Palate Susceptibility Research Articles

Association of MTHFR 1298A > C Polymorphism with Susceptibility to Non-Syndromic Cleft Lip with or without Palate: A Case-Control Study and Meta-Analysis

Background Several studies have evaluated association of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene 1298A > C polymorphism with non-syndromic cleft lip with or without palate (NSCL ± P) susceptibility, however the results are inconsistent. Materials and methods To address this issue, we performed a case-control study to evaluate the association of MTHFR 1298A > C polymorphism with NSCL ± P risk, followed by a meta-analysis. Results Including our study, a total of 22 case-control studies with 2,814 cases and 4,199 controls were selected. The results suggested that there was no significant association between MTHFR 1298A > C polymorphism and NSCL ± P risk overall. The subgroup analysis demonstrated that the polymorphism was significantly associated with NSCL ± P risk in Asians and Iranian populations, but not in Caucasians, mixed and Chinese populations. Conclusion This meta-analysis indicates that MTHFR 1298A > C polymorphism may not contribute to NSCL ± P risk in overall. However, the MTHFR 1298A > C polymorphism was significantly associated with an increased risk of NSCL ± P in Asians and Iranian populations.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Novel Mutations in the IRF6 Gene on the Background of Known Polymorphisms in Polish Patients With Orofacial Clefting.

To examine the role of the IRF6 mutations in Polish families with Van der Woude syndrome and popliteal pterygium syndrome and to determine the effect of IRF6 single nucleotide polymorphisms (rs7552506, rs2013162, and rs2235375) on cleft lip and/or palate susceptibility. IRF6 mutation screening was performed by direct sequencing of all coding exons of the gene and their flanking intronic regions. Cosegregation analysis was performed to establish the relation of single nucleotide polymorphisms and cleft lip and/or palate phenotypes. We screened the IRF6 gene in eight families with clinical recognition of Van der Woude syndrome and popliteal pterygium syndrome. In five families we identified pathogenic mutations, all affecting the DNA-binding or the protein-binding domain of IRF6. Two of the mutations were novel-a missense mutation Arg31Thr and a small deletion Trp40Glyfs*23. In most cases we found also a haplotype of three single nucleotide polymorphisms-rs7552506, rs2013162, and rs2235375. The association of the single nucleotide polymorphisms and cleft lip and/or palate susceptibility has been previously published. The variants did not cosegregate with phenotype in examined families nor did they cosegregate with pathogenic mutations. The single nucleotide polymorphisms were deemed not causative, due to their presence in unaffected family members. Two novel mutations (Arg31Thr and Trp40Glyfs*23) in the IRF6 gene were identified to be causative for Van der Woude and popliteal pterygium syndromes. In the present study no association between the single nucleotide polymorphisms rs7552506, rs2013162, and rs2235375 and the cleft lip and/or palate phenotype was found. The hypothesis, whether the haplotype of the three single nucleotide polymorphisms was correlated with IRF6 expression level, demands further investigation.

Non-syndromic cleft palate: analysis of TBX22 exon 5 gene mutation

IntroductionThis study aimed to investigate the mutation of T-box transcription factor TBX22 exon 5 in children with non-syndromic cleft palate. Four mutations in TBX22 exon 5 in X-linked cleft palate with ankyloglossia (CPX) patients had been identified in the previous studies. The study used the syndromic cleft palate susceptibility gene as a candidate gene for more common non-syndromic cleft palate.Material and methodsA family-based study with parents and their children composing parent-child trios was performed in this research. Twenty children with non-syndromic cleft palate and 38 healthy parents were enrolled. TBX22 exon 5 was examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The peaks of the sequence diagrams were analyzed using chromas221 and the results of sequencing were proofread using dnastar6.13. The index of the transmission disequilibrium test (TDT) was calculated through McNemar testing.ResultsWe have not found the presence of any mutation of TBX22 exon 5 reported in syndromic cleft palate patients in references. The index of TDT was 0.56 and showed no statistically significant difference (p<0.05). No TBX22 exon 5 mutation was found in the 20 children.ConclusionsMutation of TBX22 exon 5 is not associated with non-syndromic cleft palate in the population of Jiangzhe areas in China.

