Interaction between MTHFR 677C>T, PON1 192Q>R and PON1 55L>M polymorphisms and its effect on non-recurrent spontaneous abortion in Mexican women

Highlights

• MTHFR 677C>T polymorphisms are not associated with non-recurrent spontaneous abortion.

• PON1 55 LM+MM genotypes increased the odds of non-recurrent spontaneous abortion.

• No interactions were found between MTHFR and PON1 polymorphisms in relation to non-recurrent spontaneous abortion.

Abstract

MTHFR is a key enzyme in folate metabolism. Some genetic polymorphisms code for a less efficient enzyme, increasing serum concentrations of homocysteine. This has been associated with inadequate feto-maternal circulation and increased risk of spontaneous abortion. Paroxonase 1 (PON1) is a multifunctional enzyme that can detoxify homocysteine through its homocysteine thiolactonase activity. We evaluate the association between MTHFR 677 C>T polymorphisms and non-recurrent spontaneous abortion and its interaction with PON1 polymorphisms involved in homocysteine metabolism in women living in floricultural areas in Mexico. Sociodemographic, reproductive history, folic acid consumption during pregnancy and environmental exposure data of 264 women who had been pregnant sometime during the 10 years prior to study enrolment were collected. MTHFR 677 C>T, PON1 192Q>R and PON1 55L>M genotypes were determined by PCR amplification. Information on pregnancy outcome and maternal genotypes was obtained for 484 pregnancies: 34 non-recurrent spontaneous abortions (gestational age < 20 weeks) and 450 controls. GEE models were used to evaluate the association between MTHFR polymorphism and non-recurrent spontaneous abortion, and its interaction with PON1 polymorphisms.

After adjusting for potential confounders, no significant association was found between the MTHFR 677 C>T maternal polymorphism and non-recurrent spontaneous abortion (OR _{CT vs CC}= 0.39, 95% CI: 0.14-1.05; OR _{TT vs CC} = 0.63, 95% CI: 1.22–1.80). No interactions with PON1 192Q>R or PON1 55L>M polymorphisms were identified (p for interaction = 0.88 and 0.41, respectively). PON1 55L>M maternal polymorphism was associated with higher risk of spontaneous abortion (OR _{LM/MM vs LL} = 4.14, 95% CI: 1.49–11.54).

Our results do not demonstrate an interaction between the MTHFR 677 C>T and PON1 192Q>R or PON1 55L>M maternal polymorphisms neither an independent association of MTHFR 677 C>T polymorphism with non-recurrent spontaneous abortion, whereas PON1 55LM/MM maternal genotype increase the odds of this event.

Introduction

Spontaneous abortion (SA) is one of the most frequent complications of pregnancy, estimated to occur in 10–12% of clinically recognized pregnancies (Simpson and Carson, 1993). While approximately 50% of SA are associated with genetic abnormalities, the causes of the remaining 50% are largely unknown, although they have been associated with anatomical, endocrine, infectious, genetic, immunological and environmental factors (Cramer and Wise, 2000). Certain inherited or acquired thrombophilic mutations augment the prothrombotic state of pregnancy and can lead to inadequate feto-maternal circulation. This has been associated with complications such as pregnancy loss, fetal death, intrauterine growth restriction and pre-eclampsia (Ford and Schust, 2009; Rodger et al., 2010). One of the most common thrombophilias is homozygosity for the 677 C>T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene (Madjunkova et al., 2012). MTHFR is a key enzyme in folate metabolism, catalyzing the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for nucleotide synthesis, re-methylation of homocysteine (Hcy) to methionine, S-adenosylmethionine synthesis, and the methylation of DNA, proteins, neurotransmitters and phospholipids (Frosst et al., 1995; Yamada et al., 2001). The 677 C>T substitution results in an amino acid change from alanine to valine at codon 225, in the predicted catalytic domain of MTHFR; this change renders the enzyme thermolabile and its activity is reduced by 35% among 677CT carriers and by 50% to 70% among 677TT carriers (Zetterberg et al., 2002). Reduced MTHFR activity increases homocysteine concentrations associated with inflammation of the vascular endothelium (Mascarenhas et al., 2014) and pregnancy loss (Nelen et al., 2000a, Nelen et al., 2000b, Nelen et al., 2000c; D'Uva et al., 2007; Gris et al., 2003). At high concentrations, Hcy is first converted by methionyl-tRNA synthetase to homocysteine thiolactone (HTL), which then reacts with lysine residues in proteins, thereby causing damage in their structure and impairment in their physiological activities (Jakubowski, 2002). HTL appears to be more toxic to human cells than Hcy itself. The extent of HTL synthesis and protein homocysteinylation, which have detrimental effects on human vascular endothelial cells, mainly depends on the levels of Hcy (Dekker and van Geijn, 1996; Jakubowski et al., 2001; Isbilen et al., 2009).

