|Year : 2022 | Volume
| Issue : 1 | Page : 47-51
Association between LPA rs6415084 and rs6919346 variants and serum lipoprotein(a) levels amongst apparently healthy individuals
Dyna Ann Roby, Roopa Rani Bhandary, Sukanya Shetty, Vineet Kulkarni, Calvin Roby
Department of Biochemistry, KS Hegde Medical Academy, Nitte Deemed to be University, Deralakatte, Mangalore, Karnataka, India
|Date of Submission||21-May-2021|
|Date of Decision||13-Sep-2021|
|Date of Acceptance||23-Sep-2021|
|Date of Web Publication||31-Dec-2021|
Department of Biochemistry, KS Hegde Medical Academy, Nitte Deemed to be University, Deralakatte, Mangalore - 575 018, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Lipoprotein(a) (Lp[a]) synthesised by the liver consists of low-density lipoprotein which contains apoB-100 linked to apolipoprotein(a) (apo[a]) by disulphide bridge. LPA gene codes for highly polymorphic apo(a). The serum Lp(a) levels are highly varied amongst individuals and ethnicities, which could be attributed to the genetic polymorphisms present in LPA gene. Hence, this study aimed to analyse the association between LPA genetic variants, rs6415084 and rs6919346, with serum Lp(a) levels in apparently healthy subjects. Materials and Methods: In this cross-sectional study, 56 apparently healthy subjects were recruited after obtaining informed consent. Serum Lp(a) levels were measured by immunoturbidimetric assay, and the genotyping of the variants was determined by the real-time polymerase chain reaction. Spearman's correlation test was used to find the association between single-nucleotide polymorphism and serum Lp(a). P < 0.05 is considered statistically significant. Results: The median level of serum Lp(a) amongst the study subjects was found to be 26.75 mg/dL, which is higher in males (35.50 mg/dL) when compared to females (21.85 mg/dL). The analysis of LPA gene variant rs6415084 showed 58.9% of the subjects with computed tomography (CT) genotype, 17.85% with CC and 5.3% with TT genotype. In the case of the rs6919346 variant, 80% of the subjects had CC genotype, 8.9% with CT and 5.3% with TT genotype. Conclusion: Overall, a weak significant association was observed between heterozygous alleles of LPA gene variant rs6415084 with increased serum Lp(a) level.
Keywords: Apolipoprotein(a), lipoprotein(a), LPA gene, single-nucleotide polymorphism
|How to cite this article:|
Roby DA, Bhandary RR, Shetty S, Kulkarni V, Roby C. Association between LPA rs6415084 and rs6919346 variants and serum lipoprotein(a) levels amongst apparently healthy individuals. Adv Hum Biol 2022;12:47-51
|How to cite this URL:|
Roby DA, Bhandary RR, Shetty S, Kulkarni V, Roby C. Association between LPA rs6415084 and rs6919346 variants and serum lipoprotein(a) levels amongst apparently healthy individuals. Adv Hum Biol [serial online] 2022 [cited 2022 Aug 15];12:47-51. Available from: https://www.aihbonline.com/text.asp?2022/12/1/47/334588
| Introduction|| |
Lipoprotein(a) (Lp[a]), an atherogenic, thrombogenic and genetically regulated cardiovascular risk factor, synthesised by the liver consists of low-density lipoprotein that contains apoB100 which is linked to apolipoprotein(a) (apo[a]) through disulphide bridge. LPA gene on chromosome 6q-27 is evolved from the plasminogen gene and codes for highly polymorphic apo(a). It has been shown that serum Lp(a) levels are highly skewed amongst individuals and ethnicities, varying from 0 to 300 mg/dL. The wide variation seen may be due to different LPA gene polymorphisms such as the K-IV such as domain, copy number variation, pentanucleotide repeat and single-nucleotide polymorphisms (SNPs). Several studies conducted amongst multi-ethnic populations demonstrated the varied association of SNPs with serum Lp(a) levels and cardiovascular risk. The predominant SNPs associated with serum Lp(a) level were found to be rs10455872 in European Caucasians, rs9364559 in Chinese Han population, rs10455872 in Brazilians and rs6415084, rs10945682 and rs6919346 amongst South Asians.
