Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Analysis of candidate genes on chromosome 2 in oral cleft case-parent trios from three populations.

Isolated oral clefts, including cleft lip with/without cleft palate (CL/P) and cleft palate (CP), have a complex and heterogeneous etiology. Case-parent trios from three populations were used to study genes spanning chromosome 2, where single nucleotide polymorphic (SNP) markers were analyzed individually and as haplotypes. Case-parent trios from three populations (74 from Maryland, 64 from Singapore and 95 from Taiwan) were genotyped for 962 SNPs in 104 genes on chromosome 2, including two well-recognized candidate genes: TGFA and SATB2. Individual SNPs and haplotypes (in sliding windows of 2-5 SNPs) were used to test for linkage and disequilibrium separately in CL/P and CP trios. A novel candidate gene (ZNF533) showed consistent evidence of linkage and disequilibrium in all three populations for both CL/P and CP. SNPs in key regions of ZNF533 showed considerable variability in estimated genotypic odds ratios and their significance, suggesting allelic heterogeneity. Haplotype frequencies for regions of ZNF533 were estimated and used to partition genetic variance into among-and within-population components. Wright's fixation index, a measure of genetic diversity, showed little difference between Singapore and Taiwan compared with Maryland. The tensin-1 gene (TNS1) also showed evidence of linkage and disequilibrium among both CL/P and CP trios in all three populations, albeit at a lower level of significance. Additional genes (VAX2, GLI2, ZHFX1B on 2p; WNT6-WNT10A and COL4A3-COL4A4 on 2q) showed consistent evidence of linkage and disequilibrium only among CL/P trios in all three populations, and TGFA showed significant evidence in two of three populations.

High throughput SNP and expression analyses of candidate genes for non-syndromic oral clefts.

BACKGROUND: Recent work suggests that multiple genes and several environmental risk factors influence risk for non-syndromic oral clefts, one of the most common birth defects in humans. Advances in high-throughput genotyping technology now make it possible to test multiple markers in many candidate genes simultaneously. METHODS: We present findings from family based association tests of single nucleotide polymorphism (SNP) markers in 64 candidate genes genotyped using the BeadArray approach in 58 case-parent trios from Maryland (USA) to illustrate how multiple markers in multiple genes can be analysed. To assess whether these genes were expressed in human craniofacial structures relevant to palate and lip development, we also analysed data from the Craniofacial and Oral Gene Expression Network (COGENE) consortium, and searched public databases for expression profiles of these genes. RESULTS: Thirteen candidate genes showed significant evidence of linkage in the presence of disequilibrium, and ten of these were found to be expressed in relevant embryonic tissues: SP100, MLPH, HDAC4, LEF1, C6orf105, CD44, ALX4, ZNF202, CRHR1, and MAPT. Three other genes showing statistical evidence (ADH1C, SCN3B, and IMP5) were not expressed in the embryonic tissues examined here. CONCLUSIONS: This approach demonstrates how statistical evidence on large numbers of SNP markers typed in case-parent trios can be combined with expression data to identify candidate genes for complex disorders. Many of the genes reported here have not been previously studied as candidates for oral clefts and warrant further investigation.

Haplotype diversity in 11 candidate genes across four populations.

Analysis of haplotypes based on multiple single-nucleotide polymorphisms (SNP) is becoming common for both candidate gene and fine-mapping studies. Before embarking on studies of haplotypes from genetically distinct populations, however, it is important to consider variation both in linkage disequilibrium (LD) and in haplotype frequencies within and across populations, as both vary. Such diversity will influence the choice of "tagging" SNPs for candidate gene or whole-genome association studies because some markers will not be polymorphic in all samples and some haplotypes will be poorly represented or completely absent. Here we analyze 11 genes, originally chosen as candidate genes for oral clefts, where multiple markers were genotyped on individuals from four populations. Estimated haplotype frequencies, measures of pairwise LD, and genetic diversity were computed for 135 European-Americans, 57 Chinese-Singaporeans, 45 Malay-Singaporeans, and 46 Indian-Singaporeans. Patterns of pairwise LD were compared across these four populations and haplotype frequencies were used to assess genetic variation. Although these populations are fairly similar in allele frequencies and overall patterns of LD, both haplotype frequencies and genetic diversity varied significantly across populations. Such haplotype diversity has implications for designing studies of association involving samples from genetically distinct populations.

