Analysis of Methylenetetrahydrofolatereductase (MTHFR) Polymorphisms (C677T & A1298C) in Recurrent Pregnancy Loss (RPL) Authors - Balasubramaniam Anujan, Ranmalee Amarasekara & Subeshala Kotalawala Affiliation – BMS School of Science, Colombo 06 Country – Sri Lanka

Recurrent Pregnancy Loss (RPL) Analysis of Methylenetetrahydrofolatereductase (MTHFR) Polymorphisms (C677T & A1298C) in Recurrent Pregnancy Loss (RPL) Analysis Background Recurrent Pregnancy loss (RPL) is a multifactorial disorder responsible for 15% of pregnancy failures. WHO defined miscarriage as loss of the fetus or embryo weighting less than 500 g which would normally be at 20-22 complete weeks of gestation” (Vanilla et al., 2015). The underlying etiology of RPL remains unknown and pathogenesis of RPL is complicated. Research studies suggest genetic factors to be highly associated with recurrent miscarriages (Cao et al., 2014). Literature suggests that MTHFR polymorphisms to be associated with RPL, C677T and A1298C polymorphisms are the two common mutations involved in RPL. However the specific polymorphisms of MTHFR gene associated with RPL is still debatable. Table 1. Data Analysis Sheet Year Author Journal Place of study Sample Size Type of Sample Source Methods & Detection Mutation Found Mutation Type and Analysis 2013 Cao et al., Gene 514 (2013) 105-11 China 46 reports 3559 URPL cases, 5097 healthy controls Maternal Blood Meta-analysis MTHFR C677T 1298CC mutation was 9% in both samples East Asians people are higher risk of URPL with 677TT genotype 2015 Hubacek, J.A et al., Clin Chim Acta 440 p.104-7. Slavonia 464 samples 486 controls aborted samples PCR-RFLP MTHFR C677T MTHFR A1298C Carriers with dominant allele of these polymorphisms were associated with higher risk of abortion Distinct combination of MTHFR could be associated with higher risk of abortions in Caucasians Continues….. Figure1. Molecular location of MTHFR Gene Platelet aggregation Endothelial damage Vasculopathy in placental vasculature Influencing utero-placental circulation selectively Spontaneous Abortion Expand tHcy Decrease enzyme activity MTHFR polymorphisms C677T A1298C Results Objective Aim of the study was to determine the frequencies of MTHFR C677T and A1298C MTHFR polymorphisms associated with RPL Methodology Identification and Eligibility of Relevant Studies Articles (n=35) published between the years 2005 to 2015, were selected for this review. Case control studies (30), meta-analysis (4) and a nested case control study were taken into account. Study Design Figure 2. Ratio of the case studies A positive MTHFR polymorphisms associated with RPL were seen in 75% (26/35) of the study (p = 0.002) Eligible Studies (n=35) C677T MTHFR polymorphism in RPL 50% A1298C MTHFR polymorphism in RPL 15% Data Extraction Year Author Journal Place of study Sample Size Type of Sample Source Methods & Detection Mutation Found Mutation Type and Analysis 2013 Cao et al., Gene 514 (2013) 105-11 China 46 reports 3559 URPL cases, 5097 healthy controls Maternal Blood Meta-analysis MTHFR C677T 1298CC mutation was 9% in both samples East Asians people are higher risk of URPL with 677TT genotype 2015 Hubacek, J.A et al., Clin Chim Acta 440 p.104-7. Slavonia 464 samples 486 controls aborted samples Tissue PCR-RFLP MTHFR C677T MTHFR A1298C Carriers with dominant allele of these polymorphisms were associated with higher risk of abortion Distinct combination of MTHFR could be associated with higher risk of abortions in Caucasians Continues….. 35% Data such as year, authors, journal name, place of study, sample size, source, methodology used, mutations found, Mutation type were extracted Figure 3. Percentage of frequency of both polymorphisms in RPL Among the positive studies, 35% (9/26) had both C677T and A1298C polymorphisms. C677T polymorphism was the only polymorphism associated with RPL in 50% (13/26) of cases, while 15% (4/26) found A1298C polymorphism associated with RPL. Study subjects 17,780 cases and 22,346 controls were considered as study subjects Analysis

Significant Findings Conclusion References MTHFR polymorphisms were found to be the first common mutation to be involved in RPL ( Ozdemir et al., 2012) MTHFR polymorphisms were significantly associated with RPL ( p= 0.002). MTHFR C677T polymorphism was the most prevalent polymorphism compared to A1298C polymorphism ( p= 0.043). C677T polymorphism has been denoted as a risk factor for RPL because of expand tHcy Accordingly, the C677T polymorphism could be used as a genetic marker for early diagnosis of RPL. Study further suggests, paternal screening is equally important as maternal screening in the early diagnosis of RPL. C677 homozygous genotype was significantly higher in the RPL women in China and East Asia (p=0.003) References Cao, Y., Zhang, Z., Zheng, Z., Yuan, W., Wang, J., Liang, H., Chen, J., Du, J. & Shen, Y. (2014) The association of idiopathic recurrent early pregnancy loss with polymorphisms in folic acid metabolism-related genes, Genes Nutrition, 9, 402-410. Prevalence of MTHFR-C677T polymorphism present in the partners of women suffered from RPL Recurrent Pregnancy Loss (RPL) Ozdemir, O., Yenicesu, G.I., Silan, F., KoKsal, B., Atik, S., Ozen, F., Gol, M. & Cetin, A. (2012) Recurrent Pregnancy Loss and Its Relation to Combined Parental Thrombophilic Gene Mutations, Genetic Testing and Molecular Biomarkers, 16, 279-286. Supplementation of enriched folate diet, 5-MTHF (200 µg per day), folic acid (200 µg per day) will significantly decrease the pregnancy complications Vanilla, S., Kutty, M. & Vegi, P.K. (2015) Evidence of Paternal N5, N10 - (MTHFR) C677T Gene Polymorphism in Couples with Recurrent Spontaneous Abortions (RSAs) in Kolar District- A, Journal of Clinical and Diagnostic Research, 9, BC15-BC18.