Evidence for an association between nonsyndromic cleft lip with or without cleft palate and a gene located on the long arm of chromosome 4.

Recent studies suggest that the familial aggregation of nonsyndromic cleft lip with or without cleft palate (CL +/- P) is likely to be attributable to the effects of several susceptibility loci, acting in a multiplicative fashion. Two potential CL +/- P susceptibility loci (CSL), transforming growth factor alpha (TGFA) and retinoic acid receptor (RARA), have been identified through association studies. In addition, recent evidence of linkage between CL +/- P and two markers (D4S175 and D4S192) in the region 4q25-4q31.3 raised the possibility that a CSL, with a larger effect than either TGFA or RARA, may reside within this region of the human genome. The present analyses were undertaken to determine whether D4S175 or D4S192 is significantly associated with CL +/- P in a sample of unrelated patients that have previously provided evidence of associations between CL +/- P and both TGFA and RARA. The results of these analyses provide further, tentative, evidence for the presence of a CSL locus on the long arm of chromosome 4 and help to refine the location of this locus in the region of D4S175 and D4S192.     

Genome scan for loci involved in cleft lip with or without cleft palate,in Chinese multiplex families

Cleft lip with or without cleft palate (CL/P) is a common congenital anomaly. Birth prevalences range from 1/500 to 1/1,000 and are consistently higher in Asian populations than in populations of European descent. Therefore, it is of interest to determine whether the CL/P etiological factors in Asian populations differ from those in white populations. A sample of 36 multiplex families were ascertained through probands with CL/P who were from Shanghai. This is the first reported genome-scan study of CL/P in any Asian population. Genotyping of Weber Screening Set 9 (387 short tandem-repeat polymorphisms with average spacing approximately 9 cM [range 1-19 cM]) was performed by the Mammalian Genotyping Service of Marshfield Laboratory. Presented here are the results for the 366 autosomal markers. Linkage between each marker and CL/P was assessed by two-point and multipoint LOD scores, as well as with multipoint heterogeneity LOD scores (HLODs) plus model-free identity-by-descent statistics and the multipoint NPL statistic. In addition, association was assessed via the transmission/disequilibrium test. LOD-score and HLOD calculations were performed under a range of models of inheritance of CL/P. The following regions had positive multipoint results (HLOD > or =1.0 and/or NPL P< or =.05): chromosomes 1 (90-110 cM), 2 (220-250 cM), 3 (130-150 cM), 4 (140-170 cM), 6 (70-100 cM), 18 (110 cM), and 21 (30-50 cM). The most significant multipoint linkage results (HLOD > or =2.0; alpha=0.37) were for chromosomes 3q and 4q. Associations with P< or =.05 were found for loci on chromosomes 3, 5-7, 9, 11, 12, 16, 20, and 21. The most significant association result (P=.009) was found with D16S769 (51 cM).     

SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate

Small ubiquitin-like modifier 1 (SUMO1) haploinsufficiency results in cleft lip and palate in animal models. However, no studies have linked SUMO1 to non-syndromic cleft lip with or without cleft palate (NSCLP) in humans. In the present study, we investigated the potential association between SUMO1 single nucleotide polymorphisms (SNPs) and risk for human NSCLP. From 181 patients and 162 healthy controls, we found statistically significant correlations between a 4-SNP SUMO1 haplotype and NSCLP. These data are the first to suggest a role for SUMO1 gene variation in human NSCLP development.     

Evidence of a sex-dependent association between the MSX1 locus and nonsyndromic cleft lip with or without cleft palate in the Chilean population

Prior studies have implicated an involvement of the Msx1 homeobox gene in cleft palate in mice and its homolog in humans (called MSX1 in the HOX7 gene, located on chromosome 4). In this study we present evidence of a sex-dependent association between MSX1 and non-syndromic cleft lip/palate (NSCLP) in the Chilean population. The sample included 73 NSCLP cases, 37 from multiplex families (Mx), 36 from simplex families (Sx), and 87 controls. Polymerase chain reaction amplification of the MSX1 intragenic microsatellite (CA)n-sequence shows significant (p = 0.035) differences in the allele frequencies between NSCLP-Mx males and control males. These differences are mainly due to frequency differences in allele *2 (173 base pairs) among cases (21.9%) and controls (13.2%). When the NSCLP cases are subdivided by sex and positive family history (Mx versus Sx), the Mx males (27.8%) as well as the total NSCLP-Mx cases (25.7%) showed significantly higher frequencies of allele *2, compared to controls (11.4% and 13.2%, respectively). Analysis of the genotype data indicates that the relative risk for NSCLP is greater for persons carrying allele *2 (i.e., odds ratio [OR] larger than 1), reaching significance for all Mx cases (OR = 2.67; 95% confidence interval [CI], 1.10 to 6.52) and even more pronounced for Mx males (OR = 3.33; 95% CI, 1.08 to 10.32). Taken together, these findings support the hypothesis that the genetic variation at the MSX1 locus is a predisposing gene involved in sex-dependent susceptibility to clefting and that it also differentiates simplex from multiplex families.     

