Oral Diseases (2014) doi:10.1111/odi.
12292
ORIGINAL ARTICLE
Genetic and non-genetic factors that increase the risk of
non-syndromic cleft lip and/or palate development
JF Bezerra1, GHM Oliveira1, CD Soares1, ML Cardoso1, MAG Ururahy1, FPF Neto1, LG Lima-Neto4,
AD Luchessi1, VN Silbiger1, CM Fajardo2, SR de Oliveira3, M das G Almeida1, RDC Hirata2, AA de
Rezende1, MH Hirata2
1
Department of Clinical and Toxicological Analysis, Federal University of Rio Grande do Norte, Natal, RN; 2Department of Clinical
and Toxicological Analysis, School of Pharmaceutical Sciences, University of S~
Cleft lip and Palate, Pediatric Hospital Professor Heriberto Ferreira Bezerra, Federal University of Rio Grande do Norte, Natal, RN;
4
Universidade CEUMA, S~ao Luis, MA, Brazil
OBJECTIVES: We investigated the relationship between
non-syndromic cleft lip/palate (NSCLP) and polymor-
phisms in methylenetetrahydrofolate reductase (MTHFR),
methionine synthase (MTR), methionine synthase reduc-
tase (MTRR), and RFC1, as well as the corresponding
interactions with environmental factors.
SUBJECTS AND METHODS: One hundred and forty
NSCLP patients and their mothers, as well as 175 control
individuals and their mothers, were recruited. Informa-
tion regarding smoking and alcohol consumption was
recorded. Blood samples were obtained in order to mea-
sure serum folate and cobalamin, as well as, plasma total
homocysteine concentrations and to extract DNA. Poly-
morphisms in MTHFR(677C>T and 1298A>C), MTR
(2756A>G), MTR(66A>G), and RFC1(80A>G) were ana-
lyzed by PCR–restriction fragment length polymorphism.
RESULTS: Among the patients, 59.5% had cleft lip and
palate, 22.0% had cleft palate, and 18.5% had cleft lip
only. Maternal alcohol consumption and reduced folic
acid concentrations in both children and mothers
(P < 0.001 and P = 0.003, respectively) were risk factors
for NSCLP. Patients and their mothers carrying the
MTHFR 667T allele showed lower serum folate than CC
(P = 0.011 and P = 0.030, respectively). Mothers who car-
ried the MTHFR 1298C allele exhibited increased risk of
having a child with NSCLP, after adjusting for alcohol
consumption (OR: 1.75, 95% CI: 1.03–2.99, P = 0.038).
CONCLUSIONS: Reduced folic acid levels, alcohol con-
sumption, and the MTHFR 677T and 1298C alleles may
have contributed to NSCLP development in this sample
population from Rio Grande do Norte.
Correspondence: Adriana Augusto de Rezende, Department of Clinical
and Toxicological Analysis, University of Rio Grande do Norte, Av.
General Gustavo de Farias, S/N, Petropolis, 59012-570 Natal, RN, Brazil.
Tel: +55 84 3342 9807, Fax: +55 84 3342 9733, E-mail: adrirezende@
yahoo.com
Received 20 September 2013; revised 22 July 2014; accepted 10 August
2014
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
All rights reserved
www.wiley.com
ao Paulo, SP; 3Program for Children with
ao Paulo, S~
Oral Diseases (2014) doi: 10.1111/odi.12292
Keywords: cleft lip and/or palate; gene polymorphisms; folic
acid; alcohol consumption
Introduction
Non-syndromic cleft lip and/or palate (NSCLP) represents
the most common birth defect in humans (Dixon et al,
2011; Kim et al, 2013). The birth prevalence for NSCLP
is high worldwide, with an estimated average of 1:1000
(Bhaskar et al, 2011; Kim et al, 2013). The birth preva-
lence of NSCLP in Brazil was estimated to be 0.19 in
1000 liveborns during the period from 1975 to 1994 and
0.36 in 1000 liveborns from 1998 to 2002 (Rodrigues
et al, 2009; Wettergren et al, 2010). A previous study
reported a prevalence of 0.49 per 1000 liveborns between
