Dysregulation of Rho GTPases in orofacial cleft patients-derived primary cells leads to impaired cell migration, a potential cause of cleft/lip palate development.

Cleft lip and/or palate are a split in the lip, the palate or both. This results from the inability of lip buds and palatal shelves to properly migrate and assemble during embryogenesis. By extracting primary cells from a cleft patient, we aimed at offering a better understanding of the signaling mechanisms and interacting molecules involved in the lip and palate formation and fusion. With Rho GTPases being indirectly associated with cleft occurrence, we investigated the role of the latter in both. First, whole exome sequencing was conducted in a patient with cleft lip and palate. Primary fibroblastic cells originating from the upper right gingiva region were extracted and distinct cellular populations from two individuals were obtained: a control with no cleft phenotype and a patient with a cleft lip and palate. The genetic data showed three candidate variables in ARHGEF18, EPDR1, and CUL7. Next, the molecular data showed no significant change in proliferation rates between healthy patient cells and CL/P patient cells. However, CL/P patient cells showed decreased migration, increased adhesion and presented with a more elongated phenotype. Additionally, RhoA activity was upregulated in these cells, whereas Cdc42 activity was downregulated, resulting in loss of polarity. Our results are suggestive of a possible correlation between a dysregulation of Rho GTPases and the observed phenotype of cleft lip and palate patient cells. This insight into the intramolecular aspect of this disorder helps link the genetic defect with the observed phenotype and offers a possible mechanism by which CL/P occurs.

Peters plus syndrome mutations affect the function and stability of human ß1,3-glucosyltransferase.

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding ß1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

Association between cystathionine beta-synthase c.844ins68 polymorphism and risk of non-syndromic cleft lip/palate: A meta-analysis of family-based and case-control studies.

OBJECTIVE: Both genetics and environmental factors play a role in the occurrence of non-syndromic cleft lip/palate (NSCL/P). This meta-analysis evaluated the association between cystathionine beta-synthase (CBS) c.844ins68 polymorphism and risk of NSCL/P in family-based and case-control studies. MATERIALS AND METHODS: The PubMed, Web of Science, Scopus, and Cochrane Library databases were searched for articles published until September 2018. RevMan 5.3 software was used to calculate the odds ratio (OR) for the association between CBS c.844ins68 polymorphism and risk of NSCL/P by using five genetic models in the studies. The transmission disequilibrium test (TDT) was conducted for family-based studies. RESULTS: Three case-control and three family-based studies were evaluated. Based on the analysis of five genetic models, risk of NSCL/P was not related to CBS c.844ins68 polymorphism in case-control studies. The results of family-based studies did not show any association between the CBS c.844ins68 allele and NSCL/P either. CONCLUSIONS: The results of this meta-analysis showed that there was no association between CBS c.844ins68 polymorphism and risk of NSCL/P; therefore, this polymorphism does not play a role in susceptibility to NSCL/P.

Association of Betaine-Homocysteine S-Methyl Transferase (rs3797546 and rs3733890) polymorphisms with non-syndromic cleft lip/palate: A meta-analysis.

OBJECTIVE: Non-syndromic cleft lip/palate (NSCL/P) has a multifactorial and polygenic aetiology. The role of genetics in its occurrence has not been fully clarified. The present meta-analysis aimed to evaluate the association of betaine-homocysteine S-methyltransferase (BHMT) polymorphisms (rs3797546 and rs3733890) with the risk of NSCL/P. MATERIALS AND METHODS: PubMed/Medline, Scopus, Cochrane Library, and Web of Science databases were systematically searched for articles published up until December 2018 with no language restriction. Quality evaluation of each study was performed by the Newcastle-Ottawa Scale (NOS). The crude odds ratio (OR) and 95% confidence interval (CI) were calculated for each study by RevMan 5.3 software, and a funnel plot analysis was performed by the CMA 2.0 software using the Egger's and Begg's tests. RESULTS: Review of the four selected studies revealed that the CC genotype of rs3797546 polymorphism significantly increased the risk of NSCL/P. No association was noted between NSCL/P risk and rs3733890 polymorphism except in Chinese (elevated risk of NSCL/P) and Polish (decreased risk of NSCL/P) populations. CONCLUSIONS: According to the present meta-analysis, rs3733890 polymorphism does not play a role in susceptibility to NSCL/P; whereas, rs3797546 polymorphism may play a role in susceptibility to NSCL/P. Future studies are required to examine the association between BHMT polymorphisms and the NSCL/P risk in different ethnicities with a larger sample size.

