Journal of Clinical Chemistry and Laboratory Medicine
Open Access
ISSN: 2736-6588
ISSN: 2736-6588
Research Article - (2018) Volume 1, Issue 2
Background: The long noncoding RNAs (lncRNAs) have gradually been reported to be an important class of RNAs with pivotal roles in the development and progression of myocardial infarction (MI). In this study, we hypothesized that genetic variant of cyclin-dependent kinase inhibitor 2B antisense RNA (ANRIL) and metastasisassociated lung adenocarcinoma transcript 1 (MALAT1) may affect the prognosis of MI patients.
Methods: The study included 401 Han Chinese MI patients and 409 controls. Four lncRNA tag single nucleotide polymorphisms (SNPs)-ANRIL rs9632884 and rs1537373, MALAT1 rs619586 and rs3200401were selected. SNP genotyping was performed by an improved multiplex ligation detection reaction assay. A systematic review and metaanalysis of studies including 9,807 cases and 9.326 controls on the association of five ANRIL SNPs and the overall risk of MI or coronary artery disease (CAD) was performed.
Results: rs9632884 and rs3200401 SNPs were significantly associated with lipid levels in both controls and MI patients (P<0.003-0.046). Several SNPs interacted with sex and age to modify total cholesterol, low-density lipoprotein cholesterol, and creatinine levels to modify the risk of MI. No association between the lncRNAs SNPs and susceptibility to MI was found (P>0.05 for all). In the meta-analysis, rs4977574 A>G and rs1333049 G>C ANRIL polymorphisms, but not rs1333040, rs1333042 or rs10757274, were correlated with overall MI or CAD risk.
Conclusions: Taken together, this study provides additional evidence that genetic variation of the ANRIL rs9632884 and MALAT1 rs3200401 can mediate lipid levels in MI patients.
Keywords: Cyclin-dependent kinase inhibitor 2B antisense RNA; Metastasis associated lung adenocarcinoma transcript 1; Single nucleotide polymorphism; Myocardial infarction; Coronary artery disease
Morbidity and mortality associated with coronary artery disease (CAD) result in significant economic and social burdens [1]. Hypertension, smoking, hypercholesterolemia, and diabetes are estimated to contribute to 50%-60% of disease susceptibility and genetic variation may account for predisposition in 40%-50% of sporadic cases [2-5]. Atherosclerosis contributes to the pathophysiology of CAD, but the effects of variation in the molecular and genetic determinants are not yet clear [6]. Recent evidence of the association of single nucleotide polymorphisms (SNPs) and increased risk of CAD indicates that genetic polymorphisms have a key role in the pathogenesis of CAD [7].
Long noncoding (lnc)RNAs are transcripts of at least 200 base pairs in length that do not code for proteins [8]. Studies of the functions of lncRNAs in disease, including CAD, are ongoing [9-12]. ANRIL, also known as CDK2BAS (CDKN2B antisense RNA) overlaps with CDKN2B on human chromosome 9p21 [13], a locus associated with genetic susceptibility for cardiovascular diseases (CVD). It spans 50 kb of DNA that express the ANRIL transcripts [14], which modulate expression of CDKN2A/B and influence cellular activities. ANRIL expression is also modulated by several CAD-associated SNPs in the 9p21 locus. The reduction of ANRIL expression leads to inhibition of vascular smooth muscle cell growth, which in turn increases the risk of atherosclerotic vascular disease [13]. MALAT1, a transcript first described in metastatic lung adenocarcinoma, regulates hyperglycemiainduced inflammation in endothelial cells. In several studies, MALAT1 inhibition was shown to decrease endothelial cell proliferation both in vitro and in vivo. MALAT1 sequesters serine/arginine splicing factors in nuclear speckle domains, thereby regulating alternative splicing [15], and it may also have immune-regulatory activity [16]. This is the first cardiovascular study to describe MALAT1 polymorphisms. In this study, the association of four tag SNPs (ANRIL rs9632884 and rs1537373, MALAT1 rs619586 and rs3200401) and myocardial infarction (MI) was investigated in a case-control study of 401 Han Chinese patients with MI [9].
