Astanand Jugessur

Disruption of an AP-2α binding site in an IRF6 enhancer is associated with cleft lip

Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P) is strongly associated with SNPs in IRF6 (interferon regulatory factor 6). Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A allele is significantly overtransmitted (P = 1 × 10−11) in families with NSCL/P, in particular those with cleft lip but not cleft palate. Further, there is a dosage effect of the A allele, with a relative risk for cleft lip of 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high-frequency variant in a regulatory element contributing substantially to a common, complex disorder.

Mutations in BMP4 Are Associated with Subepithelial, Microform, and Overt Cleft Lip

Cleft lip with or without cleft palate (CL/P) is a complex trait with evidence that the clinical spectrum includes both microform and subepithelial lip defects. We identified missense and nonsense mutations in the BMP4 gene in 1 of 30 cases of microform clefts, 2 of 87 cases with subepithelial defects in the orbicularis oris muscle (OOM), 5 of 968 cases of overt CL/P, and 0 of 529 controls. These results provide confirmation that microforms and subepithelial OOM defects are part of the spectrum of CL/P and should be considered during clinical evaluation of families with clefts. Furthermore, we suggest a role for BMP4 in wound healing.

Genetics of nonsyndromic orofacial clefts.

With an average worldwide prevalence of approximately 1.2/1000 live births, orofacial clefts are the most common craniofacial birth defects in humans. Like other complex disorders, these birth defects are thought to result from the complex interplay of multiple genes and environmental factors. Significant progress in the identification of underlying genes and pathways has benefited from large populations available for study, increased international collaboration, rapid advances in genotyping technology, and major improvements in analytic approaches. Here we review recent advances in genetic epidemiological approaches to complex traits and their applications to studies of nonsyndromic orofacial clefts. Our main aim is to bring together a discussion of new and previously identified candidate genes to create a more cohesive picture of interacting pathways that shape the human craniofacial region. In future directions, we highlight the need to search for copy number variants that affect gene dosage and rare variants that are possibly associated with a higher disease penetrance. In addition, sequencing of protein-coding regions in candidate genes and screening for genetic variation in noncoding regulatory elements will help advance this important area of research.

Genome Scan, Fine-Mapping, and Candidate Gene Analysis of Non-Syndromic Cleft Lip with or without Cleft Palate Reveals Phenotype-Specific Differences in Linkage and Association Results

Objectives: Non-syndromic orofacial clefts, i.e. cleft lip (CL) and cleft palate (CP), are among the most common birth defects. The goal of this study was to identify genomic regions and genes for CL with or without CP (CL/P). Methods: We performed linkage analyses of a 10 cM genome scan in 820 multiplex CL/P families (6,565 individuals). Significant linkage results were followed by association analyses of 1,476 SNPs in candidate genes and regions, utilizing a weighted false discovery rate (wFDR) approach to control for multiple testing and incorporate the genome scan results. Results: Significant (multipoint HLOD ≥3.2) or genome-wide-significant (HLOD ≥4.02) linkage results were found for regions 1q32, 2p13, 3q27-28, 9q21, 12p11, 14q21-24 and 16q24. SNPs in IRF6 (1q32) and in or near FOXE1 (9q21) reached formal genome-wide wFDR-adjusted significance. Further, results were phenotype dependent in that the IRF6 region results were most significant for families in which affected individuals have CL alone, and the FOXE1 region results were most significant in families in which some or all of the affected individuals have CL with CP. Conclusions: These results highlight the importance of careful phenotypic delineation in large samples of families for genetic analyses of complex, heterogeneous traits such as CL/P.

Genetic variants in IRF6 and the risk of facial clefts: single-marker and haplotype-based analyses in a population-based case-control study of facial clefts in Norway.

