BHMT G742A and MTHFD1 G1958A Polymorphismsand Down Syndrome Risk in the Brazilian Population

Bruna Lancia Zampieri, Joice Matos Biselli, Eny Maria Goloni-Bertollo, and Erika Cristina Pavarino

Background: Mechanisms underlying meiotic nondisjunction are poorly understood. Attempts to elucidate thecauses of Down syndrome (DS) have analyzed the relationship between polymorphism in folate metabolism andDS. Aim: The role of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) G1958A and betaine-homocysteinemethyltransferase (BHMT) G742A polymorphisms in DS risk was investigated. Methods: Blood samples werecollected from a total of 86 DS mothers and from 161 control mothers. The investigation of the MTHFD1 G1958Apolymorphism was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and by real-time PCR for the BHMT G742A polymorphism. Results: The median maternal age of casemothers (30.40; 12.9–46.3 years) was significantly higher ( p < 0.0005) than in the control group (26.60; 15.4–57.9years). The frequency of BHMT variant genotypes was significantly lower in DS mothers compared with controls( p = 0.047). A significant decreased risk for BHMT 742 AA genotype (odds ratio [OR] = 0.30; 95% confidenceinterval [CI]: 0.10–0.93; p = 0.037) was observed. Moreover, when the dominant model was applied (BHMT742GA or 7428AA versus 742GG), there was also a significant decrease in DS risk (OR = 0.58; 95% CI: 0.37–0.98;p = 0.042). MTHFD1 G1958A genotype frequencies were not significantly altered in DS mothers ( p = 0.206).Conclusions: Our study suggests that the polymorphism BHMT G742A may modulate the DS risk in Brazilianmothers.

Introduction

Down syndrome (DS) is the most common genetic dis-order occurring with a prevalence of 1 in 600 live births

( Jones, 2006). It stems from the presence of three copies ofchromosome 21, and it is mainly due to abnormal segregation(maternal nondisjunction in 90% of the cases) (Freeman et al.,2007). Although the chromosomal basis of DS has beenknown for many years, the mechanisms underlying meioticnondisjuntion are poorly understood, and the only well-established risk factor is maternal age (Allen et al., 2009).Attempts to elucidate the causes of DS have analyzed therelationship between common polymorphisms in folate me-tabolism and trisomy 21.

Folate metabolism, in one of its pathways, results in theconversion of methionine into S-adenosylmethionine (SAM),the major methyl donor for DNA methylation reactions. En-zymes of folate metabolism play an important role in main-tenance of genomic stability by regulating DNA biosynthesis,repair, and methylation (Fig. 1). Folate deficiency has beenshown to result in DNA hypomethylation, chromosomebreakage, aneuploidy, and abnormal chromosome segrega-tion (Fenech, 2001; Wang et al., 2004). Several studies havehypothesized that impairments in folate and homocysteine

(Hcy) metabolism due to genetic polymorphisms of metabolicenzymes could predispose an individual to chromosomedamage events and act as a maternal risk factor for DS preg-nancy (Biselli et al., 2008; Meguid et al., 2008; Wang et al., 2008;Pozzi et al., 2009; Brandalize et al., 2010; Marucci et al., 2011;Sadiq et al., 2011). Thus, the present study reports the analy-sis of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1)G1958A and betaine-homocysteine methyltransferase (BHMT)G742A polymorphisms involved in Hcy/folate metabolismand the maternal risk of DS. According to our knowledge, thisis the first study that has investigated the influence of BHMTG742A polymorphism on maternal risk for DS.