Sample collection
A total of 401 hospitalized MI patients were enrolled at the Second Affiliated Hospital, Harbin Medical University (China) between September 2016 and November 2017. The study protocol was approved by the local ethics review board; all participants provided written informed consent. The diagnosis of MI was based on the Pakistan Risk of Myocardial Infarction Study (PROMIS) international guideline. MI was diagnosed by symptoms within 24 h of hospital admission, an electrocardiogram consistent with MI, and positive troponin-I. Patients with recent illnesses or infections were not eligible [17,18]. A group of 409 age-(5-year bands) and sex-matched medical center patients without a history of CAD or symptoms of MI were selected as controls. Patients with cerebrovascular, neurological, or kidney disease, blood disorders, cancer, peripheral vascular disease, or autoimmune diseases were excluded from the control group. The controls were free of MI by questionnaires, history-taking, and clinical examination. The examination comprised physical examination, blood sampling, electrocardiography, chest X-ray, and Doppler echocardiography. Participant age, sex, blood pressure, lipid profile, fasting glucose, medical, drug, smoking, and alcohol histories were collected.
SNP selection
Four ANRIL and MALAT1 lncRNA loci were selected by a tagSNP method using Haploview version 4.2 bioinformatics software (Broad Institute, Cambridge, MA, USA); assuming a minor allele frequency >0.05 and a squared correlation between genotypes (r2)>0.8 for the SNPs in the Han Chinese population (CHB+CHS). The SNP information was retrieved from the 1000 Genomes Project and included those associated with cardiovascular disease in recent studies.
SNP genotyping
The genomic DNA was extracted using a GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, USA) as per the product instruction. The SNP genotyping work was performed using an improved multiplex ligation detection reaction (iMLDR) technique developed by Genesky Biotechnologies Inc. (Shanghai, China). A multiplex PCR-ligase detection reaction method was used in the iMLDR. For each SNP, the alleles were distinguished by different fluorescent labels of allele-specific oligonucleotide probe pairs. Different SNPs were further distinguished by different extended lengths at the 3’end. Two negative controls were set: one with double-distilled water as template and the other with DNA sample without primers while keeping all other conditions the same in one plate. Duplicate tests were designed and the results were consistent. A random sample accounting for ~5% (n=40) of the total DNA samples was directly sequenced using Big Dye-terminator version 3.1 and an ABI3730XL automated sequencer (Applied Biosystems) to confirm the results of iMLDR.
Selection of relevant studies
Study reports published before April 25, 2018 were retrieved from PubMed and the Web of Science using the search terms (coronary artery disease, coronary heart disease, or myocardial infarction), (“ANRIL” or “CDKN2B-AS”), and (polymorphism, variant, or mutation). Casecontrol studies of the relationship between ANRIL polymorphism and CAD or MI were eligible. At least two studies of ANRIL polymorphism reporting the genotype frequencies of each included ANRIL SNP (i.e. rs4977574, rs1333040, rs1333042, rs10757274 or rs1333049) were desired. Only studies published in English were eligible. Studies were excluded if they were not investigations of ANRIL SNPs (rs4977574, rs1333040, rs1333042, rs10757274 or rs1333049), were duplicate publications of the same population, or did not include a control group. Thirteen articles including 9,807 cases and 9.326 controls were selected (Figure 1).
Figure 1: Thirteen articles including 9,807 cases and 9.326 controls were selected for analysis.
Statistical analysis
All statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 2016 (Microsoft Corp., Redmond, WA, USA). All tests were two-sided and P-values <0.05 were considered significant. Between-group differences in demographic characteristics and genotype frequencies of the four SNPs were evaluated by Student’s t-test for continuous variables and χ2 tests for categorical variables. The Hardy-Weinberg equilibrium was assessed for controls using the goodness-of-fit χ2 test. Associations of genotypes and alleles and the risk of MI were estimated by odds ratios (ORs) and 95% confidence intervals (CIs). Haploview software (version 4.2) was used for the analysis of pairwise linkage disequilibrium, haplotype structure, and genetic association of polymorphism loci. Differences of lipid levels and genotypes were determined by the Kruskal-Wallis test. Significant interactions of the four SNPs with alcohol consumption, cigarette smoking, age, sex, and hypertension with lipid levels and the risk of MI were detected by the independent-samples t-test for categorical variables and linear regression analysis for continuous variables after controlling for potential confounders, a p-value <0.002 after the Bonferroni correction was considered statistically significant.