Several researchers have revealed that SNPs of the LPA gene had a significant influence on the variation of serum Lp(a) levels and their association with cardiovascular diseases. Amongst them, the common variants studied extensively having a strong association with coronary heart disease was found to be rs6415084 and rs6919346. The rs6415084 is a short variation in the 5' haplotype block of the LPA gene, whereas rs6919346 is a variation in the 3' LPA haplotype block.,,,, Not many studies have been researched in India, wherein the variation of SNPs was examined. Out of the few previous studies, predominant SNPs associated with Lp(a) level amongst South Asians were rs6415084 and rs6919346. As we also belong to the same ethnicity, the same SNPs were selected for this study. In addition, there is a paucity of data amongst the Indian population with regard to the association between SNPs and serum Lp(a) level. Hence, the present study intended to investigate if SNP rs6415084 and rs6919346 of LPA gene are associated with serum Lp(a) in apparently healthy Indian subjects.
| Materials and Methods|| |
Fifty-six healthy individuals of both genders, aged 20–60 years, who attended outpatient department for the general health check-up under health plan scheme of Justice K.S. Hegde Charitable Hospital, Deralakatte, Mangalore, India, between November 2016 and August 2018 were recruited for the study. The study was approved by the Institutional Ethical Committee (#INST.EC/EC/114/2016-17 dt. January 27, 2018). Demographic details were obtained from the subjects after obtaining their informed consent. Subjects with coronary artery disease, smoking, alcohol, chronic inflammatory diseases, diabetes mellitus, renal disease, liver disease and malignancy were excluded from the study. The sample size was calculated using the formula based on the estimated standard deviation (σ = 22.5) obtained from the pilot study with an alpha (α) and error estimate (d) values of 0.05 and 6, respectively.
Collection of samples
Two millilitres each of fasting venous blood samples was collected from the participants in plain (red top) and ethylenediaminetetraacetic acid (EDTA) vacutainers, respectively. Samples collected in plain vacutainers were centrifuged at 3000 rpm for 10 min to obtain serum. Aliquots of serum were stored at −20°C for analysis of serum Lp(a). EDTA samples were also stored for DNA polymorphism at −20°C.
Measurement of serum lipoprotein(a) levels
Serum Lp(a) was measured by the immunoturbidimetric assay. Human Lp(a) agglutinates with latex particles coated with anti-Lp(a) antibodies. The precipitate obtained is determined turbidimetrically at 800/660 nm.
Genotyping of LPA gene
Genomic DNA was extracted and purified from all participants, according to manufacturer instructions, with the Qiagen DNA purification kit (Qiagen India Pvt. Ltd.). This method used 2 mL of peripheral blood obtained in EDTA anticoagulant blood tubes. After photometric concentration and purity measurement, DNA aliquots with 100 ng/μL concentration were stored at −20°C until further use. Incidence of LPA variants rs6415084 and rs6919346 was determined for each participant using reverse transcription–polymerase chain reaction (QuantStudio 5.0, Thermo Fisher Scientific, USA) through Applied BioSystems™ TaqMan® SNP Genotyping Assay (Thermo Fisher Scientific, USA).
TheIBM SPSS statistics for windows, version 20, Armonk,NY;IBM corp was employed for all statistical analyses. Serum Lp(a) levels were expressed as a median and interquartile range because of skewness. The distribution of frequency of SNP is represented as a percentage. Spearman's correlation was used to test the association between SNP and serum Lp(a). The significance level of all the statistical tests is P = 0.05.