Materials and Methods

The study protocol was approved by the Research EthicsCommittee of the Sao Jose do Rio Preto Medical School (CEP-FAMERP), in the State of Sao Paulo, and by the NationalResearch Commission in Brasilia, DF (CONEP). Blood sam-ples were collected from a total of 86 women (case mothers)who had given birth to children with karyotypically confirmedfull trisomy 21. Mothers of DS individuals with translocation ormosaicism were not included from the study. Case motherswere recruited from the Genetics Outpatient Service of

Department of Biologia Molecular, Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP), Unidade de Pesquisa em Genetica eBiologia Molecular (UPGEM), Sao Jose do Rio Preto, SP, Brazil.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 16, Number 6, 2012ª Mary Ann Liebert, Inc.Pp. 628–631DOI: 10.1089/gtmb.2011.0257

628

Hospital de Base (HB) of the Sao Jose do Rio Preto MedicalSchool (FAMERP), Sao Paulo, Brazil. The control group wascomposed of 161 mothers with at least one healthy offspringand no history of miscarriage; they were enrolled at the FA-MERP Campus and at the HB Clinical Analysis Laboratory.Maternal age was calculated considering the age of the motherat the birth of the DS child for case mothers, and the age at thebirth of the last child for the control group. Written informedconsent was obtained from all participants.

Genomic DNA was extracted from blood leukocytes inwhole blood according to Miller et al. (1988) or using theGFX� Genomic Blood DNA Purification Kit (GE Healthcare).The investigation of the polymorphism BHMT G742A wasperformed by allelic discrimination by real-time PCR (Taq-man SNP Genotyping Assay ID C__11646606_20, AppliedBiosystems). The MTHFD1 G1958A polymorphism assay wasperformed by polymerase chain reaction–restriction fragmentlength polymorphism (PCR-RFLP) using Msp I as previouslydescribed (Brody et al., 2002).

The Hardy–Weinberg equilibrium test was determined bythe chi-square test using the BioEstat program. To compare theages of mothers at the birth of the child (maternal age), theMood median test was used. Differences in genotype fre-quencies in the DS and control mothers were compared by thelikelihood ratio test. Logistic regression considering either theco-dominant model (the three genotypes studied separately) orthe dominant (grouping homozygous variant and heterozy-gous genotypes compared with homozygous wild genotype)model for the effect of the polymorphisms was perfomed.

All statistical analyses were carried out using Minitab forWindows program (Release 14), with the level of significanceset at p £ 0.05.

Results

Both the MTHFD1 G1958A and BHMT G742A genotypefrequencies were in Hardy–Weinberg equilibrium ( p = 0.1667

and p = 0.5099, respectively). The median age of case mothers(30.40; 12.9–46.3 years) when giving birth to a child with DSwas significantly higher ( p < 0.0005) than in the control group(26.60; 15.4–57.9 years).

The MTHFD1 G1958A and BHMT G742A genotypes andallele distributions in mothers of trisomy 21 probands andcontrol mothers are shown in Table 1. The distribution ofBHMT genotypes (G742A) was significantly altered inmothers of children with DS compared with controls in bothco-dominant ( p = 0.047) and dominant models ( p = 0.042).

MTHFD1 G1958A genotype frequencies were not signifi-cantly altered in mothers of children with DS in both co-dominant ( p = 0.206) and dominant models ( p = 0.401).

To evaluate the impact of the MTHFD1 G1958A and BHMTG742A variants on DS risk, we calculated the odds ratio (OR)and 95% confidence interval (CI; Table 2). We found a sig-nificantly decreased risk for DS in the presence of BHMT 742AA genotype (OR = 0.30; 95% CI: 0.10–0.93; p = 0.037). More-over, when the dominant model was applied (BHMT 742GAor 7428AA versus 742GG), there was also a nominally sig-nificantly decreased risk for bearing a child with DS in thepresence of BHMT 742 GA or AA genotypes (OR = 0.58; 95%CI: 0.37–0.98; p = 0.042). No association between MTHFD1G1958A polymorphism and maternal risk for DS was ob-served.

Discussion

DS is a complex genetic disorder that stems from the failureof normal segregation during meiosis. Besides maternal age,impairments of folate metabolism have been implicated asone of the causes of chromosome nondisjunction (Biselli et al.,2008; Meguid et al., 2008; Wang et al., 2008; Pozzi et al., 2009;Brandalize et al., 2010; Marucci et al., 2011; Sadiq et al., 2011).Accordingly, the present study reports the analysis oftwo polymorphic MTHFD1 and BHMT genes of folatemetabolism.