2000 and 2005 in the state of Rio Grande do Norte (Figu-
eir^edo et al, 2011).
Oral clefts are multifactorial birth defects attributed to
both environmental and genetic factors (Jianyan et al,
2010; Dixon et al, 2011). It has been suggested that
NSCLP is related to maternal exposure to environmental
risk factors, including medications, alcohol consumption,
cigarette smoking, and folate and vitamin deficiencies,
during the first trimester of pregnancy (Jianyan et al,
2010; Wehby and Murray, 2010).
Folate plays a major role in one-carbon metabolism in
the synthesis of nucleotides and amino acids and in DNA
methylation, which is essential for chromatin dynamics
and consequent gene expression (de Arruda et al, 2013).
Therefore, folic acid supplementation has been proposed
for the prevention of neural tube defects. The importance
of folic acid supplementation was demonstrated in a Nor-
wegian study that described a decrease in the risk of
NSCLP from 36% to 75% in mothers who had received
folate supplementation (Wilcox et al, 2007).
 Factors involved in NSCLP development
JF Bezerra et al
2
Moreover, it has been shown that alcohol consumption
can inhibit retinoic acid production, thereby increasing
susceptibility to NSCLP (DeRoo et al, 2008). The palate
and the lip are formed from cranial neural crest cells,
which require retinoic acid for normal development and
function. Alcohol intake affects folate homeostasis by
reducing folate absorption, thereby increasing folate
excretion and causing inhibition of enzymes that are
involved in folate metabolism and are essential for
embryogenesis and early fetal development, such as
methionine synthase (MTR) and methylenetetrahydrofo-
late reductase (MTHFR) (Platek et al, 2009; Boyles et al,
2010; Kim et al, 2013).
Methylenetetrahydrofolate reductase plays a central role
in folate metabolism by irreversibly converting 5,10-
methyl-tetrahydrofolate (THF) to 5-methylenetetrahydrofo-
late – the primary circulating form of folate. This product
of folate metabolism provides the methyl groups required
for methionine synthesis, which in turn are involved in the
synthesis of S-adenosylmethionine (SAM) – the primary
methyl group donor in one-carbon metabolism (de Arruda
et al, 2013).
Another important enzyme in folate metabolism is
MTR, which catalyzes the methylation of homocysteine
to methionine with simultaneous conversion of 5-methyl-
enetetrahydrofolate to THF. THF is necessary for
the synthesis of SAM and is required for methylation
reactions and nucleotide synthesis (Platek et al,
2009). In addition, the remethylation of homocysteine
requires methionine synthase reductase (MTRR) to main-
tain the levels of activated vitamin B12 needed for the
MTR-catalyzed remethylation reaction of homocysteine.
Consequently, alterations in MTR and MTRR enzymes
result in decrease in SAM formation with an accompa-
nying increase in the homocysteine level (Ho et al,
2013).
Furthermore, it has been suggested that polymorphisms
in genes that encode the enzymes of the folate pathway
may increase the susceptibility to orofacial clefts (Han
et al, 2011). Two common polymorphisms in
the MTHFR gene – 677C>T (rs1801133) and 1298A>C
(rs1801131) – generate a thermolabile version of the
enzyme (Dixon et al, 2011; Han et al, 2011; Kim et al,
2013). Additionally, the polymorphism 2756A>G
(rs1801394) in MTR could lead to a reduction in enzyme
activity, associated with decreased homocysteine concen-
trations. Likewise, the polymorphism 66A>G (rs1805087)
in the MTRR gene leads to a fourfold reduction in the
activity of the codified protein, in comparison with the
