A functional variant in the cystathionine β-synthase gene promoter significantly reduces congenital heart disease susceptibility in a Han Chinese population

Abstract

Homocysteine is an independent risk factor for various cardiovascular diseases. There are two ways to remove homocysteine from embryonic cardiac cells: remethylation to form methionine or transsulfuration to form cysteine. Cystathionine β-synthase (CBS) catalyzes the first step of homocysteine transsulfuration as a rate-limiting enzyme. In this study, we identified a functional variant -4673C>G (rs2850144) in the CBS gene promoter region that significantly reduces the susceptibility to congenital heart disease (CHD) in a Han Chinese population consisting of 2 340 CHD patients and 2 270 controls. Individuals carrying the heterozygous CG and homozygous GG genotypes had a 15% (odds ratio (OR) = 0.85, 95% confidence interval (CI) = 0.75-0.96, P = 0.011) and 40% (OR = 0.60, 95% CI = 0.49-0.73, P = 1.78 × 10(-7)) reduced risk to develop CHD than the wild-type CC genotype carriers in the combined samples, respectively. Additional stratified analyses demonstrated that CBS -4673C>G is significantly related to septation defects and conotruncal defects. In vivo detection of CBS mRNA levels in human cardiac tissues and in vitro luciferase assays consistently showed that the minor G allele significantly increased CBS transcription. A functional analysis revealed that both the attenuated transcription suppressor SP1 binding affinity and the CBS promoter hypomethylation specifically linked with the minor G allele contributed to the remarkably upregulated CBS expression. Consequently, the carriers with genetically increased CBS expression would benefit from the protection due to the low homocysteine levels maintained by CBS in certain cells during the critical heart development stages. These results shed light on unexpected role of CBS and highlight the importance of homocysteine removal in cardiac development.Cell Research advance online publication 18 September 2012; doi:10.1038/cr.2012.135.

Figure 1

The CBS −4673C>G polymorphism upregulates CBS expression at the transcriptional level. (A) Schematic graph indicates the position of CBS −4673C>G polymorphism and the constructs for reporter gene assays in the CBS promoter. (B) Quantitative real-time PCR analysis of CBS in vivo mRNA level in 28 cardiovascular tissue samples with different −4673C>G genotypes. The actual values for each genotype group were as follows: CC = 1.26 ± 0.98; CG = 2.26 ± 1.10; GG = 6.04 ± 0.54. All values were normalized to the level of GAPDH and represented means ± SD of three independent experiments. (C) Luciferase expression was significantly increased in the minor G allelic construct compared with the major C construct in different cells (52% increases in HEK293 and 42% in H9C2 cells). The actual values in HEK293 cells were as follows: pGL3-basic = 0.70 ± 0.38; pGL3-C allele = 2.14 ± 0.23; and pGL3-G allele = 3.27 ± 0.90. The values in H9C2 cells were the following: pGL3-basic = 0.46 ± 0.22; pGL3-C allele = 1.85 ± 0.22; and pGL3-G allele = 2.63 ± 0.22. Each value represents mean ± SD of three independent experiments, and each experiment was performed in triplicate.

Figure 2

The CBS −4673 G allele attenuates transcription factor binding affinity. (A) SPR analysis comparing the binding affinity of nuclear extracts to DNA probes containing either the −4673 C or G alleles. The SPR-binding activity of the A-allele (|logKD| = 9.75 ± 0.65) was more than 1 000-fold higher than that of the C-allele (|logKD| = 6.12 ± 0.05). Each value represents the mean ± SD of three experiments. (B) Competition SPR assays performed in the presence of 5-fold excess non-biotinylated C- or G-allele probes. (C) ChIP assays using HEK293 and cardiovascular tissue samples. The presence of the SP1-binding CBS promoter was verified by PCR. (D) A luciferase construct containing either the C or G allele was co-transfected with pcDNA3.1 (control) or pcDNA3.1-SP1 expression plasmids. The actual values in HEK293 cells were as follows: pcDNA3.1-basic group: pGL3-basic = 0.34 ± 0.08, pGL3-C allele = 2.27 ± 0.16, and pGL3-G allele = 3.54 ± 0.23; pcDNA3.1-SP1 group: pGL3-basic = 0.43 ± 0.13, pGL3-C allele = 1.46 ± 0.12, and pGL3-G allele = 3.10 ± 0.16. The actual values in H9C2 cells were the following: pcDNA3.1 group: pGL3-basic = 0.31 ± 0.08, pGL3-C allele = 1.73 ± 0.14, and pGL3-G allele = 2.74 ± 0.10; pcDNA3.1-SP1 group: pGL3-basic = 0.27 ± 0.03, pGL3-C allele = 1.14 ± 0.12, and pGL3-G allele = 2.21 ± 0.18. Each value represented mean ± SD of three experiments, and each experiment was performed in triplicate.

Figure 3

Polymorphism CBS −4673C>G is related with CBS gene promoter hypomethylation. (A) After blocking DNA methylation in HEK293 cells using 5-Aza, CBS mRNA increased significantly compared with the control group. The actual value of 5-Aza group is 2.02 ± 0.22, and value of control group is 1.02 ± 0.19. Each value represents the mean ± SD of three experiments, and each experiment was performed in triplicate. (B) The methylation pattern of 20 CpG sites around the −4673 polymorphism (from −4 803 to −4 516) in 28 cardiovascular tissue samples. (C) Polymorphism CBS −4673C>G was related with allelic-specific methylation. (D) Nearly 70% of C alleles had at least one CpG site methylated, but almost 80% G alleles were not methylated at any CpG sites.

Figure 4

CBS −4673C>G polymorphism and human plasma homocysteine level. The actual values for each genotype group were as follows: CC = 8.21 ± 2.52; CG = 8.10 ± 2.31; and GG = 7.80 ± 2.33. Data shown were mean ± SD.