FIG. 1. Folate metabolism. BHMT,betaine-homocysteine methyl-transferase; CBS, cystathionine-beta-synthase; CH3, methyl; 5,10-MTHF,5,10-methylenetetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate;dATP, deoxyadenosine 5¢-tripho-sphate; dGTP, deoxyguanosine 5¢-triphosphate; dTTP, deoxy-thymidine 5¢-triphosphate; Hcy,homocysteine; MMA, methylma-lonic acid; MTHFD1, methylenetet-rahydrofolate dehydrogenase 1;MTHFR, methylenetrahydrofolatereductase; MTR, methionine syn-thase; MTRR, 5- methionine syn-thase reductase; RFC1, reducedfolate carrier 1; SAH, S-adenosyl-homocysteine; SAM, S-adeno-sylmethionine; TCN2, transcobala-min II; THF, tetrahydrofolate.

BHMT AND MTHFD1 GENE POLYMORPHISMS AND DOWN SYNDROME RISK 629

The MTHFD1 gene encodes a trifunctional enzyme thatcatalyzes the sequential interconversion of tetrahydrofolateinto the corresponding 10-formyl, 5,10-methenyl, and 5,10-methylene derivatives responsible for de novo purine andpyrimidine biosynthesis and, thus, the biosynthesis of DNA(Hum et al., 1988). This gene presents a functional polymor-phism, a guanine to adenine substitution at position 1958(1958 G/A), that has been shown to reduce the activity andstability of the variant enzyme (Christensen et al., 2009). Therewere only two studies until the present on the influence of thispolymorphism on maternal risk for DS. Scala et al. (2006)

showed an association of the MTHFD1 1958 AA genotypewith DS risk only when combined with reduced folate carrier1 (RFC1) 80 GG genotype. More recently, Neagos et al. (2010)failed to find any association between the MTHFD1 G1958Apolymorphism and the risk of having a DS child, a findingsimilar to ours. Thus, given the poor published literatureabout this polymorphism on DS risk, further investigation isnecessary to clarify the role of MTHFD1 G1958A on chro-mosome 21 nondisjunction.

With regard to the BHMT gene, this is the first group thatevaluates the role of the BHMT G742A polymorphism on therisk of bearing a DS child. This polymorphism is found inexon 6 of the BHMT gene and results in a substitution ofglutamine to arginine at nucleotide position 742. Since vari-ants in folate and Hcy metabolism could confer risk for DSand BHMT possibly plays a critical role in Hcy homeostasis,studies investigating the role of BHMT polymorphism in DSbecome very important. Once Hcy is formed, it can either gotrough transulfuration, which is catalyzed by the enzymecystathionine ß-synthase, requiring vitamin B6 and formingcystathionine, or it can be remethylated into methionine,which is catalyzed by the vitamin B12-dependent enzymemethionine synthase. This last reaction is important for thesynthesis of SAM, the major intracellular methyl donor forDNA, protein, and lipid methylation. When methioninesynthesis is compromised by dietary or genetic influences,BHMT plays a crucial role in catalyzing the reaction.

Weisberg et al. (2003), in order to analyze the effect of thispolymorphism, evaluated the enzyme activity; a substantialchange in its enzymatic activity was not identified, although itis possible that mild and variant enzymes have different sta-bilities. More recently, Li et al., (2008) observed that the poly-morphism produces two distinct alloenzymes, which exhibitsignificant differences in Km values for Hcy and betaine, whichis lower for the variant alloenzyme compared with the wildtype. The low Km of the alloenzyme can be responsible for anincreased efficiency of Hcy remethylation using betaine as amethyl group donor (Ueland, 2011). Our analyses providedevidence that the BHMT G742A variant could influence the riskof having a DS child, and suggested that the BHMT 742 GA orAA genotypes are associated with a decreased risk of DS. Thedecreased effect of the BHMT 742A allele on DS risk could beexpected, once maternal AA genotype was associated withprotection against neural tube defects (NTD) in offspring(Morin et al., 2003; Mostowska et al., 2010), and NTD and DSseem to be influenced by the same genetic factors involved infolate metabolism (Barkai et al., 2003).

