X‐linked markers in the Duchenne muscular dystrophy gene associated with oral clefts

As part of an international consortium, case–parent trios were collected for a genome‐wide association study of isolated, non‐syndromic oral clefts, including cleft lip (CL), cleft palate (CP), and cleft lip and palate (CLP). Non‐syndromic oral clefts have a complex and heterogeneous etiology. Risk is influenced by genes and environmental factors, and differs markedly by gender. Family‐based association tests (FBAT) were used on 14,486 single nucleotide polymorphisms (SNPs) spanning the X chromosome, stratified by type of cleft and racial group. Significant results, even after multiple‐comparisons correction, were obtained for the Duchenne muscular dystrophy (DMD) gene, the largest single gene in the human genome, among CL/P (i.e. both CL and CLP combined) trios. When stratified into groups of European and Asian ancestry, stronger signals were obtained for Asian subjects. Although conventional sliding‐window haplotype analysis showed no increase in significance, selected combinations of the 25 most significant SNPs in the DMD gene identified four SNPs together that attained genome‐wide significance among Asian CL/P trios, raising the possibility of interaction between distant SNPs within the DMD gene.     

Genes causing clefting syndromes as candidates for non‐syndromic cleft lip with or without cleft palate: a family‐based association study

Clefts of the orofacial region are among the most common congenital defects, caused by abnormal facial development during gestation. Non‐syndromic cleft lip with or without cleft palate (NSCLP) is a complex trait most probably caused by multiple interacting loci, with possible additional environmental factors. As facial clefts form part of more than 300 syndromes, one strategy for identifying the genetic causes of NSCLP could be to study candidate genes responsible for clefting syndromes. Three genes were selected for this investigation: TP63, which codes for the tumour protein p63 and causes Ectrodactyly‐Ectodermal dysplasia‐orofacial Cleft syndrome; JAG2, a downstream gene of TP63; and MID1, which is responsible for Opitz syndrome. A linkage disequilibrium investigation was performed with intragenic single nucleotide polymorphisms on each of these genes in a sample study of 239 patients/parents trios. Evidence which suggests that JAG2 and MID1 may play a role in NSCLP was obtained.     

Susceptibility locus for non‐syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients

Nikopensius T, Birnbaum S, Ludwig KU, Jagomägi T, Saag M, Herms S, Knapp M, Hoffmann P, Nöthen MM, Metspalu A, Mangold E. Susceptibility locus for non‐syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonian patients. Eur J Oral Sci 2010; 118: 317–319. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental factors. Recently, two novel susceptibility loci and three suggestive loci for NSCL/P were identified by a genome‐wide association scan (GWAS) in a German population with subsequent independent replication in a mixed European population. The aim of the present study was to investigate whether these newly detected loci confer similar effects in the North‐East European Baltic population. A total of 101 NSCL/P patients and 254 controls from Estonia were included. A significant association was observed for rs7078160 (P = 0.0016) at chromosome 10q25, which confirms the association of this locus with NSCL/P in the Baltic population. No significant association was found for the other four loci, a result that may have been attributable to the limited power of the sample.     

Analysis of the genetic ancestry of patients with oral clefts from South American admixed populations

Increased susceptibility to cleft lip, with or without cleft palate (CL±P) has been observed in South America, as related to Amerindian ancestry, using epidemiological data, uniparental markers, and blood groups. In this study, it was evaluated whether this increased risk remains when Amerindian ancestry is estimated using autosomal markers and considered in the predictive model. Ancestry was estimated through genotyping 62 insertion and deletion (INDEL) markers in sample sets of patients with CL±P, patients with cleft palate (CP), and controls, from Patagonia in southern Argentina and Belém in northern Brazil. The Amerindian ancestry in patients from Patagonia with CL±P was greater than in controls although it did not reach statistical significance. The European ancestry in patients with CL±P from Belém and in patients with CP from Belém and Patagonia was higher than in controls and statistically significant for patients with CP who were from Belém. This high contribution of European genetic ancestry among patients with CP who were from Belém has not been previously observed in American populations. Our results do not corroborate the currently accepted risks for CL±P and CP estimated by epidemiological studies in the North American populations and probably reflect the higher admixture found in South American ethnic groups when compared with the same ethnic groups from the North American populations.     

