Multi-ARMS-PCR Versus Optimized PCP-RFLP For Apolipoprotein E Genotyping Of Majorly Depressed Patients

BackgroundMajor depressive disorder (MDD, major depression) is one of affective disorders with the main clinical features of being significant and lasting depressed which is caused by a variety of reasons. It has a lifetime prevalence of 16% that contributes to increased rates of disability and suicide.Apolipoprotein E (apolipoprotein E, apo E) is an important component of plasma lipoproteins and plays a key role in the transport and metabolism of lipids. Apo E protein has three isoforms:apoE2, apoE3 and apoE4, the difference is that the cysteine at position 112 (Cys) and the arginine at position 158 (Arg). The 112 site of apoE3 polypeptide chain is cysteine (Cys), the 158 site is Arginine (Arg); The two sites of apoE2 polypeptide chain are cysteine (Cys); The two sites of of apoE4 polypeptide chain are Arginine (Arg). The gene polymorphism of Apo E gene sequences on exon 4 at 112 site and 158 site determines the polymorphisms of apo E protein. Namely, Cys112 (TGC) and Arg158 (CGC)form apoE3,Cys112 (TGC) and Cys 158 (TGC) form apoE2, Arg112 (CGC) and Arg158 (CGC) form apoE4.With the development of technology and scientific research, the scientists found many kinds of APOE genotyping methods. There are several common PCR-based genotyping techniques currently in use, including amplification refractory mutation system PCR (ARMS-PCR), PCR restriction-fragment length polymorphism (PCR-RFLP), single-stranded conformational polymorphism (SSCP), and real-time PCR. The PCR-RFLP method has single restriction digestion and double restriction digestion:Restriction endonuclease Hha I or Cfo I is often used in single enzyme digestion method, Restriction endonuclease Afl III and Hae II are often used in double enzyme digestion method. However, not all PCR-based genotyping techniques are equally efficacious; it’s necessary to find a more accurate and cost-effective APOE genotyping method for MDD screening in large populations and studying the relationship between APOE gene polymorphism and depression. At the same time, the detection of APOE genotypes as an aid to the diagnosis of depression also has a certain value.ObjectiveIn the study, to find a more accurate and cost-effective APOE genotyping method for MDD screening in large populations, we comparatively evaluated two genotyping methods-multi-amplification refractory mutation system PCR (multi-ARMS-PCR) and optimized PCR restriction-fragment length polymorphism (optimized PCR-RFLP)Methods1. We take the Han people in the southwest area as the research object, random sampling from the normal group and depression group separately, 708 cases of blood samples were selected as the research object, including 358 cases of normal group,350 cases of depression group, Then extract the genomic DNA, detect the APOE genotype by multi-ARMS-PCR and optimized PCR-RFLP.2. Take DNA sequencing as the gold standard, we comparatively evaluated the accuracy of two genotyping methods-multi-amplification refractory mutation system PCR (multi-ARMS-PCR) and optimized PCR restriction-fragment length polymorphism (optimized PCR-RFLP) by Pearson chi square test.ResultsAlthough both methods were able to detect all six unique APOE genotypes, comparisons of both methods with S anger sequencing demonstrated that multi-ARMS-PCR (94%) was significantly more accurate than optimized PCR-RFLP (82%).ConclusionsMulti-ARMS-PCR should prove useful in quickly verifying ambiguous results obtained by other APOE genotyping methods and can be cost-effectively performed in the setting of a small laboratory or a population-based screening program.