wild type (Wettergren et al, 2010). The polymorphism
80A>G (rs1051266) in the reduced folate carrier 1
(RFC1), which is responsible for the intracellular uptake
of 5-methyltetrahydrofolate, results in decreased intracellu-
lar availability of folate (Koppen et al, 2010).
In this study, we investigated the association between
NSCLP and variants of MTHFR, MTR, MTRR, and
RFC1, and their corresponding interactions with environ-
mental factors in a case–control study involving individu-
als from the population of Rio Grande do Norte (RN),
Brazil.
Oral Diseases
Methods
Subjects
Samples in this study consisted of 630 individuals distrib-
uted across four groups, including 140 NSCLP patients
and their mothers and 175 control individuals and their
mothers. NSCLP patients of both genders who were
between 6 and 21 years of age were recruited at the Pedi-
atric Hospital Professor Heriberto Bezerra (HOSPED) and
the Pediatric Hospital Varela Santiago, Natal, RN, Brazil.
The control group was recruited from public schools in
Natal and consisted of children who were aged 2 years
and above and did not have a family history of clefts.
Non-syndromic cleft lip/palate patients were classified
according to the Fogh-Andersen (1942) classification as
cleft lip and palate (CLP), cleft palate (CP), and cleft lip
(CL). Patients were evaluated by a geneticist, and those
with characteristics of any syndrome were excluded. Sub-
jects with creatinine and ALT and AST values out of the
reference range were excluded from all groups. A reported
family history of clefts was also set as an exclusion crite-
rion for the control group.
A qualitative questionnaire (included as Supporting
Information) was used to interview the mothers or the
legal guardians of the subjects. The questionnaire covered
retrospective information regarding the habits and behav-
iors of smoking, the use and intake of alcoholic beverages
in first 3 months of pregnancy, and whether there was a
family history of oral clefts. Diet information was not
requested. This study was approved by the Ethics Com-
mittee on Human Research of the Federal University of
Rio Grande do Norte under the number n°136/08.
Informed consent was obtained from all adult subjects and
the parents or legal guardians of underage patients.
Blood collection and biochemical measurements
Fasting peripheral blood samples were collected in tubes
with EDTA for total plasma homocysteine (tHcy) mea-
surement, hematological analyses, and DNA extraction; in
addition, a tube without anti-coagulant was collected to
determine serum folate, cobalamin and creatinine concen-
trations, and AST and ALT activities.
Serum folate, cobalamin, and plasma total homocysteine
(tHcy) concentrations were determined by a chemilumi-
nescent method (Siemens Healthcare Diagnostics Inc,
Deerfield, IL, USA) with the IMMULITE 1000 Immuno-
assay System (Siemens Healthcare Diagnostics Incâ) for
the first 50 subjects included in each group.
The creatinine concentration and ALT and AST activi-
ties were measured by routine laboratory methods (Biosys-
tems Reagents & Instruments, Barcelona, Catalu~na, Spain)
using the RA 50 spectrophotometer (Bayer Diagnostics,
Dublin, Leinster, Ireland).
Hematological parameters (erythrocyte count, hemato-
crit, hemoglobin level, mean corpuscular volume, corpus-
cular hemoglobin concentration, mean corpuscular
hemoglobin concentration, and anisocytosis index) were
analyzed to check for possible cases of anemia associated
with folate deficiency, using automated ABX Micros 60
equipment (ABX Diagnostics, Kyoto, Japan).