Homozygote C/C at rs12543318 was risk factor for non‐syndromic cleft lip only from Western Han Chinese population

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder, and it results from both of the genetic modifiers and environmental factors, with genetic modifiers contributes to it more than environmental factors. GWASs made great progress in identifying the candidate genes for NSCL/P, but the findings need to be replicated in other populations. In this study, we selected eleven SNPs from recent GWASs and GWAS meta‐analysis to investigate their associations among 308 NSCL/P trios (134 non‐syndromic cleft lip only (NSCLO) trios and 174 non‐syndromic cleft lip with cleft palate (NSCLP) trios) from Han Chinese population. All SNPs were genotyped using SNPscan method and analyzed the data with FBAT, PLINK, and R package. Allelic TDT analysis showed that allele A at rs12543318 was associated with NSCLO trios (P = .0032, OR = 0.57, 95% CI: 0.39‐0.83), and parent‐of‐origin effect analysis indicated that allele A at rs12543318 was significantly maternally undertransmitted among NSCLO (P = .0046), which implied the potential influence of genomic imprinting; global TDT further confirmed this association. Individual genotypic TDT showed homozygote C/C at rs12543318 was overtransmitted among NSCLO (Z = 3.79, P = .00015) and NSCL/P groups (Z = 3.83, P = .00013), which indicated that it could increase the risk to have cleft babies. Our findings indicated that rs12543318 was associated with NSCLO from Western Han Chinese population, which will give new scientific evidence for later researches in the etiology of NSOCs.     

CRISPLD2 polymorphisms are associated with non‐syndromic cleft lip with or without cleft palate in a northern Chinese population

Shi J, Jiao X, Song T, Zhang B, Qin C, Cao F. CRISPLD2 polymorphisms are associated with non‐syndromic cleft lip with or without cleft palate in a northern Chinese population. Eur J Oral Sci 2010; 118: 430–433. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Non‐syndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect. This complex genetic disorder results from interactions between genes and environmental factors. Numerous genes have been reported in studies demonstrating association between the cleft lip and palate phenotypes and the alleles at single‐nucleotide polymorphisms (SNPs) within specific genes. Recently, the cysteine‐rich secretory protein LCCL domain containing 2 (CRISPLD2) has been revealed to be a novel candidate gene for NSCLP. The SNPs rs1546124, rs4783099 and rs16974880 in CRISPLD2 were highly significant in Caucasian and Hispanic multiplex families but showed no association in Colombian and Irish populations. In the current study, we examined these three SNPs in a northern Chinese population and found an association between these polymorphisms and NSCLP in both single‐marker and haplotype analyses. Our data further strengthen the conclusion that altered CRISPLD2 is associated with NSCLP susceptibility.     

Haplotype‐based gene–gene interaction of bone morphogenetic protein 4 and interferon regulatory factor 6 in the etiology of non‐syndromic cleft lip with or without cleft palate in a Chilean population

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect in humans, the etiology of which can be dependent on the interactions of multiple genes. We previously reported haplotype associations for polymorphic variants of interferon regulatory factor 6 (IRF6), msh homeobox 1 (MSX1), bone morphogenetic protein 4 (BMP4), and transforming growth factor beta 3 (TGFB3) in Chile. Here, we analyzed the haplotype‐based gene–gene interaction for markers of these genes and NSCL/P risk in the Chilean population. We genotyped 15 single nucleoptide polymorphisms (SNPs) in 152 Chilean patients and 164 controls. Linkage disequilibrium (LD) blocks were determined using the Haploview software, and phase reconstruction was performed by the Phase program. Haplotype‐based interactions were evaluated using the multifactor dimensionality reduction (MDR) method. We detected two LD blocks composed of two SNPs from BMP4 (Block 1) and three SNPs from IRF6 (Block 2). Although MDR showed no statistical significance for the global interaction model involving these blocks, we found four combinations conferring a statistically significantly increased NSCL/P risk (Block 1–Block 2): T‐T/T‐G C‐G‐T/G‐A‐T; T‐T/T‐G C‐G‐C/C‐G‐C; T‐T/T‐G G‐A‐T/G‐A‐T; and T‐T/C‐G G‐A‐T/G‐A‐T. These findings may reflect the presence of a genomic region containing potential causal variants interacting in the etiology of NSCL/P and may contribute to disentangling the complex etiology of this birth defect.     

