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

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.

Figure 1

CBS map showing the location of the mutations found. Exons are represented by solid gray boxes. Novel mutations are shown in bold. An asterisk (*) indicates mutations found in more than one unrelated patient. Adapted from Kraus, 2017 (Kraus, 2017)

Figure 2

CBS mRNA expression as determined by qRT‐PCR. Genotypes: P1 (c.209+1delG; c.572C>T); P2 (c.2T>C; c.828+1G>A); P3 (c.828+1G>A; c.1126G>A); P4 (c.833T>C; c.989_991delAGG); P5 (c.1058C>T; c.146C>T); and P6 (c.444delG; c.444delG). Fold change in gene expression is calculated through the 2^{−∆∆ C}_T method, where ∆C_T = (C_{T ,Time x} ‐ C_{T ,Time 0}) (Schmittgen & Livak, 2008)