed diagnosis of HCU. All exons and exon‐intron boundaries of CBS gene were sequenced. Gene expression analysis by qRT‐PCR was performed in six patients. Novel missense point mutations were expressed in E. coli by site‐directed mutagenesis.ResultsParental consanguinity was reported in 16 families, and pyridoxine responsiveness in five (15%) patients. Among individuals from the same family, all presented the same phenotype. Both pathogenic mutations were identified in 29/30 patients. Twenty‐one different mutations were detected in nine exons and three introns; being six common mutations. Most prevalent were p.Ile278Thr (18.2%), p.Trp323Ter (11.3%), p.Thr191Met (11.3%), and c.828+1G>A (11.3%). Eight novel mutations were found [c.2T>C, c.209+1delG, c.284T>C, c.329A>T, c.444delG, c.864_868delGAG c.989_991delAGG, and c.1223+5G>T]. Enzyme activity in E. coli‐expressed mutations was 1.5% for c.329A>T and 17.5% for c.284T>C. qRT‐PCR analysis revealed reduced gene expression in all evaluated genotypes: [c.209+1delG; c.572C>T]; [c.2T>C; c.828+1G>A]; [c.828+1G>A; c.1126G>A]; [c.833T>C; c.989_991delAGG]; [c.1058C>T; c.146C>T]; and [c.444delG; c.444delG]. The expected phenotype according to the genotype (pyridoxine responsiveness) matched in all cases.ConclusionsMost patients studied were pyridoxine nonresponsive and presented early manifestations, suggesting severe phenotypes. Many private mutations were observed, but the four most prevalent mutations together accounted for over 50% of mutated alleles. A good genotype–phenotype relationship was observed within families and for the four most common mutations.