Genetic analysis
Genomic DNA was isolated from peripheral blood leuko-
cytes using the QIAamp DNA Blood Mini Kit (Qiagen,
Valencia, CA, USA). Genotyping was carried out by poly-
merase chain reaction (PCR) and restriction fragment
length polymorphism (RFLP) analyses.
The presence of the rs1801133 (677C>T) and
rs1801131 (1298A>C) polymorphisms in MTHFR was
determined using the method of Frosst et al (1995) and
Van der Put et al (1998), respectively. The rs1801394
(2756 A>G) polymorphism in MTR and rs1805087
(66A>G) polymorphism in MTRR were detected using the
method of Leclerc et al (1996) and Wilson et al (1999),
respectively. The RFC1 (rs1051266) 80A>G polymor-
phism was detected using the protocol of O’leary et al
(2006).
PCR was carried out in a thermal cycler (MyCyclerTM
Thermal Cycler; Bio-Rad Laboratories Inc., Foster City,
CA, USA). Amplified products were detected on 1.0%
agarose gels stained with GelRedâ (Biotium, Hayward,
CA, USA) and photo-documented using the Gel Logic
100 system (Kodak Carestream Health, Rochester, NY,
USA).
Restriction fragments were separated by electrophoresis
in agarose gels stained with GelRedâ (Biotium) and
photo-documented by the Gel Logic 100 system.
As quality control in the genotyping protocol, 10% of
randomly selected samples were re-genotyped, while sam-
ples with the three specified genotypes were included in
PCR and restriction reactions.
Statistical analysis
Hardy–Weinberg equilibrium was evaluated by the chi-
square test. Categorical variables were compared by the
chi-square and Fisher’s exact tests. Normal distribution of
continuous variables was evaluated using the Kolmogo-
rov–Smirnov test and further compared by the t-test.
Multivariate logistic regression analysis was used to
evaluate the genetic and non-genetic risk factors associated
with NSCLP using the following features as covariates:
smoking, alcohol consumption, and family history of
clefts.
Statistical tests were performed using the Graph Pad
Prism 5.0 software (GraphPad Software Inc., La Jolla,
CA, USA) and the R statistical suite with the SNPassoc
Table 1 Clinical data of the studied groups
Children
Variables Control (175) NSCLP (140) OR (95% CI) P-value
Age, years 11.7  3.8 7.9  7.4 1.34 (0.95–3.45) 0.104
Gender, male 49.2% (86) 62.0% (87) 1.69 (1.08–2.65) 0.072
Smoking – – – –
Alcohol – – – –
consumption
Family history 0.0% (0) 35.5% (50) – –
of NSCLP
NSCLP, non-syndromic cleft lip and palate. *represent significant result.
Categorical variables were compared by chi-square test.
Factors involved in NSCLP development
JF Bezerra et al
3
package (Gonzalez et al, 2007). P-values lower than 0.05
were considered statistically significant.
Results
Sample characterization
The cohort of 140 NSCLP patients was grouped according
to the Fogh-Andersen classification: 59.5% (81) had CLP
[71.6% (58) were male and 28.4% (23) were female],
22.0% (30) had CP [40% (12) were male and 60% (18)
were female], and 18.5% (25) had CL [68.0% (17) were
male and 32.0% (08) were female]. Male gender preva-
lence was 62.0%. No differences were observed between
age and gender distribution among the groups, indicating
relative homogeneity and allowing for better downstream
comparative analysis. Alcohol consumption during preg-
nancy was more common in NSCLP mothers than in con-
trols (OR: 3.64, 95% CI: 1.6–8.3, P < 0.001). Thirty-five
percent of cleft patients reported positive family histories
of oral clefts (Table 1).
Folic acid concentrations were lower in NSCLP cases,
both in the children and the mothers (P < 0.001 and
P = 0.003, respectively), than in the controls (Figure 1a).
Low serum levels in children and mothers were associated
with increased risks of NSCLP (children: OR: 2.56; 95%
CI: 1.09–5.89; P > 0.001; mothers: OR: 2.18; 95% CI:
1.12–5.67; P = 0.003).
Cobalamin and tHcy levels were similar between
NSCLP cases and controls, in both children and mothers
(Figure 1b,c). No differences in hemogram parameters
were found between the groups.
Genetic analysis
The distribution of genotypes in the NSCLP patients and
controls (children and mothers) did not deviate from that
expected under the Hardy–Weinberg equilibrium. The fre-
quencies of MTHFR, MTR, MTRR, and RFC1 genotypes
were similar between NSCLP and control groups
(P > 0.05; Table 2).
We further evaluated the relationship between gene
polymorphisms and vitamin and tHcy levels in NSCLP
children and mothers. Carriers of MTHFR 677 CT and TT
genotypes (T allele) had lower serum folate concentrations
than the CC genotype in the NSCLP children group
(P = 0.011; Table 3) and their mothers (P = 0.030;
Mothers
Control (175) NSCLP (140) OR (95% CI) P-value
37.2  7.4 29.9  4.9 1.89 (1.01–4.75) 0.198
0.0% (0) 0.0% (0) – –
13.1% (23) 15.0% (20) 0.92 (0.45–1.89) 0.816
4.0% (07) 15.0% (21) 3.64 (1.6–8.3) <0.001*
– – – –
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 Factors involved in NSCLP development
JF Bezerra et al