Mouse genetic models of cleft lip with or without cleft palate

Nonsyndromic cleft lip and palate (CLP) is among the most common human birth defects. Transmission patterns suggest that the causes are "multifactorial" combinations of genetic and nongenetic factors, mostly distinct from those causing cleft secondary palate (CP). The major etiological factors are largely unknown, and the embryological mechanisms are not well understood. In contrast to CP or neural tube defects (NTD), CLP is uncommon in mouse mutants. Fourteen known mutants or strains express CLP, often as part of a severe syndrome, whereas nonsyndromic CLP is found in two conditional mutants and in two multifactorial models based on a hypomorphic variant with an epigenetic factor. This pattern suggests that human nonsyndromic CLP is likely caused by regulatory and hypomorphic gene variants, and may also involve epigenetics. The developmental pathogenic mechanism varies among mutants and includes deficiencies of growth of the medial, lateral or maxillary facial prominences, defects in the fusion process itself, and shifted midline position of the medial prominences. Several CLP mutants also have NTD, suggesting potential genetic overlap of the traits in humans. The mutants may reflect two interacting sets of genetic signaling pathways: Bmp4, Bmpr1a, Sp8, and Wnt9b may be in one set, and Tcfap2a and Sox11 may be in another. Combining the results of chromosomal linkage studies of unidentified human CLP genes with insights from the mouse models, the following previously unexamined genes are identified as strong candidate genes for causative roles in human nonsyndromic CLP: BMP4, BMPR1B, TFAP2A, SOX4, WNT9B, WNT3, and SP8.     

Familial recurrence-pattern analysis of cleft lip with or without cleft palate.

Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with an incidence in European white populations of about 1/1,000. The familial clustering of CL/P has been extensively characterized, and epidemiological studies have proposed monogenic models (with reduced penetrance), multifactorial/threshold models, and mixed major-gene/multifactorial models to explain its inheritance. The recognition of an association between two RFLPs at the transforming growth factor alpha (TGFA) locus and CL/P supports a major-gene component to the etiology of CL/P. Risch has shown that the recurrence risk ratio lambda R (risk to relatives, vs. population prevalence) is a useful pointer to the mode of inheritance. Here we further develop the use of lambda R to analyze recurrence-risk data for CL/P. Recurrence risks for first-, second-, and third-degree relatives equate well with oligogenic models with as few as four loci. A monogenic/additive model is strongly rejected. The limited available twin data are also consistent with this model. A "major gene" interacting epistatically with an oligogenic background is shown to be a plausible alternative. Power calculations for a linkage study to map the CL/P major-risk locus suggest that a sample of 50 affected sib pairs will be adequate, but linkage to minor-risk loci will require very much larger samples.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Genetic risk factors for nonsyndromic cleft lip with or without cleft palate in a Mesoamerican population: Evidence for IRF6 and variants at 8q24 and 10q25

INTRODUCTION: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common of all birth defects. NSCL/P has a multifactorial etiology that includes both genetic and environmental factors. The IRF6 gene and three further susceptibility loci at 8q24, 10q25, and 17q22, which were identified by a recent genome‐wide association scan (GWAS), are confirmed genetic risk factors for NSCL/P in patients of European descent. METHODS: A case‐control association study was performed to investigate whether these four risk loci contribute to NSCL/P in a Mesoamerican population using four single nucleotide polymorphisms to represent IRF6 and the three novel susceptibility loci. A total of 149 NSCL/P patients and 303 controls of Mayan origin were included. RESULTS: Single marker analysis revealed a significant association between NSCL/P and risk variants in IRF6 and the 8q24 and 10q25 loci. In contrast to previous findings, the association at the 8q24 locus was driven solely by homozygote carriers of the risk allele. This suggests that this locus might act in a recessive manner in the Mayan population. No evidence for association was found at the 17q22 locus. This may have been attributable to the limited power of the sample. CONCLUSION: These results suggest that IRF6 and the 10q25 and 8q24 loci confer a risk for the development of NSCL/P in persons of Mayan origin. Birth Defects Research (Part A), 2010. © 2010 Wiley‐Liss, Inc.     

