Letter to the Editor

Role of the C677T Polymorphism at the MTHFRGene on Risk to Nonsyndromic Cleft LipWith/Without Cleft Palate: Results From aCase-Control Study in Brazil

To the Editor:

Nonsyndromic cleft lip with or without palate (CL/P)is one of the most common birth defects with a preva-lence at birth of approximately 1 per 1,000 Caucasiannew-born infants [Gorlin et al., 1990]. The inheritanceof CL/P is complex and many models have been pro-posed to explain the familial cases. Approximately 20genes interacting multiplicatively could be responsiblefor CL/P, including a possible major gene that mightaccount for 10–50% of the cases [Mitchell and Risch,1992; FitzPatrick and Farral, 1993; Christensen andMitchell, 1996]. Several candidate regions have beenproposed to be involved in CL/P in different humanpopulations on the basis of linkage or association re-sults. The regions of interest include tumor growth fac-tor-a (TGF-a) at 2p13, MSX1 at 4p16, markers on6p23, TGF-b3 at 14q24, and BCL3 at 19q13. Theseresults, however, still need to be replicated [Murray,1995; Wyszynski et al., 1997; Scapoli et al., 1997; Li-dral et al., 1998]. Recently, homozygosity of the C677Tpolymorphism in the 5,10-methylenetetrahydrofolatereductase (MTHFR) gene has been found associatedwith CL/P among Argentinean patients [Tolarova etal., 1998]. These findings, however, were not replicatedby Shaw et al. [1998]. With data from the CaliforniaBirth Defects Registry, they did not find any evidencethat C677T homozygous Caucasian, Hispanic, or Blackchildren are at increased risk of disease. The C677Tmutation is associated with elevated plasma homocys-teine levels and lowered plasma folate because of re-duced MTHFR activity. It has been shown that thismutation is a predisposing factor for neural tube de-

fects (NTD) in several populations with increased riskwhen both the mother and child have the TT genotype[Van der Put et al., 1996, 1998].

We analyzed the C677T polymorphism by comparing77 Caucasian CL/P patients (50 males and 27 females)and 59 white mothers of CL/P patients to 113 controls(90 healthy mothers of patients with different neuro-muscular disorders and 23 unrelated children withneuromuscular diseases). No significant difference wasfound between children and mother controls for theC677T polymorphism. Thus, these two samples werepooled to constitute our control group. The controlswere matched by socioeconomic status and ethnic back-ground to the CL/P families. The T allele was similaramong all subgroups and highly prevalent (35%)among our Caucasian controls (Tables I and II). Thisfinding is consistent with previous estimates amongBrazilian healthy Caucasians [Arruda et al., 1998;Franco et al., 1998] and European and North-Americancontrols [Van der Put et al., 1995, 1997]. The TT geno-type was slightly more prevalent in our control samplethan in other published studies, although this differ-ence was not statistically significant [Van der Put etal., 1997; Arruda et al., 1998; Franco et al., 1998]. Aslightly higher proportion of TT and CC homozygoteswere observed among mothers of CL/P patients (17 and51%, respectively) as compared with controls (14 and41%, respectively), but these differences were not sta-tistically significant (Table I). Among CL/P patients,the lack of association between the TT genotype andthe phenotype was further corroborated by the trans-mission disequilibrium test (TDT), which suggestedthat the C677T allele and a putative gene for CL/Pmight be in linkage equilibrium (Table III). The fre-quency of the TT genotype did not differ when maleswere compared with females in both case and controlgroups (data not shown). Therefore, our data does notsupport the notion that there is a reduction in femalefetuses with the TT genotype as recently suggested byRozen et al. [1999]. No departure from Hardy-Weinberg (H-W) equilibrium was observed amongadult controls and CL/P children (X2 4 0.21; P> 0.05).

Contract grant sponsor: FAPESP; Contract grant sponsor:PRONEX; Contract grant sponsor: CNPq; Contract grant spon-sor: Howard Hughes Medical Institute.

*Correspondence to: M. R. Passos-Bueno, Centro de Estudos doGenoma Humano, Departamento de Biologia, Instituto de Bio-ciencias, Universidade de Sao Paulo, Sao Paulo, Brazil.

Received 20 May 1999; Accepted 30 July 1999

American Journal of Medical Genetics 87:197–199 (1999)

© 1999 Wiley-Liss, Inc.

Surprisingly, however, this polymorphic system was inH-W disequilibrium among mothers of CL/P children(X2 4 4.3; P < 0.05) even after we excluded 15 mothersof CL/P children from Southern Brazil (X2 4 4.84; P <0.05).