| Results|| |
Demographic and biochemical parameters
A total of 56 apparently healthy subjects were recruited for the study. There was an equal distribution of male and female subjects amongst the study subjects, with a mean and median age group found to be 38.70 ± 9.66 years and 36.50 years (31.00–45.75), respectively. The mean and median of serum Lp(a) levels amongst the study subjects were found to be 31.02 ± 21.08 mg/dL and 26.75 mg/dL (13.09–47.75), respectively. The median Lp(a) level in males is 35.5 mg/dL, which is higher when compared to females, which has 21.85 mg/dL [Table 1]. The distribution analysis of serum Lp(a) levels amongst the study subjects found that the 47% (23 subjects) have 20 mg/dL, 28.60% (16 subjects) have 21–40 mg/dL, 17.85% (10 subjects) have 41–60 mg/dL and 12.50% (7 subjects) are with >61 mg/dL [Figure 1]. Overall, it was found that 51.78% (n = 29) of the subjects had <30 mg/dL and 48.21% (n = 27) had >30 mg/dL of serum Lp(a) levels, in which 44.8% (n = 13) of the males and 55.2% (n = 16) of the females have shown serum Lp(a) levels <30 mg/dL. Correspondingly, 51.85% (n = 14) of the males and 48.15% (n = 13) of the females have shown >30 mg/dL of serum Lp(a) levels [Figure 2].
|Figure 1: The distribution of serum lipoprotein(a) levels amongst the study subjects.|
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|Figure 2: The distribution of serum lipoprotein(a) levels amongst the male and female study subjects.|
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Analysis of single-nucleotide polymorphism of LPA gene
The frequency distribution analysis of the LPA genotype and their SNP variants, rs6415084, showed that 58.9% of the subjects were with computed tomography (CT), 17.85% with CC and 5.3% with TT genotype, and the variant, rs6919346, showed that 80% of the subjects had CC, 8.9% with CT and 5.3% with TT genotype [Table 2]. However, the gender-wise comparison did not show any significant difference in the genotype distribution.
Association of LPA genetic variants with serum lipoprotein(a) levels in the study subjects
The distribution of study subjects based on serum Lp(a) cut-off level was analysed, i.e., <30 mg/dL and >30 mg/dL [Table 3]. Out of the 56 subjects, 51.78% (n = 29) of the subjects had serum Lp(a) <30 mg/dL and 48.22% (n = 27) of the subjects had >30 mg/dL. For rs6415084 SNP, the majority of the subjects, i.e., 75.75% (n = 25), with CT genotype had serum Lp(a) level >30 mg/dL. Majority of the subjects with CC and TT genotype had <30 mg/dL. These results indicate that there is a positive correlation between heterozygous alleles of rs6415084 (P > 0.05). For rs6919346 SNP, 55.55% (n = 25) and 44.44% (n = 20) CC genotype had <30 mg/dL and >30 mg/dL of serum Lp(a), respectively. An equal number of TT genotype subjects had <30 mg/dL and >30 mg/dL, respectively. However, amongst five subjects with CT genotype of rs6919346 variant, four subjects had >30 mg/dL of serum Lp(a). Overall, there was a weak positive correlation between heterozygous alleles of rs6915084 and a negative correlation between homozygous alleles of variant rs6919346 (P > 0.05).
|Table 3: Genotype distribution amongst study subjects based on serum lipoprotein(a) level (mg/dL)|
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| Discussion|| |
Lp(a) is heterogeneous in nature, having many different isoforms amongst the different populations. The serum concentration of Lp(a) is highly skewed and differs amongst the different ethnic populations. The result of the present study also showed a skewed distribution of serum Lp(a) level [Figure 1]. A similar pattern of skewed distribution was found in one of the studies conducted in the North Indian population. The median value of serum Lp(a) level of the study subjects in the present study was 26.75 mg/dL and mean value 31.02 mg/dL [Table 1]. The mean and median values obtained in this study were found to be higher when compared to other Indian studies. The upper limit of serum Lp(a) for apparently healthy subjects has not been defined for the Indian population. The mean and median values defined by each population are found to be different.
