Identification and Functional Analysis of Cystathionine Beta-Synthase Gene Mutations in Chinese Families with Classical Homocystinuria

Abstract

Background: Homocystinuria caused by cystathionine β-synthase (CBS) deficiency is the most common congenital disorder related to sulfur amino acid metabolism, manifested by neurological, vascular, and connective tissue involvement. Methods: This study analyzed the pathogenic gene and molecular mechanism of two classic homocystinuria families through whole exome sequencing and in vitro experiments including minigene assay and expression analysis. Results: Both probands presented with ectopia lentis, high myopia, and abnormally elevated homocysteine level, but one of them had more severe clinical manifestations, including general growth retardation, mild intellectual disability, and severe pectus excavatum. Their family members were phenotypically normal but presented slightly higher levels of homocysteine in plasma. Whole exome sequencing revealed that the two probands carried c.833T>C (p.Ile278Thr) and c.1359-1G>C, and c.919G>A (p.Gly307Ser) and c.131delT (p.Tle44Thrfs*38) compound heterozygous mutations in the CBS gene, respectively. Bioinformatics and in vitro functional analysis showed that the c.1359-1G>C mutation affects the normal splicing of CBS gene, resulting in the production of two abnormal transcripts and the production of two truncated proteins. One of the c.1359-1G>C splicing events (c.1359_1467del) and c.131delT (p.Tle44Thrfs*38) both lead to a significant decrease in CBS mRNA and protein levels. Conclusions: Accurate diagnosis of patients with homocystinuria is of great importance for timely and effective treatment, as well as for the provision of appropriate genetic counseling and prenatal diagnosis guidance to the affected families.

Keywords: CBS; RNA splicing; exome; homocystinuria; minigene; pathomechanism.

Figure 1

Pedigree of the family and the clinical features of the proband. (A) The pedigree of the family 1. The proband (II: 2) is marked with a red arrow. (B) An anterior segment photograph of the proband (II: 2) in family 1 illustrated ectopia lentis. (C) Sanger sequencing chromatogram of the CBS mutations from the proband in family 1 and her parents and sister. The red arrow represents the location of the mutation site. (D) The pedigree of family 2. The proband (II: 1) is marked with a red arrow. (E) Clinical characteristics of the proband (II: 1) in family 2. (F) Sanger sequencing chromatogram of the CBS mutations from the proband in family 2 and his parents and sister. The red arrow represents the location of the mutation site. (G) Plasma homocysteine levels quantified by ELISA showing abnormal increase in two probands. (H) Plasma CBS enzyme levels quantified by ELISA showing a decrease in two probands compared to the average control level.

Figure 2

Identification and analysis of mutations in the CBS gene. (A) Location of four CBS mutations c.833T>C, c.1359-1G>C, c.919G>A, and c.131delT at the nucleotide and protein levels (the protein and DNA structures are not drawn in scale) [12]. (B) Multiple species sequence alignments of CBS proteins by the Geneious Prime (version 2022.2.1) showed the high conservations of residue 278 (Ile) and residue 307 (Gly), respectively. The CBS protein UniportKB IDs (Uniport database URL: https://www.uniprot.org/) for eight species are displayed.

Figure 3

Minigene assay and Sanger sequencing of spliced transcripts based on pcMINI-N-CBS-wt/mut recombinant vectors. (A) Sanger sequencing results of the minigene constructs. (B) RT-PCR products of the minigenes expressed in 293T and MCF-7 cells. (C) Minigene splicing diagram and Sanger sequencing of the amplified products. Exon B is a component of the pcMINI-N vector.

Figure 4

Minigene assay and Sanger sequencing of spliced transcripts based on pcDNA3.1-CBS-wt/mut recombinant vectors. (A) Sanger sequencing results of the minigene constructs. (B) RT-PCR products of the minigenes expressed in 293T and MCF-7 cells. (C) Minigene splicing diagram and Sanger sequencing of the amplified products.

Figure 5

Structural models for wild-type and mutant CBS proteins were constructed using AlphaFold software (version 2.1) and visualized using PyMOL software (version 3.1, https://www.pymol.org/). (A) Structural models for wild-type and CBS proteins with I278T mutation. (B) Structural models for wild-type and CBS proteins with G307S mutations. (C) Structural models for CBS protein with V454Afs*9 mutation. (D) Structural model for CBS protein with V454Afs*51 mutation. (E) Structural model for CBS protein with I44Tfs*38 mutation. The amino acids located before and after the mutation are indicated in blue and red, respectively.

Figure 6

In vitro expression experiments of wild-type and mutant CBS in HEK-293T cells. (A) Detection of CBS mRNA expression of wild-type versus two mutant CBS by qRT-PCR analysis. (B) Detection of protein expressions of wild-type versus two mutant CBS by Western blotting. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.