Revman 5.3 software (Nordic Cochrane Centre, Cochrane Collaboration, Copenhagen, Denmark) was used to for the metaanalysis. ORs and their 95% CIs were calculated to determine the significance of associations between ANRIL allele genotypes and susceptibility to MI or CAD. Heterogeneity was tested with the χ2-based Q and I2 tests. The pooled OR was calculated using a fixed effect model in the absence of heterogeneity (P>0.05, I2<50%). Otherwise, a random effect model was used. The stability of the pooled ORs was determined by one-way sensitivity analysis.
Population characteristics
The clinical characteristics of the 810 study participants are shown in Table 1. The majority (82.0%) were men (60.3%) were current smokers, and 26.2% drank alcohol. Except for hypercholesterolemia and triglycerides, the factors associated with increased risk of MI or CAD occurred more frequently in the case than in the control group.
| Variables | MI (n=401) | Controls (n=409) | P-valuea |
|---|---|---|---|
| Age, years | 58.20 (11.65) | 56.34 (9.52) | 0.223 |
| Male sex, No. (%) | 329 (82.0%) | 339 (82.9%) | 0.753 |
| Smoking, No. (%) | 242 (60.3%) | 187 (45.7%) | 3.04E-05b |
| Alcohol, No. (%) | 105 (26.2%) | 82 (20.0%) | 0.038 |
| Diabetes, No. (%) | 99 (26.7%) | 50 (12.2%) | 4.71E-06 |
| Hypertension, No. (%) | 192 (47.9%) | 143 (35.0%) | 1.90E-4 |
| Hypercholesterolemia, No. (%) | 55 (13.7%) | 39 (9.5%) | 0.063 |
| WBC, 109/L | 11.96 (3.81) | 6.84 (1.90) | 1.78E-80 |
| FBG, mmol/L | 7.13 (3.86) | 5.93 (1.93) | 1.25E-7 |
| CRE, μmol/L | 85.67 (30.17) | 70.83 (13.30) | 3.22E-18 |
| AST, U/L | 80.44 (85.24) | 25.12 (9.43) | 2.54E-32 |
| TC, mmol/L | 4.56 (1.05) | 4.96 (0.84) | 7.03E-9 |
| Triglycerides, mmol/L | 1.65 (1.12) | 1.67 (1.23) | 0.764 |
| HDL cholesterol, mmol/L | 1.28 (0.43) | 1.37 (0.36) | 1.65E-3 |
| LDL cholesterol, mmol/L | 2.77 (0.83) | 3.30 (0.73) | 1.92E-20 |
Values are means ± SD or n (%). aTwo-sided chi-square test or independent-samples
The study investigated the influence of genetic variation in two lncRNAs (ANRIL and MALAT1) on MI risk in a series of Han Chinese patients and controls. The two rs9632884 and rs3200401 SNPs identified in the study population were significantly associated with lipid levels in both controls and MI patients (P<0.003-0.046). However, no associations of the lncRNA SNPs and susceptibility to MI were found (P>0.05 for all). The two lncRNAs were chosen for study because ANRIL is the best replicated genetic risk locus of CAD and regulates genes involved in glucose and fatty acid metabolism [22,23]. The variants within this locus have been significantly associated with an increased risk of CAD and diabetes in European and some other populations [24-26]. MALAT1, recently renamed nuclear-enriched noncoding transcript 2 because it accumulates in the nucleus, was selected because it regulates alternative splicing that can result in gene expression of multiple proteins in healthy and diseased states [27]. MALAT1 is highly abundant in mammalian cells, but MALAT1 polymorphism has not been associated with MI [28].
