Re-sequencing of the APOAI promoter region and the genetic association of the -75G > A polymorphism with increased cholesterol and low density lipoprotein levels among a sample of the Kuwaiti population

BACKGROUND: APOAI, a member of the APOAI/CIII/IV/V gene cluster on chromosome 11q23-24, encodes a major protein component of HDL that has been associated with serum lipid levels. The aim of this study was to determine the genetic association of polymorphisms in the APOAI promoter region with plasma lipid levels in a cohort of healthy Kuwaiti volunteers. METHODS: A 435 bp region of the APOAI promoter was analyzed by re-sequencing in 549 Kuwaiti samples. DNA was extracted from blood taken from 549 healthy Kuwaiti volunteers who had fasted for the previous 12 h. Univariate and multivariate analysis was used to determine allele association with serum lipid levels. RESULTS: The target sequence included a partial segment of the promoter region, 5’UTR and exon 1 located between nucleotides −141 to +294 upstream of the APOAI gene on chromosome 11. No novel single nucleotide polymorphisms (SNPs) were observed. The sequences obtained were deposited with the NCBI GenBank with accession number [GenBank: JX438706]. The allelic frequencies for the three SNPs were as follows: APOAI rs670G = 0.807; rs5069C = 0.964; rs1799837G = 0.997 and found to be in HWE. A significant association (p < 0.05) was observed for the APOAI rs670 polymorphism with increased serum LDL-C. Multivariate analysis showed that APOAI rs670 was an independent predictive factor when controlling for age, sex and BMI for both LDL-C (OR: 1.66, p = 0.014) and TC (OR: 1.77, p = 0.006) levels. CONCLUSION: This study is the first to report sequence analysis of the APOAI promoter in an Arab population. The unexpected positive association found between the APOAI rs670 polymorphism and increased levels of LDL-C and TC may be due to linkage disequilibrium with other polymorphisms in candidate and neighboring genes known to be associated with lipid metabolism and transport

Apolipoprotein E Genotyping Among the Healthy Kuwaiti Population

Apolipoproteins (lipid-free) are lipid-binding proteins that circulate in the plasma of human blood and are responsible for the clearance of lipoproteins. Apolipoprotein E (ApoE) is one of the several classes of this protein family. It acts as a ligand for the low-density lipid (LDL) receptors and is important for the clearance of very low-density lipid (VLDL) and chylomicron remnants. The APOE gene locus is polymorphic, with three major known alleles, APOE*3, *4, and *2. We investigated the distribution of the allele frequency of the APOE gene locus and describe here the genetic variation in four Kuwaiti subpopulations: Arab origin (Arabian peninsula), Arab Bedouin tribes, Iranian origin, and the heterogeneous population. We also describe the use of Spreadex gels in resolving the amplified and digested products of the APOE gene locus. DNA was extracted from whole blood and subjected to PCR and then to RFLP analysis. Allele and genotype frequencies were estimated for the total population and for each subpopulation. Statistical analysis showed no difference in the allele frequencies between the four groups. The frequency of APOE*3 in the Kuwaiti population was highest (88.4%) followed by the frequency of APOE*4 (6.5%) and APOE*2 (5.1%). The genotype and allele frequencies obtained for the Kuwaiti population fell within the reported worldwide distribution for the APOE gene locus. Moreover, the results obtained in this study showed no statistical difference ( p \u3e 0.05) between the APOE allele and genotype frequencies between the subgroups for all six genotypes and three alleles, supporting the assumption of admixture in the Kuwaiti population and that the obtained frequencies were in Hardy-Weinberg equilibrium. Finally, we found that the distribution of the APOE alleles in Kuwait differs somewhat from those reported in other Arab populations, suggesting that the Arabs originating from the Arabian peninsula are different from those of Lebanon, Morocco, and Sudan

A novel <i>LPL</i> intronic variant: g.18704C>A identified by re-sequencing Kuwaiti Arab samples is associated with high-density lipoprotein, very low-density lipoprotein and triglyceride lipid levels - Fig 4

<p>Comparison of the frequencies of rare (a) and common (b) variants identified at the <i>LPL</i> gene locus in the Kuwaiti Arab samples (n = 100), non-Hispanic whites (n = 95; Pirim et al., 2014) and American Africans (n = 95; Pirim et al., 2015). The variants identified in this study are shown based on their location across the 30Kb gene. The minor allele frequencies are based on gene build 89 and genome assembly GRCh38 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192617#pone.0192617.ref026" target="_blank">26</a>].</p

Demographic data of the Kuwaiti Arab samples sequenced at the <i>LPL</i> gene locus analyzed for genetic association of 10 novel SNPs (B).