Analysis of interactions between genetic variants of BMP4 and environmental factors with nonsyndromic cleft lip with or without cleft palate susceptibility

A hospital-based case-control study was conducted to identify interactions between the 538(T-->C) polymorphic site of bone morphogenetic protein 4 gene (BMP4T538C) and exposures in pregnancy with nonsyndromic cleft lip, with or without cleft palate (nsCL/P). Associations between offspring polymorphism of BMP4T538C, paternal smoking, paternal high-risk drinking, maternal passive smoking, and maternal multivitamin supplement with nsCL/P were analyzed by logistic regression analysis. BMP4T538C polymorphism, maternal passive smoking exposures and maternal multivitamin use were associated with the risk of nsCL/P but paternal smoking and paternal high-risk drinking were not. Gene-environment interactions were analyzed using the multifactor dimensionality reduction (MDR) method. The two-factor model including maternal passive smoking and BMP4T538C, was the best for predicting nsCL/P risk with a maximum cross-validation consistency (10/10) and a maximum average testing accuracy(0.605; P<0.0001). The findings suggested that: BMP4T538C could be used as a genetic susceptibility marker for nsCL/P; maternal passive smoking exposure is a risk factor for nsCL/P; maternal multivitamin supplements are a protective factor; the synergistic effect of BMP4T538C and maternal passive smoking could provide a new tool for identifying individuals at high risk of nsCL/P, and provides additional evidence that nsCL/P is determined by genetic and environmental factors.

Association between retinoic acid receptor alpha gene polymorphisms and nonsyndromic cleft lip with or without cleft palate susceptibility

To investigate the relationship between D17S579 microsatellite marker allelic polymorphisms in retinoic acid receptor-alpha (RARA) gene and the genetic susceptibility to nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Hunan Hans. PCR and denaturing polyacrylamide gel electrophoresis (PAGE) were used to detect the polymorphism of RARA gene marked by D17S579 among 140 patients with NSCL/P, 82 males and 58 females, aged 3 months-14 years, and 132 healthy persons, 68 males and 64 females, aged 6 months-14 years, who underwent physical examination. The A6 allele frequency of the NSCL/P patients was 8.93%, significantly higher than that of the healthy persons (4.17%, P = 0.026). The A9 allele frequency the NSCL/P patients was 13.21%, significantly higher than that of the healthy persons (5.68%, P = 0.003). There were not significant differences in the frequencies of different alleles between the patients with family history and the patients without family history (chi2 = 2.710, P = 0.978). The A6 and A9 alleles in D17S579 microsatellite marker of RARA gene may be correlated with the development of NSCL/P in Hunan Hans. There is no association between the family history and RARA polymorphism in the NSCL/P patients.

Effects of H-2 and dietary vitamin A on the frequency of dorsoventral vaginal septum.

The frequency of dorsoventral vaginal septum (DVS) in mice is determined in part by genes associated with the major histocompatibility complex H-2. Data presented here confirm that one locus (DVS-1) maps centromeric to E alpha and that the second (DVS-2), previously shown to be associated with the S-to-D region, maps to the C4:B144 interval, most likely between Dcp-2 which contributes to glucocorticoid-induced cleft palate susceptibility and Acp which enhances cleft palate susceptibility through the action of vitamin A. Comparisons of data obtained in this laboratory in the periods 1981-1983 and 1985-1990 and observations from the production colony from which many of the strains were purchased revealed minor variations in frequencies of DVS within strains which may be due to differences in ascertainment and/or to environmental factors. The addition of vitamin A to the diet of pregnant mice at a dose that increases the frequency of cleft palate in susceptible strains had no effect on the incidence of DVS.

Vitamin A-enhanced cleft palate susceptibility gene maps between C4 and B144 within the H-2 complex.

Pregnant mice congenic with C57BL/10 in the region of H-2, the major histocompatibility complex on Chromosome 17 (B10.BR, B10.A, B10.A(1R), B10.A(2R), B10.A(15R), B10.A(18R), B10.OL, or crosses between them) were fed Purina Laboratory Chow or the same diet plus 400 IU of vitamin A daily from conception and given dexamethasone (80 or 160 mg/kg) intraperitoneally on the twelfth day of pregnancy. It was found that the added vitamin A increased the frequency of isolated cleft palate only in strains that had b alleles between C4 and B144. The enhancement of susceptibility to glucocorticosteroid-induced cleft palate by vitamin A appears to be a recessive trait and the locus, called Acp, maps centromeric to another corticosteroid-induced cleft palate gene (Dcp-2) that also is in the C4:B144 interval.