In conclusion, our results among the Brazilian populationdid not support an association between MTHFD1 G1958Apolymorphism and risk for DS, but significant evidence wasobtained for association between decreased maternal DS riskand BHMT 742 GA or AA geneotypes. Replication studies toconfirm our results are necessary.

Acknowledgments

The authors are grateful to the mothers who participated inthis study, to the Ding-Down workgroup (multidisciplinarygroup of health professionals—FAMERP), and to the FA-MERP/FUNFARME for their collaboration in this work. Thisstudy was supported by the FAPESP, CAPES, CNPq, and BAP/FAMERP.

Table 1. Genotype and Allele Frequencies

of MTHFD1 G1958A and BHMT G742A Polymorphisms

in Mothers of Down Syndrome Children (Cases)

and Controls

GenotypeCase mothers

n (%)Control

mothers n (%) p-valuea

MTHFD1co-dominant model

GG 29 (33.72) 63 (39.13) 0.206GA 47 (54.65) 70 (43.48)AA 10 (11.63) 28 (17.39)

dominant model: dominant modelGG 29 (33.72) 63 (39.13) 0.401GA or AA 57 (66.28) 98 (60.87)

Allele frequencyG 0.611 0.609A 0.389 0.391

BHMTdominant model: co-dominant model

GG 48 (55.81) 68 (42.24) 0.047b

GA 34 (39.53) 74 (45.96)AA 4 (4.65) 19 (11.80)

dominant model: dominant modelGG 48 (55.81) 68 (42.24) 0.042b

GA or AA 38 (44.18) 93 (57.76)

Allele frequencyG 0.756 0.652A 0.244 0.348

aX2 test.bThe bold values indicate p £ 0.05.

Table 2. Maternal MTHFD1 G1958A and BHMT G742APolymorphisms and the Risk for Down Syndrome

Genotype OR 95% CI p-value

MTHFD1GG 1 ReferenceGA 1.46 0.82–2.59 0.198AA 0.78 0.33–1.81 0.556GA or AA 1.26 0.73–2.19 0.403

BHMTGG 1 ReferenceGA 0.65 0.38–1.13 0.125AA 0.30 0.10–0.93 0.037a

GA or AA 0.58 0.34–0.98 0.042a

aThe bold values indicate p £ 0.05.OR, odds ratio; CI, confidence interval.

630 ZAMPIERI ET AL.

Disclosure Statement

No competing financial interests exist.

References

Allen EG, Freeman SB, Druschel C, et al. (2009) Maternal age andrisk for trisomy 21 assessed by the origin of chromosomenondisjunction: a report from the Atlanta and National DownSyndrome Projects. Hum Genet 125:41–52.

Barkai G, Arbuzova S, Berkenstadt M, et al. (2003) Frequency ofDown’s syndrome and neural tube defects in the same family.Lancet 361:1331–1335.

Biselli JM, Goloni-Bertollo EM, Zampieri BL, et al. (2008) Geneticpolymorphisms involved in folate metabolism and elevatedconcentrations of plasma homocysteine: maternal risk factorsfor Down syndrome in Brazil. Genet Mol Res 7:33–42.

Brandalize AP, Bandinelli E, Dos Santos PA, et al. (2010) Ma-ternal gene polymorphisms involved in folate metabolism asrisk factors for Down syndrome offspring in Southern Brazil.Dis Markers 29:95–101.

Brody LC, Conley M, Cox C, et al. (2002) A polymorphism,R653Q, in the trifunctional enzyme methylenetetrahydro-folate dehydrogenasemethenyltetrahydrofolate cyclohydrolase-formyl-tetrahydrofolate synthetase is a maternal genetic riskfactor for neural tube defects Report of the Birth DefectsResearch Group. Am J Hum Genet 71:1207–1215.

Christensen KE, Rohlicek CV, Andelfinger GU, et al. (2009) TheMTHFD1 pArg653Gln variant alters enzyme function and in-creases risk for congenital heart defects. Hum Mutat 30:212–220.

Fenech M (2001) The role of folic acid and vitamin B12 in ge-nomic stability of human cells. Mutat Res 475:57–67.