Paraxonase 1 (PON1) is an enzyme linked to high-density proteins that reduce the generation of peroxidized lipids by hydrolyzing low-density lipoproteins (LDL) and preventing their oxidation (Mackness et al., 1993; Aviram et al., 1998; Pasqualini et al., 2005). PON1 is a multifunctional enzyme that can also detoxify organophosphate pesticides (Mackness et al., 1998) and the HTL through its homocysteine thiolactonase (HTase) activity (Jakubowski et al., 2001; Perła-Kaján and Jakubowski, 2012). Two major genetic polymorphisms of human PON have been described, due to glutamine (Q) or arginine (R) at position 192 and methionine (M) or leucine (L) at position 55. Previous studies (Jakubowski et al., 2001; Lacinski et al., 2004) reported that high HTase activity was associated with PON1 192R and PON1 55L alleles, whereas low HTase activity was associated with PON1 192Q and PON1 55M alleles.

Previously we found that, after adjusting for potential confounders, mothers with PON1 192RR genotype have 2.2 higher odds of non-recurrent spontaneous abortion than mothers with PON1 192QR/QQ genotype (95% CI: 0.93–5.17). The odds was close to 4 times higher in mothers with PON1 55MM/LM genotype than in mothers with PON1 55LL genotype (OR = 3.9, 95% CI: 1.38–11.0) (Blanco-Muñoz et al., 2013).

On the other hand, some meta-analyses have shown that Asian women carriers of the MTHFR 677T allele have a higher risk of recurrent pregnancy loss, but such association was not observed in Caucasian women or mixed subgroups although the number of studies included was low (Wu et al., 2012; Cao et al., 2013). However, few studies on the link between MTHFR polymorphisms and non-recurrent pregnancy loss have been conducted, and generally no association has been found (Zetterberg, 2004). Most of such studies have examined the effect on late pregnancy loss (after week 19), which could have a different etiology from that of early loss. A study in Mexico found that women with the 677TT genotype had a higher risk of spontaneous abortion before week 20 of pregnancy than those with the 677CT/CC genotype (OR _{677TT vs. 677CC/CT} = 5.0, 95% CI: 1.2–20.9) (Rodríguez-Guillén et al., 2009). However none of these studies has evaluated the existence of interaction with other polymorphisms involved in the metabolism of homocysteine, as is the case of the PON1 polymorphisms.

Some studies have evaluated the interaction between MTHFR and PON1 polymorphisms on the risk of cardiovascular diseases (Agirbasli et al., 2011; Pandey et al., 2011; García-González et al., 2018), nonetheless we have not found studies that have evaluated this topic in the case of reproductive effects.

According to the National Institute of Statistic and Geography (INEGI, 2017) in 2017, 20,000 fetal deaths were registered in Mexico, of which 7000 were classified as abortions; however the registry includes only fetal deaths that occurred after the 12th week of pregnancy, so SA frequency could be underestimated.

In the present study we estimated the association between MTHFR 677C>T polymorphism and non-recurrent early spontaneous abortion (defined as a pregnancy loss before 20 week) in Mexican women. We also examined whether there is an interaction between this polymorphism and the polymorphisms PON1 192Q>R and 55L>M involved in the metabolism of HTL. These women were participants in a study to evaluate the association between genetic factors and pesticide exposure and adverse reproductive effects.