Recombinants in the H-2S/H-2D interval of mouse chromosome 17 define the map position of a gene for cleft palate susceptibility.

The H-2 region of mouse chromosome 17 is known to include one or more genes that affect susceptibility to cortisone-induced cleft palate. We have now studied congenic strains that possess crossovers in the interval between H-2S and H-2D and have observed significant differences in susceptibility among recombinants that had been believed to possess the same H-2 haplotypes. Pregnant mice were injected on days 11 through 14 of gestation with 100 mg of cortisone per kg of body weight. The frequency of cleft palate in B10.A(2R) was significantly greater than in B10.A(1R), despite the fact that both have H-2a/H-2b crossovers in the interval between the S and D loci and have the same alleles at all loci that have been previously characterized. Both B10.BAR5 and B10.BAR12 were significantly more susceptible than B10.A(18R), although these strains also share the same alleles at all loci that have been previously characterized. All three of these strains have H-2b/H-2a recombinant chromosomes, with crossovers in the S/D interval. Genetic linkage between H-2 and the high-susceptibility gene of B10.BAR5 was confirmed by testing H-2 homozygotes derived by intercrossing backcross animals. These data therefore suggest that a gene coding for susceptibility, which we designate Cps-1, maps in the 350-kb interval between H-2S and H-2D, and the congenic strains that we have found to be different have different crossover points within this interval. Alleles at the Cps-1 locus have embryonic effects, but no demonstrable effects on the maternal environment.

Vitamin A-enhanced cleft palate susceptibility associated with H-2.

Pregnant mice from the congenic strains C57BL/10Sn, B10.BR, B10.A/SgSn, B10.A(5R)/SgSn, B10.A(2R)SgSn and B10.A(18R)Sg were fed Purina Laboratory Chow or the same diet plus approximately 400 IU vitamin A daily and given 80 mg/kg dexamethasone intra-peritoneally or a sham injection on the 12th day of pregnancy. It was found that only strains with b alleles between H-2S and H-2D had significantly higher frequencies of isolated cleft palate among their progeny when fed the supplemental vitamin A. The locus appears to be on the centromeric side of a dexamethasone-induced cleft palate gene which has been mapped to the same general area.

Erratum. Backcross test demonstrates the linkage of glucocorticoid‐induced cleft palate susceptibility to H‐2. J. Bonner and Marvin L. Tyan (1982) teratology 26: 213–216

TeratologyVolume 27, Issue 2 p. 287-287 ErratumFree Access Erratum. Backcross test demonstrates the linkage of glucocorticoid-induced cleft palate susceptibility to H-2. J. Bonner and Marvin L. Tyan (1982) teratology 26: 213–216 First published: April 1983 https://doi.org/10.1002/tera.1420270218AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume27, Issue2April 1983Pages 287-287 RelatedInformation

Backcross test demonstrates the linkage of glucocorticoid-induced cleft palate susceptibility to H-2.

Data in this report demonstrate a linkage between glucocorticoid-induced cleft palate susceptibility and H-2. B10.A X C57BL/10F1 females were backcrossed to C57BL/10 males. Cleft palate was induced in the BC1 fetuses by giving pregnant females 80 mg/kg of dexamethasone intraperitoneally (IP) on day 12 of gestation. The H-2a/b haplotype was detected on day 18 BC1 fetal spleen cells with an indirect immunofluorescent labeling technique. Fetuses without antigen H-2Kk were H-2b/b . H-2a/b fetuses had a cleft palate frequency of 29%; 11% of the H-2b/b fetuses had cleft palate (P less than .01), demonstrating that the higher susceptibility to cleft palate segregates with the H-2a halotype.

Cleft palate susceptibility maps in two H-2 subregions, H-2K to I-B and G to H-2D.

Data presented here defines the map of H-2 associated genes which affect the glucocorticoid-induced cleft palate frequency to the regions H-2K to I-B and G to H-2D. This was done by observing in four congenic strains the frequency of cleft palate induced by 160 mg/kg of dexamethasone administered to pregnant females on day 12 of gestation. The strains used were B10.A/Sg.Sn, C57BL/10Sn, B10.A (5R)/SgSn and B10.A(18R)/Sg. Additionally the cleft palate frequency was observed in a large number of litters from saline-treated pregnant females. The cleft palate frequency in this control was very low and the rank of strains was the same as for the glucocorticoid-induced cleft-palate frequency. This observation suggests that the induced cleft palate frequency reflects the spontaneous occurrence of isolated cleft palate in these strains.