Enas et al. were the first ones to report that 44% of the Indians had >20 mg/dL and 25% with ≥30 mg/dL amongst the Indians residing in the US. Our study found that 51.78% (n = 29) of the subjects had >30 mg/dL and 48.21% (n = 27) had <30 mg/dL of serum Lp(a). Serum Lp(a) level >30 mg/dL increases the risk for cardiovascular disease along with traditional atherogenic factors. Higher serum Lp(a) level was observed amongst South Asians when compared to Whites and Chinese. This demonstrated the racial and ethnic differences amongst the population. The present study found that the median value of serum Lp(a) for males (35.50 mg/dL) was higher when compared to females (21.85 mg/dL) [Table 1]. The lower mean values observed in females probably indicate the cardioprotective and vasoprotective effects of oestrogen amongst premenopausal women. On the contrary, the mean and median values of serum Lp(a) amongst females are higher in most Indian and Western studies. However, some of the studies did not find the difference in serum Lp(a) between males and females.
The analysis of LPA genetic variants rs6415084 and rs6919346 in this study revealed that 59% (n = 33) of the subjects had CT genotype and 80% (n = 45) of the subjects had CC genotype, respectively. There was no significant difference in the allele distribution amongst the gender [Table 2]. A study conducted by Boerwinkle et al. evaluated the segregation of apo(a) gene and plasma Lp(a) levels in 48 Caucasian families and found that all inter-individual variations in plasma Lp(a) were due to apo(a) gene polymorphism. This variation accounted for almost 91%. A comprehensive analysis of genomic variation in the LPA locus in a multi-ethnic population consisting of South Asians, Chinese and Caucasians carried out by Lanktree et al. has reported that variant rs10455872 was prevalent in Caucasians and rs6415084 was prevalent amongst South Asians Chinese and Caucasians.
In the present study, out of the 33 subjects with CT genotype of rs6415084, 23 subjects (75%) had >30 mg/dL of serum Lp(a). Out of the five subjects with CT genotype of rs6919346, four subjects had >30 mg/dL of serum Lp(a) [Table 3] and [Table 4]. However, the number is too small to comment. We also observed a significant association between allele distribution and serum Lp(a) level. A study reported that 16 SNPs of the LPA gene showed a significant association with serum Lp(a) level. Out of the 16 SNPs, rs3798220 and rs10455872 showed the strongest association with Lp(a) level. SNPs rs3798220 and rs10455872 together explained 36% of the total variation observed in the serum Lp(a) level and were significantly associated with increased risk of cardiovascular disease. One of the study reported that polymorphism in the size of apo(a) and SNPs put together showed a greater extend of variation in serum Lp(a)level in Caucasians(36%)when compared to Chinese (27%) or South Asians (21%). Another study demonstrated that two additional SNPs rs6919346 in intron 37 and rs1853021 (+ 93C/T) in the 5' untranslated region were significantly associated with elevated serum Lp(a) level but independent of the apo(a) size polymorphism.
|Table 4: Association of single-nucleotide polymorphisms rs6415084 and rs6919346 of LPA gene with lipoprotein(a)|
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A study conducted to find an association between five SNPs (rs1367211, rs3127596, rs9347438, rs6415085 and rs9364559) in the LPA gene and cardiovascular disease in the Chinese Han population study demonstrated that rs6415085 TT/TG genotype had significantly higher serum Lp(a) levels than that with the rs6415085 GG genotype. Our study on SNP rs6415084 also found that majority of those who had heterozygous allele distribution had an increased serum Lp(a) level. A number of common SNPs, strongly associated with serum Lp(a) level, accounted for up to 7% of the variation in serum Lp(a) level, as well as >70% of the African-Caucasian interethnic difference in Lp(a) level., Our results are consistent with previous studies. The limitation of the present study is the small sample size, and also, the study is not correlated with traditional serum lipid profile. Further, the study needs to be extended to a larger population to confirm the findings of the study.
| Conclusion|| |
Overall, the present study concludes that the skewed serum Lp(a) distribution amongst the study subjects was observed, and the heterozygous allele rs6415084 of the LPA gene showed a significant association with increased serum Lp(a) level.
The authors express their sincere acknowledgement to DST-FIST (Project No. SR/FST/LSI-637/2015) and Nitte (Deemed to be University) for providing the grant for development of infrastructure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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