Testing candidate genes for non-syndromic oral clefts using a case-parent trio design.

Markers in five candidate genes were examined on 269 case-parent trios ascertained through a child with an isolated, non-syndromic oral cleft (cleft lip, CL; cleft palate, CP; or cleft lip and palate, CLP). Cases and their parents were ascertained through treatment centers in Maryland. Markers at two of the five candidate genes, transforming growth factor beta3 (TGFbeta3) and MSX1, showed consistent evidence of linkage and disequilibrium due to linkage using several statistical tests (e.g., the global chi-square for TGFbeta3 was 21.1 with 12 df, P = 0.03; that for MSX1 was 8.7 with 3 df, P = 0.03). There was little evidence of heterogeneity in the role of TGFbeta3 between different types of oral clefts, but MSX1 did yield marginal evidence for such heterogeneity. MSX1 also showed evidence for interaction between infant's genotype and maternal smoking, giving a likelihood ratio test for heterogeneity between smoker and non-smoker mothers of 7.16 (2 df, P = 0.03). Using a conditional logistic model to test for gene-gene interaction showed no evidence of interaction between TGFbeta3 and MSX1, with both seeming to contribute independently to risk of isolated, non-syndromic oral clefts.

A case-control study of nonsyndromic oral clefts in Maryland.

PURPOSE: Isolated, nonsyndromic oral clefts cases (n = 171) and unaffected controls (n = 182) were used to identify both genetic and environmental risk factors. METHODS: Infants born in Maryland between 1992 to 1998 with an isolated, nonsyndromic oral cleft [cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP)] were recruited and exposure plus family history data were collected. Controls were unaffected infants. DNA was collected from all cases and their parents, plus controls. RESULTS: No statistically significant association was found between any of the following: maternal smoking, vitamin use, urinary tract infection, or recreational drug use in either univariate analysis or after adjusting for maternal age and education. More control mothers reported alcohol use during the critical time period of pregnancy (one month before conception through the first trimester) as compared to case mothers. There was a 10-fold increase in risk to siblings of cases as compared to siblings of controls. Markers at four candidate genes were examined: transforming growth factor alpha (TGF alpha), transforming growth factor beta 3 (TGF beta 3), MSX1, and BCL3. Only MSX1 showed significant differences in allele frequencies between CP cases and controls. MSX1 also showed significant evidence of linkage disequilibrium with a susceptibility gene controlling risk for CP. CONCLUSION: Most environmental risk factors examined here gave little evidence of association with risk to isolated, nonsyndromic oral clefts, although any alcohol consumption seemed protective. MSX1 showed evidence of linkage disequilibrium in both case-control and case-parent trio analysis.

Genome-wide linkage analyses of total serum IgE using variance components analysis in asthmatic families.

Variance components models were used to analyze total IgE levels in families ascertained though the Collaborative Study of the Genetics of Asthma (CSGA) using a genome-wide array of polymorphic markers. While IgE levels are known to be associated with clinical asthma and recognized to be under strong genetic control (here the heritability was estimated at 44-60% in the three racial groups), specific genes influencing this trait are still largely unknown. Multipoint analysis of 323 markers yielded little indication of specific regions containing a trait locus controlling total serum IgE levels (adjusted for age and gender). Although a number of regions showed LOD statistics above 1.5 in Caucasian families (chromosome 4) and in African-American families (chromosomes 2 and 4), none yielded consistent evidence in all three racial groups. Analysis of total IgE adjusted for gender, age and Allergy Index (a quantitative score of skin test sensitivity to 14 common aeroallergens) was conducted on these data. In this analysis, a much stronger signal for a trait locus controlling adjusted log[total IgE] was seen on the telomeric end of chromosome 18, but only in Caucasian families. This region accounted for most of the genetic variation in log[total IgE], and may represent a quantitative trait locus for IgE levels independent of atopic response. Oligogenic analysis accounting simultaneously for the contribution of this locus on chromosome 18 and other chromosomal regions showing some evidence of linkage in these Caucasian families (on chromosomes 2, 4 and 20) failed to yield significant evidence for interaction.