Susceptibility to cleft palate and the major histocompatibility complex (H-2) in the mouse.

The congenic mouse strains B10 and B10.A differ genetically only at the H-2 locus, B10 having the H-2b and B10.A the H-2a haplotype. The two strains also differ in susceptibility to cortisone-induced cleft palate, B10 being more resistant than B10.A. Since stage of palate closure is also associated with susceptibility to cortisone-induced cleft palate it was postulated that the H-2 haplotype may affect the cortisone-induced cleft palate susceptibility by altering the stage of palate closure. The present study shows that palate closure occurs at the same stage in the two strains, so this hypothesis must be rejected.

THE GENETICAL ASPECTS OF EXPERIMENTALLY INDUCED POLYCYSTIC KIDNEY DISEASE

Genetical factors play an important role in the etiology of some forms of polycystic kidney disease. However, virtually nothing is known of the molecular nature of the gene product defect(s), nor their site of action. A study of the genetically determined basis for susceptibility to steroid teratogens in the causation of the polycystic kidney disease was begun. In this model polycystic kidney disease is induced by injecting a glucocorticoid acetate on the first day of life. The cysts occur, with renal failure, over the next week. In the present study only a percentage of animals within an experimental group showed susceptibility to the teratogen and there was no sex predilection. Some litters showed complete resistance, others were totally sensitive, and yet others were partially reactive. Furthermore, our study indicates considerable variation in the susceptibility of rats of one and the same strain (Sprague Dawley) obtained from three sources. Biddle and Fraser studying steroid induced cleft palate susceptibility to the teratogen showed it was influenced by a small number of genes. It is possible that a similar situation pertains to polycystic disease of the kidney.

A glucocorticoid receptor in fetal mouse: its relationship to cleft palate formation.

Fetal mouse tissue was investigated for a glucocorticoid binding receptor which might be responsible for cleft palate formation. Fetal mouse heads contain a soluble component which binds the glucocorticoid triamcinolone acetonide in vitro with high affinity. This binding component is present in small finite amounts. Other glucocorticoids compete with triamcinolone acetonide for the binding site in a manner consistent with their potency ranking as cleft palate teratogens. Several mineralocorticoids and progestins also compete when administered in vitro but not when administered in vivo. Triamcinolone acetonide binding was determined in three mouse strains, A/J, C3H, and C57BL, which are listed in decreasing order of cleft palate susceptibility to cortisone. No positive correlation was found between cortisone cleft palate susceptibility and either triamcinolone acetonide binding affinity or binding amount in fetuses from these strains. Cleft palate dose response curves for triamcinolone acetonide were determined in these strains, but they were not parallel to each other as they were for cortisone. This suggests that triamcinolone acetonide may cause cleft palate by different mechanisms in these strains. Thus, fetal mouse tissue contains an apparent glucocorticoid receptors, but its relationship to cleft palate formation in mice is not clear.

Human foetal palatal corticoid receptors and teratogens for cleft palate.

CLEFT secondary palate can be induced experimentally in various mammalian species by the administration of glucocorticoids to pregnant animals1–4. The well known differences in susceptibility to cortisone-induced cleft palate in inbred strains of mice have been explained in terms of genetic differences in foetal facial mesenchymal glucocorticoid receptor levels5,6. The quantity of one protein in particular, with an isoelectric point (pI) of approximately 7.0, seems to be correlated rather closely with the susceptibility of various strains to cortisone-induced cleft palate6. It has also been shown that much of the difference in cleft palate susceptibility between C57BL/10 (B10) and A/J mice can be accounted for by H–2-linked genes7,8, and that there is H–2-linked variation in the level of cortisol-binding proteins obtained from the cytosol fraction of foetal palatal tissue6. As corticoids form a specific cytoplasmic cortisol receptor complex in target tissues which interacts with the genome resulting in activation or depression of transcription9,10, a product of a gene in or near the H–2 locus seems to be the glucocorticoid receptor, the level of which has to be raised for cleft palate to occur. As a first step to determine whether such a mechanism may operate in humans, we have examined whether human palatal mesenchymal cells, obtained in cultures from foetuses shortly after the critical period of secondary palatal closure, contain glucocorticoid receptors. We show here that these cells have specific glucocorticoid receptor proteins with high affinity for dexamethasone and triamcinolone acetonide, and that dexamethasone is displaced from its binding sites only by drugs which produce cleft palate in sensitive mouse strains. Thus, the human foetus contains an active glucocorticoid receptor mechanism immediately after the critical period of palatal organogenesis, which may be involved in palatal clefting.

