Role of Apolipoprotein E gene polymorphism in the risk of familial hypercholesterolemia : a case-control study

1Department of Family and Community Medicine, King Saud University, Riyadh, Saudi Arabia; 2Department of Pathology College of Medicine and University Hospitals, King Saud University, Riyadh, Kingdom of Saudi Arabia; 3Department of Biology Science, College of Science and Arts, Al-Qassim University, Al-Qassim, Saudi Arabia; 4Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, PO Box-10219, Riyadh-11433, Kingdom of Saudi Arabia


INTRODUION
Familial Hypercholesterolemia (FH-OMIM#143890) is an autosomal dominant pattern of limited genetic complexity, along with premature atherosclerotic cardiovascular disease (CVD) (Chan et al., 2016;Paquette et al., 2018).The single pathogenic variant involved is related to LDLR (60-80%), APOB (1-5%), or PCSK9 gene (upto 3%), affecting one in three people (Maurera et al., 2016).Recent studies confirmed that the FH prevalence was not 1:500 but 1:250 (van Lennep, 2016).It causes high LDL-R level from birth, due to a family history of high cholesterol (Onorato & Sturm, 2016).FH was diagnosed for the first time 50 years ago, and gained attention due to its connection to atherosclerosis and coronary heart disease (CHD).The association between genetic polymorphisms and clinical diseases was recognized earlier.Functional mutational studies from the earlier reports in specific genes (LDLR, APOB, PCSK9, and LDLRAP1) proved useful in FH diagnosis (Radovica-Spalvina et al., 2015).Due to smoking FH patients, with high triglycerides (TG) level and low blood pressure within the recommended limits (Pajak et al., 2016).Molecular diagnostic techniques revealed the relation between FH and Apolipoprotein E (ApoE) gene (Awan et al., 2013;Carmena et al., 1993;Eto et al., 1996;Jensen, 2002;Utermann et al., 1979).The ApoE gene was recognized as a genetic marker for coronary artery disease (CAD).The function of ApoE in lipid metabolism with serum glycoprotein serves as a ligand for cell-surface receptors and very low density lipoproteins (VLDL) in the liver and in the control's intestinal cholesterol absorption (Karahan et al., 2015;Yousuf & Iqbal, 2015).The ApoE gene was extensively studied in several diseases and it plays an important role in lipid metabolism (Megale et al., 2016).The ApoE allele e4 increases LDL-C levels and decreases ApoE plasma concentrations (Davignon et al., 1988).Three variable alleles (E2, E3 and E4) account for ApoE polymorphism that produces 6 genotypes: E2/ E2, E3/E3, and E4/E4, which are homozygous, and E2/E3, E2/E4, and E3/E4, which are heterozygous (Afroze et al., 2016).These alleles are associated with rs429358 (Arg-130-Cyt) and rs7412 (Cys-163-Arg) polymorphisms that appear in the coding region of missense mutations of the ApoE gene.The combinations of these SNPs alleles results in the production of the following transcripts: Arg/Cys (referred to as isoform e3), Arg/ Arg (isoform e4), Cys/Cys (isoform e2), and Cys/Arg (a rare isoform, seldom observed) (Alharbi et al., 2014).The studies on FH in relation to the genetic polymorphisms are limited and up to now no genetic study examined the FH in Saudi Arabia.Thus, the aim of the present research was to investigate the genetic association between FH cases and the ApoE gene polymorphisms (rs429358 and rs7412) in the Saudi population.
classification criteria (Lye et al., 2013).Ethical approval for this study was obtained from the Institutional Review Board at King Saud University (E-2-829).Selection of the 100 control subjects was described previously (Alharbi et al., 2015).
Blood analysis.Each patient provided 5 ml of blood for biochemical and molecular analysis.A serum sample of 3 ml was used for the biochemical assays and 2 ml of the EDTA sample was used for the molecular analysis.The serum sample was used to obtain the lipid profile comprising of total cholesterol (TC), TG, high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C).The assessment of the lipid profile was described in the earlier publication (Alharbi et al., 2013).DNA from peripheral leukocytes was isolated using Norgen DNA extraction kit (Thorold, Ontario, Canada).NanoDrop (Fisher Scientific, Waltham, MA, USA) was used to assess the quality of genomic DNA spectrophotometrically.TaqMan (Fisher Scientific, Waltham, MA, USA) assay genotyping as described (Alharbi et al., 2014).
Statistical analysis.The variables were compared between FH cases and healthy controls using the Student' t-test.Anthropometric and lipid profile data were presented as mean ± S.D. for continuous variables.Categorical variables were compared using the chi-square test and presented as percentages.Goodness of fit test was exploited to calculate the Hardy-Weinberg equilibrium (HWE) in FH cases and healthy controls.Allele frequencies were calculated with gene counting.Univariate logistic regression analysis was used to compare the genotype and allele frequencies between the cases and controls and the results were presented as odds ratio (OR) and 95% confidence interval (CI) values.The Yates correction was also applied during the analysis of the genotyping.ANOVA was performed on the lipid profiles and allele frequencies.A p<0.05 was considered statistically significant.Altogether, the statistical analysis was performed using the statistical package for social sciences (SPSS-22, Chicago, IL, USA).

