Estudo da associação entre genótipo e fenótipo na deficiência de biotinidase

A deficiência de biotinidase (DB) é uma doença autossômica recessiva na qual estão prejudicadas a obtenção da vitamina biotina a partir da dieta e a reciclagem da mesma, levando à deficiência de carboxilases dependentes de biotina. As manifestações clínicas incluem problemas neuropsicomotores, dermatológicos e metabólicos. O diagnóstico da DB pode ser feito a partir de triagem neonatal, sendo que a confirmação é dada pela medida da atividade da biotinidase sérica. A DB pode ser total (atividade da biotinidase C, c.479G>A, c.664G>A, e c.1337T>C e c.1466A>G) e da variante mais comum na DB (c.1330G>C). Etapa 3) Avaliação de metabólitos (ácidos graxos, aminoácidos, colesterol total, triglicerídeos, glicose e insulina) em plasma de 14 pacientes com DB parcial e de 19 controles, na busca de evidências que reforçariam a indicação de tratamento na DB parcial. Resultados: Etapa 1) Foram identificadas três variantes novas (c.1337T>C, c.1466A>G e c.962G>A). A concordância entre o fenótipo bioquímico esperado (conforme genótipo) e o observado ocorreu em aproximadamente 70% dos casos. As variantes encontradas na região promotora (c.-514C>T, c.-315A>G e c.-183G>A), icterícia neonatal, prematuridade e transporte da amostra de plasma parecem não ter sido as principais causas das discordâncias entre fenótipo esperado e fenótipo observado. A atividade da biotinidase pode aumentar entre a primeira e 12 segunda coleta (período neonatal). A inconsistência na associação genótipo-fenótipo bioquímico reforça a orientação de que a DB deve ser diagnosticada com base na medida da atividade da biotinidase. A análise genética tem utilidade principalmente na diferenciação entre DB parcial e heterozigose. Etapa 2) Os resultados sugerem que as variantes novas c.119T>C, c.479G>A e c.1337T>C são deletérias, e que as variantes c.664G>A, c.1466A>G e c.1330G>C não são deletérias. Apesar disso, a variante c.1466A>G pode estar associada a DB parcial e o mecanismo de patogenicidade da c.1330G>C pode estar relacionado à instabilidade da enzima. Etapa 3) Dentre os analitos pesquisados, não foram identificadas alterações metabólicas em indivíduos com DB parcial e, portanto, o estudo falhou em mostrar evidências que reforcem o caráter patogênico da DB parcial.Biotinidase deficiency (BD) is an autosomal recessive disorder in which both absorption of biotin from dietary sources and its reuse/recycle are impaired, leading to a deficiency of biotin-dependent carboxylases. Clinical manifestations include neurological, dermatological and metabolic abnormalities. The diagnosis of BD can be made by neonatal screening, and the confirmation is given by the measurement of serum biotinidase activity. BD may be profound (biotinidase activity C, c.479G>A, c.664G>A, c.1337T>C and c.1466A>G) and the most common variant in BD(c.1330G>C). Step 3) Evaluation of metabolites (fatty acids, amino acids, total cholesterol, triglycerides, glucose and insulin) in plasma samples of 14 patients with partial BD and 19 control individuals, in order to search for evidences that would reinforce the indication of treatment in partial BD. Results: Step 1) Three novel variants were identified (c.1337T>C, c.1466A>G e c.962G>A). The agreement between the expected and the observed biochemical phenotype occurred in approximately 70% of cases. The variants found in the promoter region (c.-514C>T, c.-315A>G and c.-183G>A), neonatal jaundice, prematurity, and transport of the plasma sample were not the major causes of disagreement between expected and observed phenotype. Biotinidase activity may increase between the first and second collection (neonatal period). The inconsistency in the genotype-biochemical phenotype association reinforces the orientation that the BD should be diagnosed based 14 on the measurement of biotinidase activity. Genetic analysis has utility mainly in the differentiation between partial BD and heterozygosis. Step 2) The results suggest that the novel variants c.119T>C, c.479G>A and c.1337T>C are deleterious, and that the c.664G>A, c.1466A>G and c.1330G>C variants are not deleterious. Despite that, the c.1466A>G variant may be associated with partial DB and the mechanism of pathogenicity of c.1330G>C may be related to enzyme instability. Step 3) No metabolic changes were verified in individuals with partial BD, therefore the study failed to show evidence to reinforce the pathogenicity of partial DB

CBS mutations are good predictors for B6-responsiveness : a study based on the analysis of 35 brazilian classical homocystinuria patients

Background Classical 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. Results Parental 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. Conclusions Most 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

Biotinidase deficiency: Genotype-biochemical phenotype association in Brazilian patients