Hum DW, Bell AW, Rozen R, et al. (1988) Primary structure of ahuman trifunctional enzyme. Isolation of a cDNA encodingmethylenetetrahydrofolate dehydrogenase-methenyltetrahy-drofolate cyclohydrolase-formyltetrahydrofolate synthetase.J Biol Chem 263:15946–15950.

Jones, KL (2006) Smith’s Recognizable Patterns of Human Mal-formation, 6th edition. Elsevier Saunders, Philadelphia, p 7.

Li F, Feng Q, Lee C, et al. (2008) Human betaine-homocysteinemethyltransferase (BHMT) and BHMT2: common gene se-quence variation and functional characterization. Mol GenetMetab 94:326–335.

Marucci GH, Zampieri BL, Biselli JM, et al. (2011) PolymorphismC1420T of serine hydroxymethyltransferase gene on maternalrisk for Down syndrome. Mol Biol Rep [Epub ahead of print];DOI: 10.1007/s11033-011-1008-7.

Meguid NA, Dardir AA, Khass M, et al. (2008) MTHFR geneticpolymorphism as a risk factor in Egyptian mothers withDown syndrome children. Dis Markers 24:19–26.

Miller SA, Dykes DD, Polesky HF (1988) A simple salting outprocedure for extracting DNA from human nucleated cells.Nucleic Acids Res 16:1215.

Morin I, Platt R, Weisberg I, et al. (2003) Common variant inbetaine-homocysteine methyltransferase (BHMT) and risk forspina bifida. Am J Med Genet 119A:172–176.

Mostowska A, Hozyasz KK, Wojcicki P, et al. (2010) Associationsof folate and choline metabolism gene polymorphisms withorofacial clefts. J Med Genet 47:809–815.

Neagos D, Cretu R, Tutulan-Cunita A, et al. (2010) Methylene-tetrahydrofolate dehydrogenase (MTHFD) enzyme polymor-phism as a maternal risk factor for trisomy 21: a clinical study.J Med Life 3:454–457.

Pozzi E, Vergani P, Dalpra L, et al. (2009) Maternal polymor-phisms for methyltetrahydrofolate reductase and methioninesynthetase reductase and risk of children with Down syn-drome. Am J Obstet Gynecol 200:636e1–636e6.

Sadiq MF, Al-Refai EA, Al-Nasser A, et al. (2011) Methylenete-trahydrofolate reductase polymorphisms C677T and A1298Cas maternal risk factors for Down syndrome in Jordan. GenetTest Mol Biomarkers 15:51–57.

Scala I, Granese B, Sellitto M, et al. (2006) Analysis of sevenmaternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring.Genet Med 8:409–416.

Ueland PM (2011) Choline and betaine in health and disease.J Inherit Metab Dis 34:3–15.

Wang SS, Qiao FY, Feng L, et al. (2008) Polymorphisms in genesinvolved in folate metabolism as maternal risk factors forDown syndrome in China. J Zhejiang Univ Sci B 9:93–99.

Wang X, Thomas P, Xue J, et al. (2004) Folate deficiency inducesaneuploidy in human lymphocytes in vitro-evidence usingcytokinesis-blocked cells and probes specific for chromosomes17 and 21. Mutat Res 551:167–180.

Weisberg IS, Park E, Ballman KV, et al. (2003) Investigations of acommon genetic variant in betaine-homocysteine methyl-transferase (BHMT) in coronary artery disease. Atherosclerosis167:205–214.

Address correspondence to:Erika Cristina Pavarino, Ph.D.

Department of Biologia MolecularFaculdade de Medicina de Sao Jose do Rio Preto (FAMERP)

Unidade de Pesquisa em Genetica e Biologia Molecular (UPGEM)Av. Brigadeiro Faria Lima, n.� 416-Bloco U-6

Sao Jose do Rio Preto, SP, CEP: 15.090-000Brazil

E-mail: erika@famerp.br

BHMT AND MTHFD1 GENE POLYMORPHISMS AND DOWN SYNDROME RISK 631