RESULTS
Overall, 204 subjects were included in this study, divided into 104 FH cases and 100 controls.Table 1 presents the anthropometric and biochemical characteristics of all the study participants and the results of the t-test.The mean age of FH cases and controls was 57.76±9.94 and 44.02±6.29 years, respectively.The BMI value of the FH cases equaled 27.1±1.91kg/m 2 .However, comparison between FH and control cases included age, TC, TG, HDL-C and LDL-C, which were positively associated with statistical significance (p<0.05).Gender parameter did not show any gender association (p=0.58);one of the reasons could be unequal gender proportions in the studied groups i.e., 71.2% of males in FH cases and only 60% in controls.
In this case-control study, two specific SNPs (rs429358 and rs7412) of the ApoE gene were analyzed, in relation to them with FH in the Saudi population.HWE showed the association in the Saudi population.The genotype and allele distribution in FH cases and controls was pre-   3.However, for all four lipid profile parameters the levels were higher in the e2 allele bearers than in the case of alleles e3 and e4.

DISCUSSION
The presented study evaluated the relation between FH disease and the ApoE gene polymorphisms in the Saudi population.It revealed a nominal association i.e., association with the e4 allele (p=0.02).We did not find any genotype association, which may be due to the small sample size.Genetic susceptibility is thought to contribute to the pathogenesis of FH disease.However, FH prevalence in Saudi Arabia is still not well-documented, with too few studies focused on the subject (Al-Allaf et al., 2017;Al-Allaf et al., 2016;Al-Allaf et al., 2014;Al-Allaf et al., 2015;Alallaf et al., 2017;Alharbi et al., 2013;Alharbi et al., 2015;Nuglozeh, 2017).Moreover, FH disease may have a high prevalence may be due to connection with obesity (68%) and consanguineous marriages (~50-60%) in both the Saudi and Arab populations.In addition, the number of T2DM cases has been increasing (Alharbi et al., 2016), while FH disease is still underdiagnosed, but actively being evaluated.A previous study on 69 016 subjects from the Danish population revealed the prevalence of ~1 in 137 for diagnosed basing on the Dutch lipid clinic criteria, suggesting that FH is possibly underdiagnosed.The FH samples selected in this study has been selected through the Dutch group criteria, as only clinicians has diagnose FH disease (Benn et al., 2012).
Identification of SNPs in the DNA coding regions enables a better understanding of the pathophysiology of the diseases and may result in improved diagnosis, prevention and, treatment.Approximately, 90% of DNA sequence variants in humans are localized in coding regions (Khan et al., 2016).A significant progress regarding the detection of genetic diseases was documented (Risch & Merikangas, 1996).The techniques for sequencing of that complete exome are used to analyze the genetic variation in the affected patients with decreasing costs and increasing accuracy (Katsanis & Katsanis, 2013).The whole-exome sequencing (WES) technique identified the disease-causing marker and is a promising tool for understanding the molecular mechanisms of the diseases and for personalized treatment (Braenne et al., 2014).Earlier studies implemented exome sequencing in 125 FH-diagnosed patients in the UK population.The results did not reveal any novel genetic variants due to the selection of a limited number of genes.Exome sequencing analysis also failed to identify disease-related loci in both British and German population (Braenne et al., 2014;Futema et al., 2014).However, in the Saudi population, only specific genes were selected for the next-generation sequencing and a novel variant (c.2026delG, p. Gly676fs) identified in the LDLR gene in exon 14 (Al-Allaf et al., 2015).More number of studies have been published with the advanced technology in genotyping and detection of huge variant numbers in the human genome, which did not provide a better understanding of the disease mechanisms.Meta-analysis studies are defined as statistical procedures that integrates the results of several independent studies, thereby playing a central role in evidence-based medicine (Haidich, 2010;Salanti et al., 2005).
Meta-analysis of GWAS data can expand the associated data documented from earlier studies with assigned genotype data.However, the initial selection should be performed for the combinational results of GWAS and meta-analyses, along with genetic association with strong statistical support; at the same time, GWAS and metaanalyses showed convincing sizes effects are moderate in current platforms, as sample sizes can still explain the majority of large genetic risk for most common diseases (Zeggini & Ioannidis, 2009).
Our results showed that the ε4 allele is associated with FH cases in the Saudi population.The ε4 allele proved an important and reliable marker and did not appear to have a significant association with lipid profiles.Future studies on FH should be implemented with large sample sizes, comprised of global ethnicities and confirm the relation between the FH and ApoE gene.Exome-and next-generation sequencing analysis should be performed to identify a reliable diagnostic marker linking FH disease with specific SNP/variations.