Multilocus linkage analysis of the German asthma data.

We analyzed data from the German Asthma Genetics Group with three methods that utilize pedigree-specific nonparametric linkage scores to facilitate the search for multiple independent and interacting susceptibility loci. The three methods included a conditional analysis, logistic regression, and neural networks. Although there were differences, the three methods identified many of the same susceptibility loci. The most consistent evidence was provided for loci on chromosomes 1, 2, 6, 9, and 15. Both the conditional and the logistic regression analyses suggested an epistatic relationship between loci on chromosomes 2 and 9. The logistic regression analysis further revealed evidence for locus heterogeneity between loci on chromosomes 6 and 15. Finally, the neural network analysis identified a potential locus on chromosome 17 that was not identified in the other analyses.

A study of contemporary levels and temporal trends in inbreeding in the Tangier Island, Virginia, population using pedigree data and isonymy.

In this study we describe inbreeding in a large pedigree from Tangier Island, Virginia, in which we compare two commonly used methods to estimate inbreeding in humans: pedigree and isonymy (identical surnames of spouses). Genealogical data on 3,512 individuals dating back to 1722 were used. Using the pedigree method, we determined an average inbreeding coefficient (F) of 0.00873 for the community as a whole, and 0.018 for inbred individuals. Analysis of temporal trends showed that inbreeding began around 1800 and peaked at 0.0109 in 1824-1849 and 1875-1899. Thereafter, inbreeding steadily declined to 0.00565 in 1975-1997. Analysis of pedigree structure complexity over time showed that close consanguinity contributes to inbreeding in the earlier cohorts, and remote consanguinity accounts for much of the inbreeding in the later cohorts. The number of common ancestors increases over time, as does the number of paths connecting inbred individuals to these common ancestors. Inbreeding estimates based on the isonymy approach yielded a 2.2-fold higher value of F (0.01945) compared to the pedigree method. Total isonymy estimates over 25-year cohorts overestimated inbreeding values from pedigree data between 1. 5-8-fold. We speculate that the overestimation is probably due to the inability of our data to satisfy the method's assumption of monophyletic origin of each surname. In conclusion, inbreeding in the Tangier Island population is consistent with the isolated nature of its population, and temporal trends reflect patterns in emigration and a breakdown in isolation over time.

Testing for interaction between maternal smoking and TGFA genotype among oral cleft cases born in Maryland 1992-1996.

OBJECTIVE: Infants born in Maryland between June 1992 and June 1996 were used in a case-control study of nonsyndromic oral clefts to test for effects of maternal smoking and a polymorphic genetic marker at the transforming growth factor alpha (TGFA) locus, both of which have been reported to be risk factors for these common birth defects. DESIGN AND SETTING: Cases were infants with an oral cleft ascertained through three comprehensive treatment centers, with additional ascertainment through a registry of birth defects maintained by the Maryland Health Department. Controls were healthy infants. Medical history information on infants and mothers were collected, along with DNA samples. PATIENTS, PARTICIPANTS: Among 286 cases contacted (72% ascertainment), there were 192 nonsyndromic isolated oral clefts (106 M; 86 F) available for this case-control study. MAIN OUTCOME MEASURES: The largest group of 149 Caucasian nonsyndromic cases and 86 controls was used to test for association with maternal smoking and genotype at the Taq1 polymorphism in TGFA. RESULTS: While this modest sample had limited statistical power to detect gene-environment interaction, there was a significant marginal increase in risk of having an oral cleft if the mother smoked (odds ratio = 1.75, 95% CI = 1.01 to 3.02). We could not demonstrate statistical interaction between maternal smoking and TGFA genotype in this study, however, and the observed increase in the C2 allele among cases was not statistically significant. CONCLUSIONS: We could not confirm either the reported association between oral clefts and TGFA genotype or its interaction with maternal smoking. However, these data do show an increased risk if the mother smoked during pregnancy, and this effect was greatest among infants with a bilateral cleft and no close family history of clefts.