TNS1 and NRXN1 Genes Interacting With Early-Life Smoking Exposure in Asthma-Plus-Eczema Susceptibility.

PURPOSE: Numerous genes have been associated with allergic diseases (asthma, allergic rhinitis, and eczema), but they explain only part of their heritability. This is partly because most previous studies ignored complex mechanisms such as gene-environment (G-E) interactions and complex phenotypes such as co-morbidity. However, it was recently evidenced that the co-morbidity of asthma-plus-eczema appears as a sub-entity depending on specific genetic factors. Besides, evidence also suggest that gene-by-early life environmental tobacco smoke (ETS) exposure interactions play a role in asthma, but were never investigated for asthma-plus-eczema. To identify genetic variants interacting with ETS exposure that influence asthma-plus-eczema susceptibility. METHODS: To conduct a genome-wide interaction study (GWIS) of asthma-plus-eczema according to ETS exposure, we applied a 2-stage strategy with a first selection of single nucleotide polymorphisms (SNPs) from genome-wide association meta-analysis to be tested at a second stage by interaction meta-analysis. All meta-analyses were conducted across 4 studies including a total of 5,516 European-ancestry individuals, of whom 1,164 had both asthma and eczema. RESULTS: Two SNPs showed significant interactions with ETS exposure. They were located in 2 genes, NRXN1 (2p16) and TNS1 (2q35), never reported associated and/or interacting with ETS exposure for asthma, eczema or more generally for allergic diseases. TNS1 is a promising candidate gene because of its link to lung and skin diseases with possible interactive effect with tobacco smoke exposure. CONCLUSIONS: This first GWIS of asthma-plus-eczema with ETS exposure underlines the importance of studying sub-phenotypes such as co-morbidities as well as G-E interactions to detect new susceptibility genes.

Identification of novel genes influencing eosinophil-specific protein levels in asthma families.

BACKGROUND: Eosinophils play a key role in the asthma allergic response by releasing cytotoxic molecules such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) that generate epithelium damages. OBJECTIVE: We sought to identify genetic variants influencing ECP and EDN levels in asthma-ascertained families. METHODS: We performed univariate and bivariate genome-wide association analyses of ECP and EDN levels in 1018 subjects from the EGEA study with follow-up in 153 subjects from the Saguenay-Lac-Saint-Jean study and combined the results of these 2 studies through meta-analysis. We then conducted Bayesian statistical fine mapping together with quantitative trait locus and functional annotation analyses to identify the most likely functional genetic variants and candidate genes. RESULTS: We identified 5 genome-wide significant loci (P < 5 × 10^-8) including 7 distinct signals associated with ECP and/or EDN levels. The genes targeted by our fine mapping and functional search include RNASE2 and RNASE3 (14q11), which encode EDN and ECP, respectively, and 4 other genes that regulate ECP and EDN levels. These 4 genes were JAK1 (1p31), a transcription factor that plays a key role in the immune response and acts as a potential therapeutic target for eosinophilic asthma; ARHGAP25 (2p13), which is involved in leukocyte recruitment to inflammatory sites; NDUFA4 (7p21), which encodes a component of the mitochondrial respiratory chain and is involved in cellular response to stress; and CTSL (9q22), which is involved in immune response, extracellular remodeling, and allergic inflammation. CONCLUSION: Analysis of specific phenotypes produced by eosinophils allows the identification of genes that play a major role in allergic response and inflammation, and offers potential therapeutic targets for asthma.

Genome-Wide Association Study of Fluorescent Oxidation Products Accounting for Tobacco Smoking Status in Adults from the French EGEA Study.

Oxidative stress (OS) is the main pathophysiological mechanism involved in several chronic diseases, including asthma. Fluorescent oxidation products (FlOPs), a global biomarker of damage due to OS, is of growing interest in epidemiological studies. We conducted a genome-wide association study (GWAS) of the FlOPs level in 1216 adults from the case-control and family-based EGEA study (mean age 43 years old, 51% women, and 23% current smokers) to identify genetic variants associated with FlOPs. The GWAS was first conducted in the whole sample and then stratified according to smoking status, the main exogenous source of reactive oxygen species. Among the top genetic variants identified by the three GWAS, those located in BMP6 (p = 3 × 10-6), near BMPER (p = 9 × 10-6), in GABRG3 (p = 4 × 10-7), and near ATG5 (p = 2 × 10-9) are the most relevant because of both their link to biological pathways related to OS and their association with several chronic diseases for which the role of OS in their pathophysiology has been pointed out. BMP6 and BMPER are of particular interest due to their involvement in the same biological pathways related to OS and their functional interaction. To conclude, this study, which is the first GWAS of FlOPs, provides new insights into the pathophysiology of chronic OS-related diseases.