Mutations in the gene encoding interferon regulatory factor 6 (IRF6) underlie a common form of syndromic clefting known as Van der Woude syndrome. Lip pits and missing teeth are the only additional features distinguishing the syndrome from isolated clefts. Van der Woude syndrome, therefore, provides an excellent model for studying the isolated forms of clefting. From a population‐based case‐control study of facial clefts in Norway (1996–2001), we selected 377 cleft lip with or without cleft palate (CL/P), 196 cleft palate only (CPO), and 763 control infant‐parent triads for analysis. We genotyped six single nucleotide polymorphisms within the IRF6 locus and estimated the relative risks (RR) conferred on the child by alleles and haplotypes of the child and of the mother. On the whole, there were strong statistical associations with CL/P but not CPO in our data. In single‐marker analyses, mothers with a double‐dose of the ‘a’‐allele at rs4844880 had an increased risk of having a child with CL/P (RR=1.85, 95% confidence interval: 1.04–3.25; P=0.036). An RR of 0.38 (95% confidence interval: 0.16–0.92; P=0.031) was obtained when the child carried a single‐dose of the ‘a’‐allele at rs2235371 (the p.V274I polymorphism). The P‐value for the overall test was <0.001. In haplotype analyses, several of the fetal and maternal haplotype relative risks were statistically significant individually but were not strong enough to show up on the overall test (P=0.113). Taken together, these findings further support a role for IRF6 variants in clefting of the lip and provide specific risk estimates in a Norwegian population. Genet. Epidemiol. 2008.

Genetic Determinants of Facial Clefting: Analysis of 357 Candidate Genes Using Two National Cleft Studies from Scandinavia

Background Facial clefts are common birth defects with a strong genetic component. To identify fetal genetic risk factors for clefting, 1536 SNPs in 357 candidate genes were genotyped in two population-based samples from Scandinavia (Norway: 562 case-parent and 592 control-parent triads; Denmark: 235 case-parent triads). Methodology/Principal Findings We used two complementary statistical methods, TRIMM and HAPLIN, to look for associations across these two national samples. TRIMM tests for association in each gene by using multi-SNP genotypes from case-parent triads directly without the need to infer haplotypes. HAPLIN on the other hand estimates the full haplotype distribution over a set of SNPs and estimates relative risks associated with each haplotype. For isolated cleft lip with or without cleft palate (I-CL/P), TRIMM and HAPLIN both identified significant associations with IRF6 and ADH1C in both populations, but only HAPLIN found an association with FGF12. For isolated cleft palate (I-CP), TRIMM found associations with ALX3, MKX, and PDGFC in both populations, but only the association with PDGFC was identified by HAPLIN. In addition, HAPLIN identified an association with ETV5 that was not detected by TRIMM. Conclusion/Significance Strong associations with seven genes were replicated in the Scandinavian samples and our approach effectively replicated the strongest previously known association in clefting—with IRF6. Based on two national cleft cohorts of similar ancestry, two robust statistical methods and a large panel of SNPs in the most promising cleft candidate genes to date, this study identified a previously unknown association with clefting for ADH1C and provides additional candidates and analytic approaches to advance the field.

Resting heart rate and physical activity as risk factors for lone atrial fibrillation: a prospective study of 309 540 men and women

Objective To study the impact of resting heart rate and leisure time physical activity at middle age on long term risk of drug treated lone atrial fibrillation (AF). Design Longitudinal cohort study of 309 540 Norwegian men and women aged 40–45 years examined during 1985–1999 followed from 2005 through 2009. Setting Data from a national health screening programme were linked to the Norwegian Prescription Database (NorPD). Patients The cohort comprised 162 078 women and 147 462 men; 575 (0.4%) men and 288 women (0.2%) received flecainide and 568 men and 256 women sotalol and were defined as patients with AF. Interventions No interventions. Main outcome measures The outcome was lone fibrillation defined by having at least one prescription of flecainide or sotalol registered in NorPD between 2005 and 2009. Cox proportional hazard regression models were used to assess time to first prescription. Results The risk for being prescribed these drugs increased with decreasing baseline resting heart. Adjusted hazard ratio (HR) per 10 beats/min decrease in resting heart rate for flecainide prescription was 1.26 in men (95% CI 1.17 to 1.35) and 1.15 (95% CI 1.05 to 1.27) in women. Similar effects were seen for sotalol in men, but not in women. Men who reported intensive physical activity were more often prescribed flecainide than those in the sedentary group (adjusted HR=3.14, 95% CI 2.17 to 4.54). Conclusions This population based study supports the hypothesis that the risk of drug treated lone AF increases with declining resting heart rate in both sexes, and with increasing levels of self-reported physical activity in men.