In the study patients, rs9632884 was associated with TG levels but not with MI. In previous studies in Asian patients Song et al. found that ANRIL expression increased serum lipids and the atherogenic index [29], and Chen et al. showed that coronary atherosclerosis patients had elevated total LDLs and TGs [30]. We did not find a significant association of ANRIL rs9632884 and rs1537373 SNPs with MI in Han Chinese, but rs9632884 has been associated with the risk of CAD in European studies [31], which is inconsistent with our findings. To avoid false-negative results, we performed a systematic meta-analysis to confirm the results. SNP rs4977574 on chromosome 9p21.3 is located in ANRIL. This region has been considered as the most widely and consistently replicated risk locus for CHD and MI. We demonstrated that the variant G allele of rs4977574 exhibited a significant decreased risk for developing CAD, and the C allele of rs1333049 was associated with a significant increased CAD risk in Asians. In contrast, the rs1333040, rs1333042 and rs10757274 variant alleles exhibited no significant association with CAD risks. The differences in these study findings may be explained by ethnic, environmental, or lifestyle differences that affected the development of MI. Another explanation is that the participants in this study had experienced their first MI, and their risk factor profiles may differ from the profiles of patients with recurrent CAD. The way in which these SNP loci contribute to susceptibility to MI requires further study.
To the best of our knowledge, no previous studies have investigated the association of MALAT1 lncRNA polymorphisms and acute MI. rs3200401 was associated with TC levels, but the nature of any interaction was not determined. Some lncRNAs act as competing endogenous RNAs and function as micro (mi) RNA sponges that sequester miRNA regulates transcript expression by sharing common miRNA response elements [32]. Variant miRNA binding sites could result in gain or loss of function of miRNA-lncRNA interactions, ultimately affecting expression of other miRNA-targeted mRNAs [33,34]. According to the lncRNASNP [35], the C>T of the MALAT1 rs3200401 SNP results in a 1.62 kcal/mol minimal free energy (MFE, ΔG) change that may accompany loss of hsa-miR-1324 miRNA binding sites on MALAT1, leading to loss of function of miRNA-lncRNA interactions (Supplementary Figure 6). However, we did not find any associations of MALAT1 rs619586 and rs3200401 SNP and the risk of MI. Further study is needed to draw any conclusions about the role of MALAT1 rs619586 and rs3200401 in atherogenesis, and since this study included only Han Chinese participants, further studies in other ethnic groups are needed to confirm the relationships.
The study limitations include the relatively small sample size and inclusion of only one Chinese ethnicity. Second, there were differences in the clinical characteristics of the patients and controls. The statistical analyses adjusted for several confounders, but not all of the potential influences on the results were eliminated. Finally, the biological activities of the genetic variants of the two lncRNAs were not investigated. Larger studies are warranted to assess the potential association of the SNPs with more complex, clinical endpoints of MI and CAD.
Taken together, this study provides additional evidence that genetic variation of the ANRIL rs9632884 and MALAT1 rs3200401 can mediate lipid levels in MI patients, although the four SNPs in the cardiovascular-related lncRNAs were not associated with the risk of MI in this Han Chinese population. Those with the rs9632884 GG genotype had lower FBG than those with rs12762303 CC and rs12762303 GC genotypes. Those with the rs3200401 TT genotype had higher TC levels than those with rs3200401 CT and rs3200401 CC genotypes. The SNP of rs9632884 interacted with sex to modify CRE levels and to modify the risk of MI. Several SNPs interacted with age to influence TC (rs9632884) and LDL-C (rs1537373) levels. However, this study was designed as a pilot study and further investigations are needed to confirm our results and to elucidate unresolved questions. The contribution of other genetic variants of these vascular-related genes to CAD and MI cannot be excluded.
We thank all authors for their contributions and support. We are grateful to all participants in the study who provided blood samples. We would also like to thank the hospital staff who contributed to data collection for this study. We thank International Science Editing for editing this manuscript.
All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Supported by the National Natural Science Foundation of China (Grant number: 81770255), Natural Science Fund Project of Heilongjiang Province of China (Grant number: H201314). Harbin medical university innovation fund foundation research project (Grant number: YJSCX2017-40HYD).