IRF6 gene variants in Central European patients with non‐syndromic cleft lip with or without cleft palate

Variants in the interferon regulatory factor 6 (IRF6) gene have repeatedly been associated with non‐syndromic cleft lip with or without cleft palate (NSCL/P). A recent study has suggested that the functionally relevant variant rs642961 is the underlying cause of the observed associations. We genotyped rs642961 in our Central European case–control sample of 460 NSCL/P patients and 952 controls. In order to investigate whether other IRF6 variants contribute independently to the etiology of NSCL/P, we also genotyped the non‐synonymous coding variant V274I (rs2235371) and five IRF6‐haplotype tagging single nucleotide polymorphisms (SNPs). A highly significant result was observed for rs642961 (P = 1.44 × 10^?6) in our sample. The odds ratio was 1.75 [95% confidence interval (CI): 1.38–2.22] for the heterozygous genotype and 1.94 (95% CI: 1.21–3.10) for the homozygous genotype, values that are similar to those reported in a previously published family‐based study. Our results thus confirm the involvement of the IRF6 variant, rs642961, in the etiology of NSCL/P in the Central European population. We also found evidence suggestive of an independent protective effect of the coding variant V274I. In order to understand fully the genetic architecture of the IRF6 locus, it will be necessary to conduct additional SNP‐based and resequencing studies using large samples of patients.     

MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate

Jagomägi T, Nikopensius T, Krjut?kov K, Tammekivi V, Viltrop T, Saag M, Metspalu A. MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci 2010; 118: 213–220. © 2010 The Authors. Journal compilation©2010 Eur J Oral Sci Recent studies suggest that multiple interacting loci, with possible additional environmental factors, influence the risk for nonsyndromic oral clefts, one of the most common birth defects in humans. Advances in high‐throughput genotyping technology allow the testing of multiple markers, simultaneously, in many candidate genes. We tested for associations between 176 haplotype‐tagging single nucleotide polymorphisms (SNPs) in 18 candidate genes/loci and nonsyndromic clefts in a case–control study in an Estonian sample (153 patients, 205 controls). The most significant associations with nonsyndromic cleft lip with or without cleft palate (CL/P) were found for SNPs in MSX1, MTHFR, and PVRL2, including several common haplotypes in the MTHFR and MSX1 genes. The strongest association was observed for rs6446693 in the MSX1 region, which remained statistically significant after Bonferroni correction. The strongest association with nonsyndromic cleft palate (CP) was found for the SNP rs11624283 in the JAG2 gene. Epistatic interactions were observed for SNPs within PVRL2, between BCL3 and EDN1, and between IRF6 and MSX1 genes. This study provides further evidence implicating MSX1 and MTHFR in the etiology of nonsyndromic CL/P across different populations.     

Classification of oral clefts by affection site and laterality: a genotype-phenotype correlation study

Objectives – The aim of this study was to classify the phenotypes found in a series of patients with non‐syndromic cleft lip (CL) with or without cleft palate (CP) and isolated cleft palate. Additionally, the frequency distribution of cases belonging to families linked to markers on chromosomes 6 and 2 within these phenotypic patterns were estimated. Design – A retrospective examination of all the available affected cases collected in Italy. Setting and Sample Population – Ninety‐seven affected subjects aged 5–18 years belonging to 38 families were considered. Patterns were identified by variance of the cleft (lip, primary palate, secondary palate) and stratified according to the side of occurrence (right, left, or bilateral). Latent class analysis was used as main statistical tool for carrying out the results. Results – Three homogenous classes were identified (P < 0.0001) by means of latent class analysis. Individuals were assigned to the most suited class. All three variables (lip, primary and secondary cleft palate) generated a specific class. Optimal findings were reported in cases having `any isolated cleft lip' (class 1); `secondary CP with or without bilateral/right primary cleft palate + bilateral/right cleft lip' (class 2); and `left primary cleft palate + left/bilateral cleft lip with or without secondary CP' (class 3). Correspondence to the evidence of linkage to chromosome 6 showed that 9 of 10 cases presenting with `right primary CP + right CL with secondary cleft palate' (class 2) belonged to a linked family. The same combination, but occurring on the left side (class 3), revealed that only three of nine cases belong to families linked to chromosome 6 (P‐value=0.02). The two patterns (right and left) never occurred in the same family. Three reliable groups were identified based on laterality and the presence of a cleft. A single right sided pattern displayed a statistically different distribution of linkage to chromosome 6 when compared with the homologous left side. Conclusion – Non‐syndromic CL with/without CP can be classified according to laterality that can be under genetic control.     