Identification of microdeletions in candidate genes for cleft lip and/or palate

BACKGROUND Genome-wide association studies are now used routinely to identify genes implicated in complex traits. The panels used for such analyses can detect single nucleotide polymorphisms and copy number variants, both of which may help to identify small deleted regions of the genome that may contribute to a particular disease. METHODS We performed a candidate gene analysis involving 1,221 SNPs in 333 candidate genes for orofacial clefting, using 2,823 samples from 725 two- and three-generation families with a proband having cleft lip with or without cleft palate. We used SNP genotyping, DNA sequencing, high-resolution DNA microarray analysis, and long-range PCR to confirm and characterize the deletion events. RESULTS This dataset had a high duplicate reproducibility rate (99.98%), high Mendelian consistency rate (99.93%), and low missing data rate (0.55%), which provided a powerful opportunity for deletion detection. Apparent Mendelian inconsistencies between parents and children suggested deletion events in 15 individuals in 11 genomic regions. We confirmed deletions involving CYP1B1, FGF10, SP8, SUMO1, TBX1, TFAP2A, and UGT7A1, including both de novo and familial cases. Deletions of SUMO1, TBX1, and TFAP2A are likely to be etiologic. CONCLUSIONS These deletions suggest the potential roles of genes or regulatory elements contained within deleted regions in the etiology of clefting. Our analysis took advantage of genotypes from a candidate-gene-based SNP survey and proved to be an efficient analytical approach to interrogate genes potentially involved in clefting. This can serve as a model to find genes playing a role in complex traits in general.

Maternal Alcohol Consumption, Alcohol Metabolism Genes, and the Risk of Oral Clefts: A Population-based Case-Control Study in Norway, 1996–2001

Heavy maternal alcohol consumption during early pregnancy increases the risk of oral clefts, but little is known about how genetic variation in alcohol metabolism affects this association. Variants in the alcohol dehydrogenase 1C (ADH1C) gene may modify the association between alcohol and clefts. In a population-based case-control study carried out in Norway (1996-2001), the authors examined the association between maternal alcohol consumption and risk of oral clefts according to mother and infant ADH1C haplotypes encoding fast or slow alcohol-metabolizing phenotypes. Subjects were 483 infants with oral cleft malformations and 503 control infants and their mothers, randomly selected from all other livebirths taking place during the same period. Mothers who consumed 5 or more alcoholic drinks per sitting during the first trimester of pregnancy had an elevated risk of oral cleft in their offspring (odds ratio (OR) = 2.6, 95% confidence interval (CI): 1.4, 4.7). This increased risk was evident only in mothers or children who carried the ADH1C haplotype associated with reduced alcohol metabolism (OR= 3.0, 95% CI: 1.4, 6.8). There was no evidence of alcohol-related risk when both mother and infant carried only the rapid-metabolism ADH1C variant (OR = 0.9, 95% CI: 0.2, 4.1). The teratogenic effect of alcohol may depend on the genetic capacity of the mother and fetus to metabolize alcohol.

Oral facial clefts and gene polymorphisms in metabolism of folate/one-carbon and vitamin A: a pathway-wide association study.

An increased risk of facial clefts has been observed among mothers with lower intake of folic acid or vitamin A around conception. We hypothesized that the risk of clefts may be further moderated by genes involved in metabolizing folate or vitamin A. We included 425 case‐parent triads in which the child had either cleft lip with or without cleft palate (CL/P) or cleft palate only (CPO), and no other major defects. We analyzed 108 SNPs and one insertion in 29 genes involved in folate/one‐carbon metabolism and 68 SNPs from 16 genes involved in vitamin A metabolism. Using the Triad Multi‐Marker (TRIMM) approach we performed SNP, gene, chromosomal region, and pathway‐wide association tests of child or maternal genetic effects for both CL/P and CPO. We stratified these analyses on maternal intake of folic acid or vitamin A during the periconceptional period.