Novel Indel Variation of NPC1 Gene Associates With Risk of Sudden Cardiac Death

Abstract

Background and Aims: Sudden cardiac death (SCD) was defined as an unexpected death from cardiac causes during a very short duration. It has been reported that Niemann-Pick type C1 (NPC1) gene mutations might be related to cardiovascular diseases. The purpose of the study is to investigate whether common genetic variants of NPC1 is involved in SCD susceptibility. Methods: Based on a candidate-gene-based approach and systematic screening strategy, this study analyzed an 8-bp insertion/deletion polymorphism (rs150703258) within downstream of NPC1 for the association with SCD risk in Chinese populations using 158 SCD cases and 524 controls. The association of rs150703258 and SCD susceptibility was analyzed using logistic regression. Genotype-phenotype correlation analysis was performed using public database including 1000G, expression quantitative trait loci (eQTL), and further validated by human heart tissues using PCR. Dual-luciferase assay was used to explore the potential regulatory role of rs150703258. Gene expression profiling interactive analysis and transcription factors prediction were performed. Results: Logistic regression analysis exhibited that the deletion allele of rs150703258 significantly increased the risk of SCD [odds ratio (OR) = 1.329; 95% confidence interval (95%CI):1.03-1.72; p = 0.0289]. Genotype-phenotype correlation analysis showed that the risk allele was significantly associated with higher expression of NPC1 at mRNA and protein expressions level in human heart tissues. eQTL analysis showed NPC1 and C18orf8 (an adjacent gene to NPC1) are both related to rs150703258 and have higher expression level in the samples with deletion allele. Dual-luciferase activity assays indicate a significant regulatory role for rs150703258. Gene expression profiling interactive analysis revealed that NPC1 and C18orf8 seemed to be co-regulated in human blood, arteries and heart tissues. In silico analysis showed that the rs150703258 deletion variant may create transcription factor binding sites. In addition, a rare 12-bp allele (4-bp longer than the insertion allele) of rs150703258 was discovered in the current cohort. Conclusion: In summary, our study revealed that rs150703258 might contribute to SCD susceptibility by regulating NPC1 and C18orf8 expression. This indel may be a potential marker for risk stratification and molecular diagnosis of SCD. Validations in different ethnic groups with larger sample size and mechanism explorations are warranted to confirm our findings.

Keywords: NPC1; genetic susceptibility; indel polymorphism; rs150703258; sudden cardiac death.

FIGURE 1

The schematic workflow of the present study. GEO: Gene Expression Omnibus; 3′UTR: 3′untranslated region; MAF: minor allele frequency; GTEx: Genotype-Tissue expression; eQTL: expression quality trait loci; PAGE: polyacrylamide gel electrophoresis; SCD: sudden cardiac death.

FIGURE 2

Public database analysis and LD analysis. (A) NPC1 was up-regulated in ruptured plaque, metabolic syndrome and coronary artery disease (CAD) tissues. (*p < 0.05, ***p < 0.001). The diversity expression atlases (GSE41571 and GSE23561) were obtained from the Gene Expression Omnibus (GEO) database. (B) The validation of the regulatory effect of rs150703258 on expression of NPC1 in 445 lymphoblastoid cell lines in 1000 Genomes Project database. (C) Linkage disequilibrium (LD) analysis of rs150703258 and nearby genome-wide association (GWAS) loci.

FIGURE 3

Example output from sequencing and genotyping assays of rs150703258. (A) The genotyping outcomes by using 7% non-denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining (lane 7, 9 and 10, ins/ins genotype; lane 1, 2, 3, 4 and 6, ins/del genotype; lane 5 and 8, del/del genotype; lane 11, rare/ins genotype; lane 12, rare/del genotype). (B) The sequencing results of insertion, deletion and rare allele in template strands. The underlined bases indicate the “GTA​TGT​AT”/“GTA​TGT​ATG​TAT” insertion in coding strands.

FIGURE 4

Expression levels of NPC1 in human tissues among different genotypes. (A) Genotype-expression analysis between rs150703258 and mRNA expression in human heart, arteries and whole blood samples in GTEx database. (B) The mRNA level of NPC1 in human myocardium samples showed that in tissues with ins/del and del/del genotype was 2.73-fold higher than that in samples with ins/ins genotype. (****p < 0.0001). ins/ins, N = 6, ins/del and del/del, N = 9. (C) Western blot assay analysis of NPC1 at protein level. Lanes 1–2 represent del/del genotype, and lanes 3–5 represent ins/del genotype, and lanes 6-8 represent ins/ins genotype.

FIGURE 5

Gene expression profiling interactive analysis. The expression of the two genes indicates a significant correlation in human whole blood, atrial appendage, left ventricle and coronary tissues.

FIGURE 6

The effect of rs150703258 on gene transcriptional activity. The relative firefly luciferase activities were compared between insertion construct group (pGL3-MT) and deletion construct group (pGL3-WT) in 293T cell lines. Cells transfected with pGL3-WT exhibited a significantly higher luciferase activity as compared with cells transfected with pGL3-MT (*p < 0.05).

FIGURE 7

Transcription factor prediction. This UCSC Genome Browser track hub represents genome-wide predicted binding sites for transcription factor binding profiles in the JASPAR database CORE collection, this view only showed predicted binding sites with scores above 500, which indicated the p-value of the TF match to indel position was less than 0.00001.