A novel mutation in the OFD1 (Cxorf5) gene may contribute to oral phenotype in patients with oral-facial-digital syndrome type 1

Oral Diseases (2011) 17 , 610–614 Background: Oral‐facial‐digital syndrome (OFDS) type 1 (OFD1) is an X‐linked dominant condition associated with embryonic male lethality. It almost always affects the oral cavity, face, and digits. It is considered to be a ciliopathy caused by mutations in the OFD1 gene. A variety of mutations have been described, and a genotype–phenotype correlation has been suggested. Objective and Methods: The proband was an 8‐year‐old Spanish girl with suspected OFD1. We extended the pedigree to three proband’s generations, performing a thorough physical examination and screening for OFD1 mutations in nine individuals. Results: The proband, her mother, and her sister showed oral findings consistent with OFD1. Ultrasound evaluation revealed the existence of renal cysts only in the proband’s mother. The rest of the family (all male) had no relevant morphological abnormalities. A single‐base deletion in exon 16 of OFD1 (c.2183delG) leading to a frameshift was detected in the proband, her mother, and her sister. Conclusion: Because all three women had a similar oral phenotype, this new mutation might be involved in the development of the OFD1 oral manifestations. In cases of OFDS, physical examination (including the oral cavity and renal function) and genetic screening of the probands and their relatives are mandatory.     

Enamel Matrix Derivative,Alone or Associated With a Synthetic Bone Substitute,in the Treatment of 1‐ to 2‐Wall Periodontal Defects

Background: In this study, we compare the effects of enamel matrix derivative (EMD) associated with a hydroxyapatite and β‐tricalcium phosphate (HA/β‐TCP) implant to EMD alone and to open‐flap debridement (OFD) when surgically treating 1‐ to 2‐wall intrabony defects. Methods: Thirty‐four patients, exhibiting ≥3 intraosseous defects in different quadrants, were each treated by OFD, EMD, or EMD + HA/β‐TCP in each defect. At baseline and 12 and 24 months, a complete clinical and radiographic examination was done. Pre‐therapy and post‐therapy clinical (probing depth [PD], clinical attachment level [CAL], and gingival recession [GR]) and radiographic (defect bone level [DBL] and radiographic bone gain [RBG]) parameters for the different treatments were compared. Results: After 12 and 24 months, almost all the clinical and radiographic parameters showed significant changes from baseline within each group (P <0.001). Differences in PD, CAL, and DBL scores were also seen among the three groups at the 12‐ and 24‐month visits (P <0.001). At 12 and 24 months after treatment, the EMD + HA/β‐TCP group showed significantly greater PD reduction (4.00 ± 0.42 mm; 4.25 ± 0.63 mm), CAL gain (3.47 ± 0.65 mm; 3.63 ± 0.91 mm), and RBG (3.17 ± 0.69 mm; 3.35 ± 0.80 mm) and less GR increase (0.56 ± 0.37 mm; 0.63 ± 0.42 mm) compared with the OFD and EMD groups (P <0.05). Conclusion: Our data support the hypothesis that the adjunct of an HA/β‐TCP composite implant with EMD may improve the clinical and radiographic outcomes of the surgical treatment of unfavorable intrabony defects.     

Titanium‐prepared platelet‐rich fibrin provides advantages on periodontal healing: A randomized split‐mouth clinical study

1 Background

The aim of this study to evaluate the contributions of titanium‐prepared platelet‐rich fibrin (T‐PRF) combined with open flap debridement (OFD) on biological markers in gingival crevicular fluid (GCF)and periodontal outcomes.

2 Methods

Twenty‐nine participants with chronic periodontitis were treated either with autologous T‐PRF+OFD or OFD alone. GCF growth factor levels and relative receptor activator nuclear factor kappa‐B/osteoprotegerin (RANKL/OPG) ratio at baseline and 2, 4, and 6 weeks postoperatively were analyzed, and clinical parameters such as probing depth (PD), relative attachment level (RAL) and gingival margin level (GML) at baseline and 9 months after surgery were compared.