Evidence, from family studies, for linkage disequilibrium between TGFA and a gene for nonsyndromic cleft lip with or without cleft palate.

The inheritance of alleles of the transforming growth factor alpha (TGFA) locus has been studied in families affected with cleft lip with or without cleft palate (CL/P), by using the transmission/disequilibrium test described by Spielman and colleagues. Only heterozygous parents with an affected child can be included in this test, but within such families a significantly greater frequency of C2 alleles were transmitted to affected children than would be expected by chance. There was no evidence that the total number of C2 alleles transmitted to affected and unaffected children differed significantly from random segregation. These data provide evidence from within families that a gene for susceptibility to CL/P is in significant linkage disequilibrium with the C2 allele of the TGFA locus.     

Nonsyndromic cleft lip with or without cleft palate: evidence of linkage to BCL3 in 17 multigenerational families.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common craniofacial developmental defect. Recent segregation analyses have suggested that major genes play a role in the etiology of CL/P. Linkage to 22 candidate genes was tested in 11 multigenerational families with CL/P, and 21 of these candidates were excluded. APOC2, 19q13.1, which is linked to the proto-oncogene BCL3, gave suggestive evidence for linkage to CL/P. The study was expanded to include a total of 39 multigenerational CL/P families. Linkage was tested in all families, using an anonymous marker, D19S178, and intragenic markers in BCL3 and APOC2. Linkage was tested under two models, autosomal dominant with reduced penetrance and affecteds only. Homogeneity testing on the two-point data gave evidence of heterogeneity at APOC2 under the affecteds-only model. Both models showed evidence of heterogeneity, with 43% of families linked at zero recombination to BCL3 when marker data from BCL3 and APOC2 were included. A maximum multipoint LOD score of 7.00 at BCL3 was found among the 17 families that had posterior probabilities > = 50% in favor of linkage. The transmission disequilibrium test provided additional evidence for linkage with the 3 allele of BCL3 more often transmitted to affected children. These results suggest that BCL3, or a nearby gene, plays a role in the etiology of CL/P in some families.     

Racial differences in cephalometric measurements and incidence of cleft lip with or without cleft palate

A study was made to search for the morphological basis and, indirectly, the developmental basis for racial differences in risk of cleft lip with or without cleft palate CL(P) in Hawaii. A total of ten linear and three angular measurements read from anterior-posterior cephalographs were examined on 210 healthy adult Caucasian, Hawaiian, Japanese, Chinese, and Filipino subjects. Racial comparisons were made on these variables after adjusting for age and sex. Generally, the CL(P) high-risk group consisting of Japanese, Chinese, and Filipinos had smaller dimensions than Caucasians and Hawaiians in the variables (S-N, N-Ba) representing size of cranial base and the measurements of face height (N-A, N-ANS), palatal length (ANS-PNS), and mandibular length (Ar-Gn). Facial width relative to palatal length as measured by the ratio of bizygomatic diameter to palatal length showed a marked consistency with the racial differences in CL(P) risk. Possible significance of this finding is discussed in relation to development of the orofacial structure.     

Stress-coping and cortisol analysis in patients with non-syndromic cleft lip and palate: an explorative study

Background

Non-syndromic clefts of the orofacial region occur in approximately 1 per 500 to 2,500 live births, depending on geographical area and ethnicity. It can be supposed that the disruption of the normal facial structure and the long-standing pressure of treatment from birth to adulthood bring about a range of life stressors which may lead to a long-lasting impact on affected subjects throughout their lives. Therefore, the present study aimed to assess different aspects of psychosocial stress in affected individuals.

Methods

The study was divided into two parts: first, the Trier Social Stress Test which involves uncontrollability and high levels of social-evaluative stress under real conditions and second, the query of various aspects of coping with psychosocial stress. The test group consisted of 30 affected adult subjects, and an equally sized control group of unaffected volunteers. Cortisol dysregulation was determined by saliva samples before and after stress induction. Meanwhile, participants were asked to complete the SVF 120 stress-coping questionnaire.

Results

The analysis of saliva samples showed a similar baseline concentration as well as a similar increase in cortisol levels after stress induction for both groups. Subsequently, the decline in cortisol concentrations was significantly faster in the CLP group (course: p<0.001; groups: p = 0.102; interaction: p = 0.167). The evaluation of the stress-coping questionnaire revealed a significantly shorter rumination about a stressful event in individuals with CLP-related malformations (p = 0.03).

Conclusion

We conclude that adults with CLP have significantly better stress-coping strategies than their healthy peers.

Trial Registration

German Clinical Trials Organization DRKS00003466