BIOCHEMICAL AND GENETIC BASIS OF PALATAL SHELF FUSION IN MICE: H-2, CAMP-MEDIATED VERSOS H-2, CORTICOSTEROID MEDIATED EFFECTS

On a biochemical level, both steroids and CAMP are known to play a role in palate formation. CAMP levels rise prior to fusion, inducing epithelial cell death and adhesiveness. Steroid injections increase the incidence of palatal clefting; the magnitude of the effect is strain dependent, and strains which are less susceptible have been shown to have fewer steroid receptors. On a genetic level, susceptibility to steroid-induced cleft palate is H-2 linked. Basal CAMP levels in some tissues also appear to be H-2 determined. The mechanism by which steroids induce cleft palate is unknown, but CAMP may be involved. Premature cell death and/or glycocalyx formation as a result of abnormally high levels of CAMP might well prevent subsequent growth and fusion of shelves. H-2 might mediate its effect through control of basal or steroid-induced cAMP levels, through both, or neither. To determine the relationship between steroids, cAMP, palatal fusion, and H-2, we have been measuring the developmental curve of palatal shelf cAMP levels in congenic lines differing in both H-2 and cleft palate susceptibility, with and without steroid injections. To date, no difference in basal palatal shelf CAMP levels have been found. Steroid induced levels are significantly higher, but strain differences are not yet apparent. The results do not indicate whether steroids are exerting a permissive effect on cAMP levels or whether the more usual, transcriptional control mechanism is at work.

Palatal cytosol cortisol-binding protein associated with cleft palate susceptibility and H-2 genotype.

GENETICALLY different inbred strains of mice have different susceptibilities to the production of cleft palate by a standard dose regimen of corticoids at the critical period during palatal organogenesis1. Because in target tissues corticoids form a specific cytoplasmic cortisol–receptor complex which interacts with the genome, resulting in activation or depression of transcription2,3, we have postulated that susceptibility to corticoid-induced cleft palate is mediated by genetic differences in a cytosolic corticoid receptor protein(s) in palatal anlagen4. Other studies have suggested that susceptibility to cortisone-induced cleft palate is regulated by genes closely linked to the H–2 locus5. We report here that the total level of cytosolic protein binders of 3H-cortisol in maternal and foetal palates on day 11 of gestation correlates with susceptibility to cleft palate and with the H–2 genotype. After separation of macromolecular bound hormone from free hormone on columns of Sephadex G-25 and further separation by microelectrofocusing6, only one maternal and foetal palatal cytosol binder for 3H-cortisol (pI 6.9–7.0) was found to correlate with susceptibility to cleft palate and the H–2 genotype. Thus, a product of a gene in or near the H–2 locus seems to be the glucocorticoid receptor, the level of which must be increased for a congenital malformation to occur.

Cleft palate susceptibility linked to histocompatibility-2 (H-2) in the mouse

Data have been obtained indicating that cortisone-induced cleft palate in the mouse is linked to theH-2 a complex. Cortisone (2.5 mg) was administered to pregnant females on days 11 through 14 of pregnancy. On day 17 of pregnancy, the fetuses were inspected for cleft palates. Sham experiments were done by injecting sterile saline instead of cortisone. The inbred strains, A/J and C57BL/6, and the congenic strains C57BL/10ScSn and B10.A were tested for susceptibility to cleft palate. The clefting frequency was also observed in hybrids of the congenic strains. The A/J and B10.A strains showed a characteristic high susceptibility to cleft palate (i.e., 99% and 81% incidence of cleft palate, respectively) after teratogenic treatment. The C57BL/6 and C57BL/ 10ScSn demonstrated a significant resistance to the teratogen (i.e., 25% and 21 % incidence of clefting, respectively). The teratogenic treatment of congenic hybrids indicated that maternal influences significantly affected the incidence of cleft palate formation. The maternal influence appeared to depend upon the specificH-2 haplotype of the mother.