3 Results

The mean PD reduction, RAL gain, and GML change were significantly greater in the OFD+T‐PRF sites than in the OFD sites (P = 0.033, P = 0.029, and P = 0.026, respectively). Both groups demonstrated increased growth factor levels at week 2 compared with baseline, followed by reductions at weeks 4 and 6. GCF growth factor levels in the test group were seen at higher concentrations with respect to control group until 6 weeks post‐surgery. During this 6‐week period, relative RANKL/OPG ratio was found significantly lower in the OFD+T‐PRF group compared to the OFD group(P < 0.05).

4 Conclusions

Using T‐PRF membrane combined with OFD provided significantly higher concentrations of growth factors and lower RANKL/OPG ratio in GCF for approximately 4 to 6 weeks, and improved periodontal healing compared to conventional flap sites.     

Lack of association between IRF6 polymorphisms (rs2235371 and rs642961) and non‐syndromic cleft lip and/or palate in a Brazilian population

Oral Diseases (2010) 16 , 193–197 Background: Interferon regulatory factor 6 (IRF6) gene has emerged as a potential susceptibility gene for non‐syndromic cleft lip and/or palate (NSCL/P) in different populations. The aim of this study was to determine the association of IRF6 rs2235371 and rs642961 polymorphisms with NSCL/P in a Brazilian population. Methods: Two hundred and twenty‐eight patients affected by NSCL/P and 126 healthy individuals were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay. Results: Overall genotype distributions of rs2235371 and rs642961 polymorphisms were as expected by Hardy‐Weinberg equilibrium test. The rs2235371 polymorphic genotype GA was identified in 10.1% of the patients with NSCL/P and in 10.3% of the control group, revealing no statistical difference. Similarly, the frequency of rs642961 minor genotypes (GA and AA) was quite similar between control group (28.6%) and NSCL/P group (25.4%), without significant difference. Conclusion: Our findings are consistent with a lack of involvement of IRF6 rs2235371 and rs642961 polymorphisms in the NSCL/P pathogenesis in the Brazilian population.     

Epidemiological and genetic study of 121 cases of oral clefts in Kuwait

Authors – S Al‐Bustan, M El‐Zawahri, A Al‐Adsani, R Bang, I Ghunaim, B Maher, S Weinberg, M Marazita Aim – The aim of the study was to ascertain some epidemiological factors such as sex and consanguinity that may be associated with cleft lip with or without cleft palate (CL ± CP) in Kuwait as well as to conduct genetic segregation analysis of these families. Setting and sample population – A total of 113 families ascertained through 121 CL ± CP and CP surgical probands in Kuwait. The frequencies of cleft types and the epidemiological variables were calculated using SPSS version 5.0 software. Chi‐square for goodness‐of‐fit test was used to test the significance of the associated epidemiological variables to facial clefts. Genetic segregation analysis was performed on 76 families with extended pedigrees and included only those with non‐syndromic CL ± CP (NS CL ± CP). Major locus segregation analysis was used to fit models to the observed family patterns under Class A regressive models as implemented by REGD routine in S.A.G.E. release 4.0. A test for heterogeneity was also conducted to complete data set in addition to two subsets: Arabs and nomads. Results – Of the 121 patients, 34(28.1%) had CP, 30(24.8%) had CL and 57 (47.1%) had CL + CP. The male to female ratio was 0.89 for CP, 1.14 for CL, 1.35 for CL + CP and 1.2 for all the clefts. The percentage of consanguineous families among those with a positive family history (60%) was not significantly different from that of the general population (54.3%), whereas for all the families with clefts the percent consanguineous was significantly lower (38%). No evidence of heterogeneity in the results between the Arab and nomad subsets was observed. The results for the major locus segregation analysis were inconclusive. Conclusion – No definite association was observed between consanguinity and the occurrence of facial clefts in Kuwait. General transmission models in the full data set showed no evidence of heterogeneity in the results between the Arab and nomad subsets.     