4
(a) (b) (c)

Figure 1 Serum concentrations of folate (a), cobalamin (b), and plasma total homocysteine (c) for the children and the mothers: serum folate – reference
values: 7–24 ng ml 1, serum cobalamin – reference values: 210–700 pg ml 1, (c) plasma total homocysteine – reference values: 3.4–17 lmol l 1. *Sig-
nificant difference between control and non-syndromic cleft lip/palate. (children: OR: 2.56, 95% CI: 1.09–5.89, P < 0.001 and mothers: OR: 2.18, 95%
CI: 1.12–5.67, P = 0.003)

Table 2 Frequencies of polymorphisms in the studied groups

Genotypes

Polymorphism Groups CC CT TT P-value

MTHFR 677C>T NSCLP children 52.8% (74) 38.6% (54) 8.6% (12) 0.515
rs1801133 Control children 48.6% (85) 40.0% (70) 11.4% (20)
NSCLP mothers 61.4% (86) 31.4% (44) 7.2% (10) 0.189
Control mothers 52.0% (91) 36.5% (64) 11.5% (20)
AA AC CC
MTHFR 1298A>C NSCLP children 52.9% (72) 34.3% (48) 12.9% (18) 0.120
rs1801131 Control children 58.7% (91) 23.9% (37) 17.4% (27)
NSCLP mothers 58.6% (82) 37.8% (53) 3.6% (05) 0.752
Control mothers 61.1% (107) 36.6% (64) 2.3% (04)
AA AG GG
MTR 2756A>G NSCLP children 70.7% (99) 25.7% (36) 3.6% (05) 0.460
rs1805087 Control children 68.6% (120) 29.7% (52) 1.7% (03)
NSCLP mothers 63.6% (89) 35.0% (49) 1.4% (02) 0.975
Control mothers 62.8% (110) 35.4% (62) 1.8% (03)
AA AG GG
MTRR 66A>G NSCLP children 70.0% (98) 28.4% (37) 1.6% (05) 0.219
rs1801394 Control children 64.0% (112) 34.3% (60) 1.7% (03)
NSCLP mothers 68.6% (96) 30.0% (42) 1.4% (02) 0.181
Control mothers 58.8% (103) 38.2% (67) 3.0% (05)
AA AG GG
RFC1 80A>G NSCLP children 58.6% (82) 34.3% (48) 7.1% (10) 0.551
rs1051266 Control children 52.6% (92) 40.0% (70) 7.4% (13)
NSCLP mothers 69.3% (97) 27.8% (39) 2.9% (04) 0.542
Control mothers 70.3% (123) 28.6% (50) 1.1% (02)

MTHFR, methylenetetrahydrofolate reductase, MTR, methionine synthase; MTRR, methionine synthase reductase; RFC1, reduced folate carrier; NSCLP,
non-syndromic cleft lip/palate.
Number of individuals in parenthesis. Genotypes were compared by chi-square test.

Table 4). In the control groups, polymorphisms were not

associated with differences in the folate, cobalamin, and
tHcy concentrations (data not shown).
Multivariate regression analysis was carried out to evalu-
ate the relationship between polymorphisms and NSCLP in
the mothers’ group, using alcohol consumption as the co-
variate. Mothers carrying the MTHFR 1298C allele (AC
and CC genotypes) exhibited higher risks of having a child
with NSCLP than mothers with the AA genotype (OR:
1.75, 95% CI: 1.03–2.99, P = 0.038; Table 5). Moreover,
smoking and family histories of clefts were also used as co-
variates in multivariate regression analysis of all polymor-
phisms; however, the results were not statistically
significant.
Discussion
Non-syndromic cleft lip/palate presents a complex etiol-
ogy, and analysis of both environmental and genetic fac-
tors is important for understanding the normal
development of the facial structures (Jianyan et al, 2010;
Scapoli et al, 2010; Dixon et al, 2011).
Male gender was one of the first risk factors to be docu-
mented for NSCLP (Blanton et al, 2011). Our results also
indicated that the male gender was more prevalent among
CLP and CL patients, with nearly twice as many males
affected compared to females, corroborating the findings
of previous studies conducted in Brazil and in Italy (Car-
inci et al, 2005; Coutinho et al, 2009). Moreover, in this