Association between methylenetetrahydrofolate reductase polymorphisms and non-syndromic cleft lip with or without palate susceptibility: an updated systematic review and meta-analysis.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms are thought to be involved in the development of cleft lip with or without cleft palate (NSCL/P), but published results are contradictory. We therefore designed an updated meta-analysis to pool eligible studies and to evaluate further the possible relations between MTHFR polymorphisms (c.677C>T and c.1298A>C) and susceptibility to NSCL/P. A comprehensive search based on PubMed, Medline, Web of Science, and Embase databases was made up to February 2018. Twenty-three case-control and 10 case-parent trio studies (including 1149 cases and 1161 controls) were retrieved. Odds ratio (OR) with 95% CI were used to estimate the pooled strength of association under different genetic models. The Q test and I2 test were used to estimate heterogeneity among studies, the quality of which was assessed using the Newcastle-Ottawa scale. In the MTHFR c.677C>T polymorphism group, there were significant overall results for the recessive (OR 1.231, 95%CI 1.092 to 1.387) and homozygote (OR 1.252, 95%CI 1.078 to 1.456) models. Subgroup analysis by subjects and ethnicity identified only associations in European mothers for the recessive model and the homozygote model. For the c.1298A>C group, there were no significant results for either European or Asian patients for all genetic models. The MTHFR c.677C>T polymorphism might increase susceptibility to NSCL/P in European mothers, but was negatively associated in Asian patients, and the MTHFR c.1298A>C polymorphism is not involved in the development of NSCL/P in either European or Asian patients.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

MTR, MTRR, and MTHFR Gene Polymorphisms and Susceptibility to Nonsyndromic Cleft Lip With or Without Cleft Palate.

OBJECTIVE: To examine the associations of methionine synthase (MTR), methionine synthase reductase (MTRR), and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with the susceptibility to nonsyndromic cleft lip with or without cleft palate (NSCL/P). METHODS: Between May 2012 and August 2014, 147 NSCL/P patients (case group) and 129 healthy volunteers (control group) were recruited for the study. The MTR A2756G, MTRR A66G, MTHFR C677T and MTHFR A1298C polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism. Haplotype analyses were performed with SHEsis software. Logistic regression analysis was used to evaluate the possible risk factors for NSCL/P. Generalized multifactor dimensionality reduction (GMDR) was applied to detect gene-gene interactions. RESULTS: MTR A2756G, MTRR A66G, and MTHFR C677T gene polymorphisms were associated with the risk of NSCL/P (all p < 0.05). Logistic regression analysis revealed that MTR A2756G, MTR RA66G, and MTHFR C667T might increase the risk of NSCL/P (odds ratio [OR] = 0.270, 95% confidence interval [95% CI] = 0.106-0.689; OR = 0.159, 95% CI = 0.069-0.368; OR = 0.343, 95% CI = 0.139-0.844). The CA haplotype in the MTHFR gene may serve as a protective factor for NSCL/P (OR = 0.658, 95% CI = 0.470-0.923), and the TA haplotype might be a risk factor (OR = 2.001, 95% CI = 1.301-3.077). GMDR revealed that the optimal models were two- and four-dimensional models with prediction accuracies of 75.73% (p = 0.001) and 77.21% (p = 0.001) and the best cross-validation consistencies of 10/10 and 10/10, respectively. CONCLUSION: MTR A2756G, MTRR A66G, and MTHFR C677T polymorphisms may be related to NSCL/P, and interactions were detected between the MTR A2756G, MTRR A66G, and MTHFR C677T and A1298C polymorphisms.

Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80µg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

CBS c.844ins68 Polymorphism Frequencies in Control Populations: Implications on Nonsyndromic Cleft Lip With or Without Cleft Palate.

INTRODUCTION: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with substantial clinical and social impact. Folate deficiency is one of the factors that have been associated with increased risk for NSCLP. Polymorphisms in folate and homocysteine pathway genes may act as susceptibility factors. OBJECTIVE: The objective of this study was to evaluate prevalence estimates of cystathionine beta-synthase (CBS) insertion of 68-bp (c.844ins68) polymorphisms and their correlation with NSCLP. MATERIAL AND METHODS: A total of 236 unrelated individuals from seven Indian populations and an additional 355 cases with NSCLP and 357 controls without NSCLP were included in this study. We investigated the CBS c.844ins68 polymorphism in all samples. Genotyping was performed with polymerase chain reaction and electrophoresis. The data were statistically analyzed using the chi-square test. RESULTS: The CBS c.844ins68 allele is present in six of the seven populations analyzed, and allele frequencies range from 1.5% in Balija to 9.1% in Sugali populations. The CBS c.844ins68 polymorphism showed a significant protective effect on NSCLP at both genotype (WW versus WI: odds ratio [OR] = 0.54, 95% confidence interval [CI] = 0.31 to 0.95, P = .149) and allele levels (W versus I: OR = 0.56, 95% CI = 0.32 to 0.96, P = .033). CONCLUSIONS: The current study observed significant differences in the frequency of the CBS 844ins68 allele across populations. There is a significant association between CBS c.844ins68 polymorphism and cleft lip and palate in the Indian population. Additional studies are warranted to identify the functional variants in the genes controlling homocysteine as etiological contributors to the formation of oral clefts.