<p>Demographic data of the Kuwaiti Arab samples sequenced at the <i>LPL</i> gene locus analyzed for genetic association of 10 novel SNPs (B).</p

Genetic association of the two novel <i>LPL</i> variants with plasma lipid levels among the Kuwaiti cohort.

<p>Genetic association of the two novel <i>LPL</i> variants with plasma lipid levels among the Kuwaiti cohort.</p

A summary of the 47 novel variants identified by re-sequencing the full <i>LPL</i> gene locus and flanking sequences based on genome assembly GRch38.p10 [26].

<p>A summary of the 47 novel variants identified by re-sequencing the full <i>LPL</i> gene locus and flanking sequences based on genome assembly GRch38.p10 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192617#pone.0192617.ref026" target="_blank">26</a>].</p

Multivariate analysis using linear regression to predict the effect of KUA LPL-27 genotypes ss2137497749 on HDL, TG and VLDL levels in the Kuwaiti cohort (n = 702).

<p>Multivariate analysis using linear regression to predict the effect of KUA LPL-27 genotypes <a href="https://www.ncbi.nlm.nih.gov/SNP/snp_ss.cgi?ss=ss2137497750" target="_blank">ss2137497749</a> on HDL, TG and VLDL levels in the Kuwaiti cohort (n = 702).</p

Comparison of the variants based on the minor allele frequencies (MAF) identified across the <i>LPL</i> gene locus in different populations including the Kuwaiti Arab samples from this study.

<p>Comparison of the variants based on the minor allele frequencies (MAF) identified across the <i>LPL</i> gene locus in different populations including the Kuwaiti Arab samples from this study.</p

A novel <i>LPL</i> intronic variant: g.18704C>A identified by re-sequencing Kuwaiti Arab samples is associated with high-density lipoprotein, very low-density lipoprotein and triglyceride lipid levels

<div><p>The role interethnic genetic differences play in plasma lipid level variation across populations is a global health concern. Several genes involved in lipid metabolism and transport are strong candidates for the genetic association with lipid level variation especially lipoprotein lipase (<i>LPL</i>). The objective of this study was to re-sequence the full <i>LPL</i> gene in Kuwaiti Arabs, analyse the sequence variation and identify variants that could attribute to variation in plasma lipid levels for further genetic association. Samples (n = 100) of an Arab ethnic group from Kuwait were analysed for sequence variation by Sanger sequencing across the 30 Kb <i>LPL</i> gene and its flanking sequences. A total of 293 variants including 252 single nucleotide polymorphisms (SNPs) and 39 insertions/deletions (InDels) were identified among which 47 variants (32 SNPs and 15 InDels) were novel to Kuwaiti Arabs. This study is the first to report sequence data and analysis of frequencies of variants at the <i>LPL</i> gene locus in an Arab ethnic group with a novel “rare” variant (<i>LPL</i>:g.18704C>A) significantly associated to HDL (B = -0.181; 95% CI (-0.357, -0.006); p = 0.043), TG (B = 0.134; 95% CI (0.004–0.263); p = 0.044) and VLDL (B = 0.131; 95% CI (-0.001–0.263); p = 0.043) levels. Sequence variation in Kuwaiti Arabs was compared to other populations and was found to be similar with regards to the number of SNPs, InDels and distribution of the number of variants across the <i>LPL</i> gene locus and minor allele frequency (MAF). Moreover, comparison of the identified variants and their MAF with other reports provided a list of 46 potential variants across the <i>LPL</i> gene to be considered for future genetic association studies. The findings warrant further investigation into the association of g.18704C>A with lipid levels in other ethnic groups and with clinical manifestations of dyslipidemia.</p></div