Human Galectin-7 Gene LGALS7 Promoter Sequence Polymorphisms and Risk of Spontaneous Intracerebral Hemorrhage: A Prospective Study

Abstract

Background and purpose: Despite evidence for the role of genetic factors in stroke, only a small proportion of strokes have been clearly attributed to monogenic factors, due to phenotypic heterogeneity. The goal of this study was to determine whether a significant relationship exists between human galectin-7 gene LGALS7 promoter region polymorphisms and the risk of stroke due to non-traumatic intracerebral hemorrhage (ICH).

Methods: This two-stage genetic association study included an initial exploratory stage followed by a discovery stage. During the exploratory stage, transgenic galectin-7 mice or transgenic mice with the scrambled sequence of the hairpin structure -silenced down gene LGALS7-were generated and then expressed differentially expressed proteins and galectin-7-interacting proteins were identified through proteomic analysis. During the discovery stage, a single-nucleotide polymorphism (SNP) genotyping approach was used to determine associations between 2 LGALS7 SNPs and ICH stroke risk for a cohort of 24 patients with stroke of the Chinese Han population and 70 controls.

Results: During the exploratory phase, LGALS7 expression was found to be decreased in TG^LGALS-DOWN mice as compared to its expression in TG^LGALS mice. During the discovery phase, analysis of LGALS7 sequences of 24 non-traumatic ICH cases and 70 controls led to the identification of 2 ICH susceptibility loci: a genomic region on 19q13.2 containing two LGALS7 SNPs, rs567785577 and rs138945880, whereby the A allele of rs567785577 and the T allele of rs138945880 were associated with greater risk of contracting ICH [for T and A vs. C and G, unadjusted odds ratio (OR) = 13.5; 95% CI = 2.249-146.5; p = 0.002]. This is the first study to genotype the galectin-7 promoter in patients with hemorrhagic stroke. Genotype and allele association tests and preliminary analysis of patients with stroke revealed that a single locus may be a genetic risk factor for hemorrhagic stroke.

Conclusion: A and T alleles of two novel SNP loci of 19q13.2, rs567785577 and rs138945880, respectively, were evaluated for associations with susceptibility to ICH. Further studies with expanded case numbers that include subjects of other ethnic populations are needed to elucidate mechanisms underlying associations between these SNPs and ICH risk.

Keywords: cerebral hemorrhage; galectin-7; genetic; promoter; single-nucleotide polymorphism.

FIGURE 1

Detection of dual-luciferase activity of 293T cells at 48 h after transfection. Promoter activities of recombinant plasmids p-CG-Luc (p = 0.003, t = 4.88), p-TG-Luc (p < 0.05, t = 16.38), p-CA-Luc (p < 0.05, t = 8.19), and p-TA-Luc (p < 0.05, t = 39.34), p-CG-Luc, p-TG-Luc, p-CA-Luc, and p-TA-Luc were much higher than that of the negative control plasmid PGL3-control with SV40 promoter. ***P < 0.001 three asterisks signify less than 0.001, indicating a significant difference, statistically significant.

FIGURE 2

Agarose electrophoresis analysis of PCR amplification products of the galectin-7-encoding gene in the mouse of different generations of TG mice at different annealing temperatures. Each lane was loaded with 5 μl of PCR products. This is a combination diagram of agarose gel electrophoresis reactions of different individual mice, where F0, F1, and F2 represent the algebra of different mice. In the agarose gel electrophoresis figure, from left to right, the first lane on the left is a marker (DL2000-DNA-marker, with sizes of 100, 250, 500, 750, 1,000, and 2,000 bp, respectively. Second, each lane represents an individual. First built mice 12 mice, F0 has 12 mice, F1 has 10 mice, and F2 had 15 mice. Each individual uses two sets of different primers for PCR amplification, the product sizes are 323 bp and 372 bp, respectively, and the reaction system was 25 μl as described in the method. When the first band is unclear or the band is too shallow, it uses the second set of primers to detect coincidence. The positive control is the galactin-7 template (cDNA sequence) ligated in the T-vector—the positive control. A number of 4 individuals were tested two times in the F0 generation.

FIGURE 3

Differential expression of the galectin-7 protein in tissues of TG mice. Panel (A) shows transgene expression at the protein level [Tg (+) positive mice in the same litter, Tg (−) negative controls in the same litter]. WB bands are clearly visible in TG mouse samples (Tg +) as 17 kD protein bands, with expression levels presented as means ± SD. *p < 0.01 vs. that of the Tg(–) group. Panel (B) shows the expression of galectin-7 protein expression (as determined via WB) in knockdown mice and wild-type BALB/c mice, respectively. WT (BALB/c mice), Si (galectin-7 knockdown BALB/c mice). Protein bands with expression levels presented as means ± SD. *p < 0.05 vs. WT group. Panel (C) shows a positive control PCR experiment diagram of the interference sequence, and 392 bp is the product of the interfering sequence and interfering sequence vector amplification. *P < 0.05, **P < 0.01, ***P < 0.001. One asterisks signify less than 0.05, indicating a difference, statistically significant.

FIGURE 4

Homozygosity of LGALS7 SNP alleles in patients with ICH. LGALS7 gene sequencing electropherogram showing sequences in regions of SNPs rs567785577 and rs138945880. Right figure: results show successful insertion of promoter region DNA of the LGALS7 gene into the vector (750 bp). Results of electrophoresis of PCR products revealed predicted results for the patients with ICH (lane 1) and two control healthy subjects (lanes 2–3), respectively.