[EN] 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. 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. 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.SIThis study received financial support from Fundo de Incentivo à Pesquisa e Eventos/Hospital de Clínicas de Porto Alegre (FIPE-HCPA) for research materials and publication fee. Post Graduate Program in Genetics and Molecular Biology (Universidade Federal do Rio Grande do Sul) funded the translation. ECN has a commercial affiliation (CTN Diagnósticos) which did not have any role or financial contribution to this research. TB have fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). FS had fellowship from the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). IVDS, MRSC and PASF have fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). HB receives a research grant of Orphan Europe. The funders did no provide support in the form of salaries for any author, and did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

Deficiência de biotinidase : avaliação de uma amostra de pacientes brasileiros com atividade reduzida da biotinidase

A deficiência de biotinidase (DB) é uma doença autossômica recessiva na qual estão prejudicadas tanto a obtenção da biotina a partir da dieta, quanto a reutilização da mesma, levando à deficiência de determinadas carboxilases dependentes de biotina. A DB pode ser total (atividade residual menor que 10%) ou parcial (10-30% da média da atividade normal) e heterozigotos apresentam atividade enzimática intermediária entre a dos afetados e a dos homozigotos normais. No Brasil, a frequência da DB combinada (DB total + DB parcial) parece ser uma das mais altas já relatadas, entre 1:6.843-1:62.500 nascidos vivos (incidência mundial estimada: 1:60.089 nascidos vivos). A idade do aparecimento das manifestações clínicas é variada, e essas incluem problemas neuropsicomotores, dermatológicos e metabólicos. O gene que codifica a biotinidase, BTD, é composto por quatro éxons, e alguns genótipos específicos apresentam uma boa associação com o nível de atividade enzimática. A confirmação da DB é baseada na medida da atividade da biotinidase no plasma, teste que pode ser falsamente reduzido devido à labilidade da enzima, prematuridade e icterícia, o que dificulta a classificação fenotípica e a decisão sobre instituição da terapia - suplementação oral de biotina livre. No final de 2012, a pesquisa da DB foi incluída no Programa Nacional de Triagem Neonatal, e, com a disponibilidade do exame pelo sistema público de saúde em todo o país, espera-se um aumento do número de crianças identificadas com atividade da biotinidase reduzida. O objetivo deste estudo foi caracterizar o perfil clínico e genético de uma amostra de pacientes brasileiros com atividade reduzida da biotinidase. O estudo foi observacional, multicêntrico e a amostragem foi por conveniência, sendo sequenciados os éxons 2, 3 e 4 do gene BTD. Foram incluídos 38 indivíduos com fenótipos bioquímicos definidos a priori a partir dos níveis de atividade da biotinidase em soro/plasma (deficiência total= 2; deficiência parcial= 9; heterozigotos= 15; limítrofe entre deficiência parcial e heterozigoto= 1; limítrofe entre heterozigoto e normal= 2) ou papel-filtro (n= 9, todos com tipo de deficiência não discriminada), a maioria proveniente da região sul do Brasil (n= 29/38) e identificados por triagem neonatal (n= 33/38). Consanguinidade parental foi relatada em dois casos. As variantes mais frequentemente encontradas nos pacientes foram c.1330G>C (p.D444H), c.755A>G (p.D252G) e c.[1330G>C;511G>A] (p.[D444H;A171T]), cujas frequências alélicas foram 50%, 9,4% e 5,4%, respectivamente. Três novas variantes patogênicas foram identificadas (c.119T>C ou p.L40P, c.479G>A ou p.C160Y e c.664G>A ou p.D222N). Vinte e nove pacientes tiveram duas variantes patogênicas detectadas (em 26/29 o estado de cis/trans pode ser determinado), seis apenas uma variante e três nenhuma variante detectada. A genotipagem confirmou a classificação feita a partir da atividade enzimática em 16/26 casos. Em indivíduos controles, foram identificadas três variantes polimórficas, sendo duas não patogênicas (c.1171C>T ou p.P391S e c.1413T>C ou p.C471C, com frequência de 1,5% e 5,5%, respectivamente) e uma patogênica (c.1330G>C, com frequência de 4%). Os nossos dados sugerem que a DB parcial é o tipo mais comum de DB no Brasil, e ampliam a heterogeneidade alélica já descrita para essa doença.Biotinidase deficiency (BD) is an autosomal recessive disorder in which both absorption of biotin from dietary sources and its reuse/recycle are impaired, leading to a deficiency of biotin-dependent carboxylases. BD may be profound (residual activity C (p. D444H), c.755A>G (p.D252G), and c.[1330G>C;511G>A] (p.[D444H;A171T]), with allele frequencies of 50%, 9.4%, and 5.4% respectively. Three novel pathogenic variants were identified (c.119T>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 configuration 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.1413T>C or p.C471C, with a frequency of 1.5% and 5.5% respectively) and one pathogenic (c.1330G>C, frequency 4%). Our 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