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 Factors involved in NSCLP development
JF Bezerra et al

5
Table 3 Relationship of gene polymorphisms with folate, cobalamin, and homocysteine concentrations of NSCLP children

Polymorphisms Genotypes Folate, ng ml 1

Cobalamin, pg ml 1
Homocysteine, lmol l 1

MTHFR 677C>T CC (28) 16.9  2.9 609.8  316 5.5  2.6

rs1801133 CT+TT (22) 13.5  3.2 578.6  332.2 6.2  2.8
P-value 0.011a 0.803 0.320
MTHFR 1298A>C AA (30) 16.4  2.9 528.1  275.2 5.8  2.0
rs1801131 AC+CC (20) 14.2  3.7 684.6  349.6 5.7  3.3
P-value 0.073 0.189 0.970
MTR 2756A>G AA (29) 15.4  2.8 568.3  323.5 6.1  2.9
rs1801394 AG+GG (21) 14.9  4.1 627.7  305.7 5.1  2.3
P-value 0.427 0.623 0.242
MTRR 80A>G AA (31) 14.8  3.4 541.7  316.3 5.7  2.6
rs1805087 AG+GG (19) 16.4  3.3 665.5  304.4 6.0  2.9
P-value 0.221 0.301 0.671
RFC1 80A>G AA (29) 15.4  3.8 604.2  328.3 5.6  2.5
rs1051266 AG+GG (21) 15.3  2.7 576.9  301.4 6.0  2.9
P-value 0.956 0.821 0.555

MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; NSCLP, non-syndromic cleft lip/pal-
ate; RFC1, reduced folate carrier.
Values are shown as mean  s.d. and compared by t-test. Number of patients in parentheses.
a
Significant result.

Table 4 Relationship of gene polymorphisms with folate, cobalamin, and homocysteine concentrations in mothers of NSCLP patients

Polymorphisms Genotypes Folate, ng ml 1

Cobalamin, pg ml 1
Homocysteine, lmol l 1

MTHFR 677C>T CC (29) 15.0  1.98 432.1  222.1 6.2  2.73

rs1801133 CT+TT (21) 11.1  2.67 376.0  127.1 7.4  3.19
P-value 0.030a 0.447 0.180
MTHFR 1298A>C AA (40) 15.2  1.82 406.5  181.4 7.2  3.10
rs1801131 AC+CC (10) 12.7  2.61 417.4  231.2 5.6  2.50
P-value 0.193 0.909 0.063
MTR 2756A>G AA (32) 14.5  2.06 430.7  183.6 6.5  2.98
rs1801394 AG+GG (18) 13.1  3.31 368.3  194.4 7.1  2.93
P-value 0.179 0.406 0.460
MTRR 80A>G AA (34) 14.3  2.55 396.1  206.3 6.7  2.94
rs1805087 AG+GG (16) 13.7  2.81 430.6  151.7 6.7  3.10
P-value 0.553 0.648 0.972
RFC1 80A>G AA (28) 14.4  2.90 459.1  170.2 6.1  2.60
rs1051266 AG+GG (22) 13.5  2.23 350.1  193.7 7.3  3.23
P-value 0.281 0.125 0.163

MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; NSCLP, non-syndromic cleft lip/pal-
ate; RFC1: reduced folate carrier.
Values are shown as mean  s.d. and compared by t-test. Number of patients in parentheses.
a
Significant result.