Folic acid rescue of ATRA‐induced cleft palate by restoring the TGF‐β signal and inhibiting apoptosis

J Oral Pathol Med (2010) 40 : 433–439 Background: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF‐β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all‐trans retinoic acid (ATRA)‐induced cleft palate in organ culture were tested. Methods: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. Results: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase‐9 in MEPM cells were detected in the ATRA group than in the control or the ATRA + FA group. Compared with the control or the ATRA + FA group, ATRA had little effect on TGF‐β3 in MEPM cells but significantly inhibited TGF‐β receptor II. Conclusions: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA‐induced cleft palate.     

Genetic variation in the promoter region of beta-defensin 1 (DEFB 1) is associated with high caries experience in children born with cleft lip and palate

Objective. Caries is a common disease in humans and has a multifactorial etiology. It has been suggested that children born with cleft lip and/or palate (CL/P) have a higher susceptibility to caries, but data from several independent cohorts does not support this assumption. Previous work from our group suggested DEFB 1 is associated with higher caries experience. Since it is suspected that children born with CL/P have the same risk factors predisposing them to caries as other children of the same ages, the aim was to test if DEFB 1 was associated with caries experience in children born with CL/P. Materials and methods. Sixty-nine children born with CL/P (aged 2–12 years) were included. Twenty-seven males and seven females had cleft lip and palate (CLP), six males and seven females had cleft lip (CL) and 13 males and nine females had cleft palate (CP). Caries was evaluated with the DMFT/dmft index by a calibrated evaluator. Two single nucleotide polymorphisms in DEFB 1 were selected (rs11362 and rs1800972) based on being associated with higher caries experience in previous work. Genotyping were carried out by real-time PCR using the Taqman assay method. The statistical analysis was performed between ‘low-to-moderate caries experience group' and the ‘high caries experience group'. Odds ratio calculations between caries experience and variant alleles and chi-square of Fisher exact tests at a level of significance of 0.05 were used. Results. There was no significant difference for caries experience between cleft types (p = 0.551). An association was found for the marker rs11362 and genotype distribution (p = 0.047). When analyzed in a recessive model, the genotype GG in this polymorphism increased the risk for caries susceptibility by more than 3-times (p = 0.031; OR = 3.16; 95% CI = 0.97–10.62). Conclusion. The genetic variant rs11362 in DEFB 1 influences caries susceptibility in CL/P children. The results support the hypothesis that expression of DEFB 1 in saliva may serve as a biomarker for future caries risk.     

Polymorphisms located in the region containing BHMT and BHMT2 genes as maternal protective factors for orofacial clefts

Mostowska A, Hozyasz KK, Biedziak B, Misiak J, Jagodzinski PP. Polymorphisms located in the region containing BHMT and BHMT2 genes as maternal protective factors for orofacial clefts. Eur J Oral Sci 2010; 118: 325–332. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Nonsyndromic cleft lip with or without cleft palate (NCL/P) is one of the most common craniofacial malformations; however, its aetiology is still unclear. Because the effects of maternal nutrition on fetal development are well known, we decided to pursue the question of whether polymorphic variants of genes encoding enzymes involved in choline metabolism might be associated with the maternal risk of having a baby with NCL/P. Analysis of 18 single nucleotide polymorphisms (SNPs) of betaine‐homocysteine methyltransferase (BHMT), betaine‐homocysteine methyltransferase‐2 (BHMT2), choline dehydrogenase (CHDH), choline kinase (CHKA), dimethylglycine dehydrogenase (DMGDH), choline‐phosphate cytidylyltransferase A (PCYT1A), and phosphatidylethanolamine N‐methyltransferase (PEMT) provided evidence that polymorphisms located in the region containing BHMT and BHMT2 were protective factors against NCL/P affected pregnancies in our population. The strongest signal was found for the SNP located in the intronic sequence of BHMT2. Women carrying two copies of the rs625879 T allele had a significantly decreased risk of having offspring with orofacial clefts. These results were significant, even after correction for multiple comparisons. Moreover, the gene–gene interaction analysis revealed a significant epistatic interaction of BHMT2 (rs673752), PEMT (rs12325817), and PCYT1A (rs712012) with maternal NCL/P susceptibility. Altogether, our study identified a novel gene, the nucleotide variants of which were be associated with a decreased risk of having a baby with NCL/P.