Functional Significance of MMP3 and TIMP2 Polymorphisms in Cleft Lip/Palate.

Evidence from biological and human studies strongly supports a role for MMP and TIMP genes as candidate genes for non-syndromic cleft lip with or without cleft palate (NSCL/P). We previously showed the association of promoter polymorphisms in MMP3 (rs3025058 and rs522616) and TIMP2 (rs8179096) with NSCL/P. In this study, we examined the functional significance of these polymorphisms. A specific DNA-protein complex for MMP3 rs522616 A was detected, and this allele by itself showed greater promoter activity than the G allele. However, the effect of rs522616 was ultimately regulated by the rs3025058 allele on the background. For TIMP2 rs8179096, the T allele showed a 2.5-fold increase in promoter activity when compared with allele C, whereas both C and T alleles were found to bind to nuclear factor kappa B. Our results provide new evidence that promoter polymorphisms in MMP3 and TIMP2 are functional and may affect gene transcription with possible effects on craniofacial development leading to NSCL/P.

Association of common variants in PAH and LAT1 with non-syndromic cleft lip with or without cleft palate (NSCL/P) in the Polish population.

BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common structural malformation with a complex and multifactorial aetiology. Associations of abnormalities in phenylalanine metabolism and orofacial clefts have been suggested. METHODS: Eight single nucleotide polymorphisms (SNPs) of genes encoding phenylalanine hydroxylase (PAH) and large neutral l-amino acid transporter type 1 (LAT1), as well as the PAH mutation that is most common in the Polish population (rs5030858; R408W), were investigated in 263 patients with NSCL/P and 270 matched controls using high resolution melting curve analysis (HRM). RESULTS: We found that two polymorphic variants of PAH appear to be risk factors for NSCL/P. The odds ratio (OR) for individuals with the rs7485331 A allele (AC or AA) compared to CC homozygotes was 0.616 (95% confidence interval [CI]=0.437-0.868; p=0.005) and this association remains statistically significant after multiple testing correction. The PAH rs12425434, previously associated with schizophrenia, was borderline associated with orofacial clefts. Moreover, haplotype analysis of polymorphisms in the PAH gene revealed a 4-marker combination that was significantly associated with NSCL/P. The global p-value for a haplotype comprised of SNPs rs74385331, rs12425434, rs1722392, and the mutation rs5030858 was 0.032, but this association did not survive multiple testing correction. CONCLUSION: This study suggests the involvement of the PAH gene in the aetiology of NSCL/P in the tested population. Further replication will be required in separate cohorts to confirm the consistency of the observed association.

Evidence of the involvement of the DHFR gene in nonsyndromic cleft lip with or without cleft palate.

Studies aimed at evidencing genetic causes for neural tube defect (NTD) occurrence have often provided the inspiration for orofacial cleft aetiology investigations. The correlation between the two congenital malformations is provided by the similar incidence timing and the involvement of structures localized in the midline of the embryo. This connection is corroborated by the existence of a number of genes involved in both malformations. In this article, we considered the dihydrofolate reductase (DHFR) gene, previously seen implicated in NTDs, as a candidate for cleft lip with or without cleft palate (CL/P) risk. Four SNPs mapping on the DHFR gene were genotyped for 400 Italian CL/P triads, using TaqMan(®) approach. The rs1677693 provided evidence of association, even if at borderline level (P value 0.049). In particular, the variant allele seems to have a protective effect OR = 0.80 (95% C.I. 0.64-0.99). Moreover, the combination of rs1677693(A)-rs1650723(G) alleles showed a significant association OR 0.64 (95% C.I. 0.47-0.86) (P value = 0.006). This represents the first attempt to demonstrate a role for DHFR in CL/P aetiology, howbeit the study of such gene deserves a deepening.

FGFR1 mutations cause Hartsfield syndrome, the unique association of holoprosencephaly and ectrodactyly.