study, we found that the higher prevalence of females
among CP patients may be related to the longer period
required to close the palate in the developing female fetus.
The palate and the lip have different embryonic origins:
the palate is derived from the endodermal while the lip
arises from the ectodermic leaflet (Meng et al, 2009).
Folate is the basic acceptor molecule for one-carbon
metabolism, and as a result, the finding that patients with
NSCLP (and their mothers) showed lower serum folic acid
concentrations than the controls reinforces the previously
delineated association between folate metabolism and the
pathogenesis of orofacial clefts (Platek et al, 2009; Bhas-
kar et al, 2011; Liang et al, 2014). Reduced levels of
folate can cause reduction in SAM levels, leading to
impairment and likely insufficiency in DNA methylation,
which an epigenetic mechanism that regulates genomic
programming during embryogenesis (de Arruda et al,
2013). The consequent homocysteine accumulation has
been associated with thrombophilias and increased risks
for placental vasculopathies, spontaneous pregnancy loss,
intra-uterine death, placental disease, and neural tube
defects (Kim et al, 2009; Sule et al, 2012).
Dietary intake of folate was not evaluated in the current
study. It is known that the best sources of folate are viscera,
beans, and green leafy vegetables such as spinach, aspara-
gus, and broccoli. Investigation of folate intake during preg-
nancy in Brazilian women found a 63.6% prevalence of
inadequate dietary folate intake (Santos and Pereira, 2007).
A recent systematic review concluded that the use of
wheat flour fortified with folic acid was effective and dem-
onstrated a strong protective effect, significantly reducing
the incidence of neural tube defects. In Brazil, a resolution
stated that, from June 2004, wheat flour sold directly to con-
sumers and those used in industry for manufacturing other
food products must be enriched with iron and folic acid
(Kramer and Falc~ao, 2011). Currently, fortification of flour
with folic acid has become a mandatory requirement in
approximately 40 countries, including most of America and