Therefore, our data do not support the evidence thatthe frequency of TT genotype is increased among CL/Pindividuals, thus confirming the results of Shaw et al.[1998]. However, the observation that this polymorphicsystem is not in H-W equilibrium among mothers ofCL/P patients is very intriguing. The relatively lowerproportion of heterozygotes might be a random varia-tion. Alternatively, this might suggest that homozygos-ity for either the T or C allele at the C677T polymor-phism in females constitutes an important susceptibil-ity factor to CL/P. If confirmed, CT heterozygoteswould have an advantage towards the homozygotes inrelation to this trait. It is relevant to point out thatWeitkamp et al. [1998] recently described a heterozy-gote advantage of the MTHFR gene in patients withNTD and their relatives. Given the findings presentedhere, additional studies on the maternal and genotypiceffects of the C677T/MTHFR polymorphism in CL/Prisk are warranted because important preventive mea-sures can then be adopted. Shaw et al. [1998], based onthe absence of an interaction between maternal vita-min use and infant genotype for the C677T polymor-phism, have already suggested that the reduced riskfor orofacial clefts associated with vitamin intake re-lates to a correction of a maternal metabolic defectrather than that of the fetus.

ACKNOWLEDGMENTS

We thank Dr. Ana Beatriz Perez for valuable sugges-tions, Eloısa de Sa Moreira for reading the manuscript,

and Constância G. Urbani, Antonia M. P. Cerqueira,and Marta Canovas for constant help. Also, we thankall of the patients and their relatives. This work wassupported by grants from the FAPESP, PRONEX, andCNPq. M.R.P.B. is supported in part by an Interna-tional Research Scholars grant from the HowardHughes Medical Institute.

REFERENCES

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Christensen K, Mitchell LE. 1996. Familial recurrence pattern analysis ofnonsyndromic isolated cleft palate: a Danish registry study. Am J HumGenet 58:182–190.

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TABLE I. MTHFR Genotype Distribution Among CL/PPatients, Mothers of CL/P Cases, and Controls

Subjects N

Genotypea

CC CT TT

CL/P patients 77 30 39 8CL/Ps’ mothers 59 30 19 10Controls 113 49 49 15Controls’ mothersb 90 37 40 13

aCL/P patients and controls; x2 4 1.05; P 4 0.6; CL/P mothers and con-trols: x2 4 2.05; P 4 0.36.bSample used for the Hardy-Weinberg test only.

TABLE II. Frequency of the C677T/MTHFR Mutation in CL/PPatients, Mothers of CL/P Cases, and in Controls

Subjects

Allele frequency

TotalC

n (%)T

n (%)

CL/P patients 99 (64) 55 (36)CL/Ps’ mothers 79 (67) 39 (33) 118Controls 147 (65) 79 (35) 226Controls’ mothersa 114 (63) 66 (37) 180

aSample used for the Hardy-Weinberg test only.

TABLE III. TDT Test to MTHFR and CL/P Patients andTheir Parents

Allele Transmitteda Not transmitted

C 9 10T 10 9

aAllelic TDT Pearson x2 4 1.0, P 4 0.8185; genotypic marginal TDT x2 40.1, P 4 0.9881.

198 Gaspar et al.

Van Der Put NMJ, Steegers-Theunissen RPM, Frosst P, Trijbels FJM,Eskes TKAB, Van Den Heuvel LP, Mariman ECM, Den Heyer M, Ro-zen R, Blom HJ. 1995. Mutated 5-10 methylenetetrahydrofolate reduc-tase as a risk factor for spina bifida. Lancet 346:1070–1071.

Van Der Put NMJ, Van Den Heuvel L P, Steegers-Thenissen RPM, TrijbelsFJM, Eskes TKAB, Mariman ECM, Den Heyer M, Blom HJ. 1996.Decreased methylenetetrahydrofolate reductase activity due to theC677T mutation in families with spina bifida offspring. J Mol Med74:691–694.

Weitkamp LR, Tackels DC, Hunter AGW, Holmes LB, Schwartz CE. 1998.Heterozygote advantge of the MTHFR gene in patients with neuraltube defect and their relatives. Lancet 351:1554–1555.

Wyszynski DF, Maestri N, McIntosh I, Smith EA, Lewanda AF, Garcia-Delgado C, Vinageras-Guarneros E, Beaty TH. 1997. Evidence for anassociation between markers on chromosome 19q and nonsyndromiccleft lip with or without cleft palate in two groups of multiplex families.Hum Genet 99:22–26.

D. A. GasparR. C. PavanelloM. ZatzM. R. Passos-Bueno*Centro de Estudos do Genoma HumanoDepartamento de BiologiaInstituto de BiocienciasUniversidade de Sao PauloSao Paulo, Brazil

M. AndreFaculdade de OdontologiaUniversidade de Sao PauloSao Paulo, Brazil

S. StemanDepartamento de Cirurgia Plastica, Faculdade de

MedicinaUniversidade de Sao PauloSao Paulo, Brazil

D. F. WyszynskiLaboratorio de Epidemiologia GeneticaDepartamento de Salud PublicaUniversidad de Buenos AiresBuenos Aires, Argentina

S. R. MatiolliDepartamento de BiologiaInstituto de BiocienciasUniversidade de Sao PauloSao Paulo, Brazil

Letter to the Editor 199