BACKGROUND: Harstfield syndrome is the rare and unique association of holoprosencephaly (HPE) and ectrodactyly, with or without cleft lip and palate, and variable additional features. All the reported cases occurred sporadically. Although several causal genes of HPE and ectrodactyly have been identified, the genetic cause of Hartsfield syndrome remains unknown. We hypothesised that a single key developmental gene may underlie the co-occurrence of HPE and ectrodactyly. METHODS: We used whole exome sequencing in four isolated cases including one case-parents trio, and direct Sanger sequencing of three additional cases, to investigate the causative variants in Hartsfield syndrome. RESULTS: We identified a novel FGFR1 mutation in six out of seven patients. Affected residues are highly conserved and are located in the extracellular binding domain of the receptor (two homozygous mutations) or the intracellular tyrosine kinase domain (four heterozygous de novo variants). Strikingly, among the six novel mutations, three are located in close proximity to the ATP's phosphates or the coordinating magnesium, with one position required for kinase activity, and three are adjacent to known mutations involved in Kallmann syndrome plus other developmental anomalies. CONCLUSIONS: Dominant or recessive FGFR1 mutations are responsible for Hartsfield syndrome, consistent with the known roles of FGFR1 in vertebrate ontogeny and conditional Fgfr1-deficient mice. Our study shows that, in humans, lack of accurate FGFR1 activation can disrupt both brain and hand/foot midline development, and that FGFR1 loss-of-function mutations are responsible for a wider spectrum of clinical anomalies than previously thought, ranging in severity from seemingly isolated hypogonadotropic hypogonadism, through Kallmann syndrome with or without additional features, to Hartsfield syndrome at its most severe end.

[Allelic polymorphism of MTHFR, MTR and MTRR genes in patients with cleft lip and/or palate and their mothers].

The frequency of common MTHFR, MTR and MTRR genes polymorphisms was evaluated among patients with non-syndromic cleft lip and/or palate (CL/P), their mothers and healthy persons from West-Ukrainian region. MTHFR 677TT genotype was shown to increase more than three-fold risk of CL/P and for mothers the risk of having CL/P children may increase two-fold compared with homozygous carriers of MTHFR 677CC genotype (OR = 3.3, OR = 1.92, respectively). The heterozygous MTR 2756AG genotype was associated with 1.5-fold increased risk of CL/P compared with the AA genotype (OR = 1.48). The heterozygous genotype MTRR 66AG was associated with the 5.56-fold increased CL/P risk (OR = 5.56) and for mothers with 2.6-fold increased risk of delivering a CL/P offspring (OR = 2.6). The results showed that MTRR 66G allele is more prevalent than MTRR 66A (wild type) and the MTRR 66GG genotype frequency was significantly lower among CL/P patients and their mothers than in control group among Western Ukrainian inhabitants.

New evidence for the role of cystathionine beta-synthase in non-syndromic cleft lip with or without cleft palate.

Orofacial clefts have a multifactorial aetiology encompassing both genetic and environmental components. While there is wide agreement on the importance of both genetic and nutritional factors, genetic influence in particular has not been well defined. As genetic variants in folate and homocysteine metabolism have been reported to influence the risk of orofacial clefts, an Italian cleft lip with or without cleft palate (CL/P) data set was enrolled for an analysis based on family association to test betaine-homocysteine methyltransferase (BHMT and BHMT2) and cystathionine beta-synthase (CBS) variants. No significant level of association was found between BHMT and BHMT2 variants, while evidence of an allelic association with CL/P was found for the single nucleotide polymorphism rs4920037, mapping at the CBS gene. A log-linear approach indicated that the best genetic model takes into account both mother and child genotypes. This suggests that human orofacial development is influenced by CBS genotypes that possibly operate through intergenerational fetal-maternal interaction.

Polymorphic variants of genes related to arginine metabolism and the risk of orofacial clefts.

OBJECTIVE: Maternal mid-pregnancy low levels of symmetric dimethylarginine and newborn low levels of citrulline are suspected to be risk factors for orofacial clefts. This study was undertaken to investigate the involvement of polymorphic variants of genes related to arginine metabolism in the susceptibility of clefting. DESIGN: PCR-RFLP and HRM analyses were used to analyze single nucleotide polymorphisms (SNPs) of ASS1, ASL, and SLC25A13 in 172 children with non-syndromic cleft lip with or without cleft palate (CL/P) and 188 controls without congenital anomalies. The differences in allele and genotype frequencies between cases and controls were determined using standard Chi-square and Fisher exact tests. The odds ratio (OR) and associated 95% confidence intervals (95% CI) for individuals with CL/P versus controls were also calculated. Associations between the investigated polymorphisms and the risk of being born with an orofacial cleft were tested using the nonparametric and genetic model-free Multifactor Dimensionality Reduction (MDR) approach. RESULTS: Analysis of five SNPs of the ASS1 gene revealed that the G allele of rs7860909 is associated with increased CL/P risk. Compared to individuals with the AA genotype, the G allele carriers had an OR of 1.768 (95% CI: 1.133-2.759; p=0.012). For the remaining SNPs of all analysed genes, there was no overall evidence for cleft association considering the allele and genotype distribution. However, gene-by-gene interaction analysis conducted using the MDR approach revealed a significant interactive genetic effect of ASS1 (rs666174) and SLC25A13 (rs10252573) on the occurrence of clefting (p=0.002). CONCLUSION: Our results demonstrate moderate evidence for the association of polymorphic variants of genes related to arginine metabolism with abnormal palatogenesis.