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JF Bezerra et al

6
Table 5 Multivariate logistic regression analysis: relationship of gene
polymorphisms with NSCLP in mothers of NSCLP patients group
Polymorphisms Genotypes OR (95% CI) P-value
MTHFR 677C>T CC 1 0.690
rs1801133 CT+TT 0.90 (0.54–1.50)
MTHFR 1298A>C AA 1 0.038*
rs1801131 AC+CC 1.75 (1.03–2.99)
MTR 2756A>G AA 1 0.927
rs1805087 AG+GG 0.98 (0.57–1.68)
MTRR 66A>G AA 1 0.071
rs1801394 AG+GG 0.62 (0.36–1.05)
RFC1 80A>G AA 1 0.278
rs1051266 AG+GG 0.75 (0.45–1.26)
MTHFR, methylenetetrahydrofolate reductase; MTR, methionine syn-
thase; MTRR, methionine synthase reductase; NSCLP, non-syndromic
cleft lip/palate; RFC1, reduced folate carrier; OR, odds ratio; CI, confi-
dence interval. *represent significant result.
Analysis adjusted for alcohol consumption. Cleft lip and palate. Data are
shown in dominant model of inheritance.
some countries in Africa and Asia (Santos and Pereira,
2007). Among the various countries, risk reduction ranged
from 16% to 78% (Santos and Pereira, 2007).
In addition to the diet, it is important to examine the
genes involved in the folate cycle, as polymorphisms in
these genes are associated with the predisposition to an oro-
facial cleft in individuals with low folate levels (Han et al,
2011). Although no significant association was found
between SNPs and NSCLP in children and their mothers in
the present study, interestingly, patients and their mothers
who carried the MTHFR 677T allele exhibited lower serum
folate concentrations, suggesting that this variant may have
also influenced folate status during pregnancy and increased
the risks of developing orofacial clefts. Previous data
showed that pregnant women from Sorocaba city in Brazil
presented lower serum folate levels when carrying the
MTHFR 677 CT+TT genotypes. The presence of the
MTHFR 677T allele could lead to decreased enzyme activ-
ity, leading to reduced THF availability and homocysteine
remethylation, impaired de novo synthesis of nucleic acids,
and diminished methylation, all of which are essential pro-
cesses in embryogenesis (Jianyan et al, 2010; Dixon et al,
2011; Ho et al, 2013; Kim et al, 2013).
Furthermore, in our samples, we found a possible associ-
ation between the MTHFR 1298C allele and NSCLP among
mothers, after adjusting for alcohol consumption; this sug-
gests interaction between environmental and genetic factors,
which may contribute to NSCLP. This result is in accor-
dance with another larger study conducted in Norway (377
NSCLP and 196 CP infants and 763 controls), which
described higher risks of orofacial clefts in infants whose
mothers reported binge-level drinking during the first tri-
mester of pregnancy, compared with non-drinkers (DeRoo
et al, 2008).
A potential mechanism may be the effect that alcohol
has on one-carbon metabolism, where the two key
enzymes in this pathway (MTHFR and MTR) are inhibited
by alcohol consumption. This inhibition was analyzed by
Halsted et al (2002), using Yucatan micropigs that
received different diets with reduced vs supplemented folic
acids and were divided in groups that either did or did not
receive alcohol. It has been shown that alcohol can disturb
one-carbon metabolism, demonstrating a threefold increase
in Hcy serum levels, reduced hepatic MTR activity,
increased S-adenosylhomocysteine (SAH) levels, and a
corresponding decrease in the SAM/SAH ratio (Halsted
et al, 2002).
Furthermore, alcohol consumption can affect the intake,
absorption, activation, and storage of folate and other
nutrients that are methyl contributors and could potentially
affect methylation adversely, both globally and at specific
CpG sites in the promoter regions of genes. Alcohol can
also negatively influence other cellular processes essential
and critical for embryogenesis and early fetal develop-
ment, such as nucleotide synthesis and consequently DNA
synthesis and repair (Kim et al, 2009; Platek et al, 2009;
Ho et al, 2013).
The limitations of this study include the fact that die-
tary patterns were not monitored and the number of
patients was not sufficient to critically investigate the
interactions between the polymorphisms studied and the
levels of nutritional factors related to one-carbon metabo-
lism.
In this study, the MTHFR 677T allele was associated
with low serum folate levels, while the MTHFR 1298C
variant and alcohol intake were demonstrated to be rele-
vant risk factors for NSCLP in a population from the
Northwest region of Brazil. These results corroborated the
hypothesis that both genetic and non-genetic factors influ-
ence the development of NSCLP. Future studies should
extend investigations to evaluate the methylation patterns
of specific genes and also include a larger sample of
patients to improve statistical significance.
Acknowledgements
This project was supported by grants from Fundacß~ao de Amparo
a Pesquisa de S~ao Paulo (FAPESP # 2008/05064-9), Sao Paulo,
Brazil. JFB, CDS, MAGU, MHH, and RDCH are recipients of
fellowships from CNPq, Brazil. GHMO and ADL are recipients
of fellowships from CAPES, Brazil.
Author contribution
J. Felipe – designed study, sample collection, standardization of
techniques, analyzed data, statistical analyzes, carrying out ana-
lyzes. G. H. M. Oliveira– sample collection, carrying out ana-
lyzes. C. D. Soares – sample collection, carrying out analyzes.
M. L. Cardoso – sample collection, carrying out analyzes. A. A.
de Rezende – Project coordination, designed study, statistical
analyzes, drafted paper. M. H. Hirata– Project coordination,
designed study, drafted paper. R. D. C. Hirata – designed study,
statistical analyzes, drafted paper. C.M. Fajardo – Standardization
of techniques. A. D. Luchessi – Analyzed data, statistical ana-
lyzes. M. A. G. Ururahy – Analyzed data, statistical analyzes. V.
N. SILBIGER – Analyzed data, statistical analyzes. F. P. F. Neto
– Sample collection. L. G. Lima-Neto – Statistical analyzes. S.
R. de Oliveira – Screening of patients. M. das G. Almeida –
Designed study.
Conflict of interests
The authors report no conflict of interests.

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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1. Questionnaire to collect personal data.
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