CBS mutations are good predictors for B6-responsiveness: A study based on the analysis of 35 Brazilian Classical Homocystinuria patients

Mol Genet Genomic Med. 2018 Mar;6(2):160-170. doi: 10.1002/mgg3.342. Epub 2018 Jan 20.

Abstract

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.

Keywords: CBS mutations; CβS deficiency; CβS expression; classical homocystinuria; homocysteine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Alleles
  • Base Sequence / genetics
  • Biomarkers, Pharmacological / blood
  • Brazil / epidemiology
  • Child
  • Cystathionine beta-Synthase / genetics*
  • Cystathionine beta-Synthase / metabolism
  • Exons / genetics
  • Female
  • Gene Expression / genetics
  • Genetic Association Studies / methods
  • Genetic Predisposition to Disease / genetics
  • Homocystinuria / genetics*
  • Humans
  • Male
  • Mutation / genetics
  • Polymorphism, Single Nucleotide / genetics
  • Pyridoxine / genetics*
  • Pyridoxine / pharmacology

Substances

  • Biomarkers, Pharmacological
  • Cystathionine beta-Synthase
  • Pyridoxine