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nepsilon-dimethyl lysine demethylase.

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N(epsilon)-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N(epsilon)-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

Associations of folate and choline metabolism gene polymorphisms with orofacial clefts.

BACKGROUND: Non-syndromic isolated cleft lip with or without cleft palate (NCL/P) is a common congenital anomaly in humans, the aetiology of which is complex and associated with both genetic and environmental factors. It has been reported that maternal nutritional factors are likely to play a major role in development of NCL/P in the embryo. OBJECTIVE: As the mechanism by which folic acid and choline supplementation prevents NCL/P is poorly understood, the relationship between 16 polymorphic variants of 12 genes encoding enzymes involved in the metabolism of these two nutrients and the risk of facial clefts was investigated. RESULTS: It was found that individuals with the AA genotype of the BHMT rs3733890 polymorphism have a significantly lower risk of orofacial clefts (OR 0.1450, 95% CI 0.0420 to 0.4995; p=0.0005; p(corr)=0.008). It was also demonstrated that the rs7639752 polymorphism of the PCYT1A gene increases the risk of NCL/P nearly twofold in the Polish population (OR 1.891, 95% CI 1.151 to 3.107; p=0.011), but this association would not withstand correction for multiple testing (p(corr)=0.176). The genetic variations in CBS, MTHFD1, MTHFR, MTR, MTRR, TCN2, BHMT2, CHDH, CHKA, and PEMT were not separately correlated with NCL/P risk. However, the Multifactor Dimensionality Reduction (MDR) analysis showed a significant epistatic interaction between MTHFR (rs1801133), MTR (rs1805087), and PEMT (rs4646406) in NCL/P susceptibility. CONCLUSION: This study demonstrates that choline metabolism may play an important role in the aetiology of NCL/P. Polymorphic variants of BHMT and PCYT1A and interactions between genes of choline and folate metabolism might influence the risk of NCL/P in the Polish population.

The maternal homocysteine pathway is influenced by riboflavin intake and MTHFR polymorphisms without affecting the risk of orofacial clefts in the offspring.

BACKGROUND/OBJECTIVES: Riboflavin is a cofactor for the 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme involved in the homocysteine pathway. The aim of this study was to investigate the effects of maternal riboflavin intake and two MTHFR polymorphisms (677C>T; Ala222Val and 1298A>C; Glu429Ala substitutions) on the biomarkers of the homocysteine pathway, and investigate the risk of having offspring with an orofacial cleft (OFC). SUBJECTS/METHODS: In a case-control study design, dietary riboflavin intake and the MTHFR 677C>T and 1298A>C polymorphisms were evaluated in 123 OFC and 108 control mothers by using food frequency questionnaires and blood samples. Homocysteine (tHcy), folate and vitamin B12 concentrations in blood were analyzed in 70 cases and 68 controls. Linear and logistic regression analyses were applied. RESULTS: At 14 months postpartum riboflavin intake and MTHFR 677C>T and 1298A>C genotypes were not significantly different between cases and controls. The 677TT genotype showed lower folate concentrations compared to C-allele carriers with a mean difference of 2.8 nmol/l in serum and 174 nmol/l in red blood cell (both P's=0.01). Every mg per day increase of dietary riboflavin intake was positively associated with increase in vitamin B12 concentration by 52.1% (P<0.01). This effect was most pronounced in MTHFR 677TT homozygotes (205.1%, P=0.03). The riboflavin-adjusted MTHFR 677TT and 1298CC genotypes showed a trend toward an increasing risk for OFC, adjusted odds ratio 1.7 (confidence interval (95% CI), 0.7-4.5) and 1.6 (95% CI, 0.7-4.2), respectively. CONCLUSIONS: Maternal riboflavin intake is significantly associated with biomarkers of the homocysteine pathway, with the strongest effects in MTHFR 677TT homozygotes. The maternal risk of having OFC offspring, however, is not associated with dietary riboflavin intake.