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Association analysis of LDL-cholesterol with CELSR2, APOE and APOB genes through ARMS-PCR

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Author: UMAIR ZUBAIR

Citable URI : https://vspace.vu.edu.pk/detail.aspx?id=235

Publisher : Virtual University

Date Issued: 8/20/2019 12:00:00 AM

Abstract

Dyslipidemia is term to demonstrate the abnormal lipid metabolism that may lead to cardiovascular diseases. Several variants have been already reported to be associated with abnormal lipid metabolism. Four different variants CELSR2 (rs629301), APOE (rs429358 & rs7412) and APOB (rs1367117) were analyzed by Tetra Primer-Amplification Refractory Mutation System Polymerase Chain Reaction (T-ARMS PCR). This technique uses an approach that in addition to amplifying the control fragment (which surrounds the allele of interest), the mismatch, at the point of SNP on allele, on 3’ terminus in additional inner primers makes these primers specific to one allele and refractory to other one. An additional mismatch near the 3’ terminus of the inner primers strengthen the allele specificity. An amplicon is produced or not produced, in this condition, thus demonstrate the genotype of the subject. Though the optimization and development of T-ARMS PCR was difficult and time consuming but the method allows to genotype the specific variant and was validated by Sanger Sequencing method. As with other techniques, this technique has also its advantages and disadvantages. Variants having high GC content cannot be genotyped because of having high melting temperature and therefore formation of stable secondary structures thereby halting the amplification procedure even when organic additives were added. Therefore T-ARMS assay could not be developed for APOE (rs429358 & rs7412) variants. The second part of the study was to find the association analysis of the selected variants. T-ARMS PCR assay was developed for the CELSR2 (rs629301) and APOB (rs1367117) variants. Individuals having normal and abnormal lipid profiles (n=25 each) were examined for association analysis. Genotype distribution were found to be in Hardy-Weinberg Equilibrium and Chi square test indicates the significant association for both variants. Large scale genotyping approach can be employed for other along with these variants by using plate-based heat-block on PCR machine. As T-ARMS PCR Technique is robust and cost-effective, early risk detection can save the time window of treatment and would ultimately enhance the life expectancy of the patients suffering from dyslipidemia.


URI : https://vspace.vu.edu.pk/details.aspx?id=235

Citation: Zubair, U(2018). Association analysis of LDL-cholesterol with CELSR2, APOE and APOB genes through ARMS-PCR. Virtual University of Pakistan.(Lahore, Pakistan).

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