Identification of OCA2 as a novel locus for the co-morbidity of asthma-plus-eczema.

BACKGROUND: Numerous genes have been associated with the three most common allergic diseases (asthma, allergic rhinitis or eczema) but these genes explain only a part of the heritability. In the vast majority of genetic studies, complex phenotypes such as co-morbidity of two of these diseases, have not been considered. This may partly explain missing heritability. OBJECTIVE: To identify genetic variants specifically associated with the co-morbidity of asthma-plus-eczema. METHODS: We first conducted a meta-analysis of four GWAS (Genome-Wide Association Study) of the combined asthma-plus-eczema phenotype (total of 8807 European-ancestry subjects of whom 1208 subjects had both asthma and eczema). To assess whether the association with SNP(s) was specific to the co-morbidity, we also conducted a meta-analysis of homogeneity test of association according to disease status ("asthma-plus-eczema" vs. the presence of only one disease "asthma only or eczema only"). We then used a joint test by combining the two test statistics from the co-morbidity-SNP association and the phenotypic heterogeneity of SNP effect meta-analyses. RESULTS: Seven SNPs were detected for specific association to the asthma-plus-eczema co-morbidity, two with significant and five with suggestive evidence using the joint test after correction for multiple testing. The two significant SNPs are located in the OCA2 gene (Oculocutaneous Albinism II), a new locus never detected for significant evidence of association with any allergic disease. This gene is a promising candidate gene, because of its link to skin and lung diseases, and to epithelial barrier and immune mechanisms. CONCLUSION: Our study underlines the importance of studying sub-phenotypes as co-morbidities to detect new susceptibility genes.

PID1 is associated to a respiratory endotype related to occupational exposures to irritants.

BACKGROUND: Studying associations between genes and asthma endotypes and interactions with environment could help to identify new susceptibility genes. We used a previously identified asthma endotype characterized by adult-onset asthma, poor lung function, and high level of Fluorescent oxidation products, a marker of damages due to oxidative stress. This endotype was associated with high occupational exposure to irritants. We aimed to investigate the associations between genes related to oxidative stress and this endotype, and if the associations differed according to irritants exposure. METHODS: We conducted association analyses between the asthma endotype and genetic variants (4715 SNPs) located in 422 genes involved in the "response to oxidative stress" in adults from the Epidemiological study on the Genetic and Environment of Asthma. Analyses using logistic regression were conducted first in all participants, and then separately among high vs. non-exposed participants to assess whether association differs according to irritants exposure. RESULTS: An association was found between the SNP rs1419958 located in PID1 gene and the endotype (P = 2.2E-05), reaching significance level after correction for multiple testing. This association was even more significant in non-exposed participants (P = 1.06E-06) while there was no association in participants with high exposure to occupational irritants. CONCLUSION: This study showed a significant association between an asthma endotype and PID1, a promising candidate gene, the association being different according to the exposure to irritants. These results highlight the interest of studying asthma endotypes in association with genes from candidate pathways and their link with occupational irritants to decipher asthma etiology.

Interactive effect between ATPase-related genes and early-life tobacco smoke exposure on bronchial hyper-responsiveness detected in asthma-ascertained families.

BACKGROUND: A positional cloning study of bronchial hyper-responsiveness (BHR) at the 17p11 locus in the French Epidemiological study on the Genetics and Environment of Asthma (EGEA) families showed significant interaction between early-life environmental tobacco smoke (ETS) exposure and genetic variants located in DNAH9. This gene encodes the heavy chain subunit of axonemal dynein, which is involved with ATP in the motile cilia function.Our goal was to identify genetic variants at other genes interacting with ETS in BHR by investigating all genes belonging to the 'ATP-binding' and 'ATPase activity' pathways which include DNAH9, are targets of cigarette smoke and play a crucial role in the airway inflammation. METHODS: Family-based interaction tests between ETS-exposed and unexposed BHR siblings were conducted in 388 EGEA families. Twenty single-nucleotide polymorphisms (SNP) showing interaction signals (p≤5.10-3) were tested in the 253 Saguenay-Lac-Saint-Jean (SLSJ) families. RESULTS: One of these SNPs was significantly replicated for interaction with ETS in SLSJ families (p=0.003). Another SNP reached the significance threshold after correction for multiple testing in the combined analysis of the two samples (p=10-5). Results were confirmed using both a robust log-linear test and a gene-based interaction test. CONCLUSION: The SNPs showing interaction with ETS belong to the ATP8A1 and ABCA1 genes, which play a role in the maintenance of asymmetry and homeostasis of lung membrane lipids.

A comprehensive genome-wide analysis of melanoma Breslow thickness identifies interaction between CDC42 and SCIN genetic variants.

Breslow thickness (BT) is a major prognostic factor of cutaneous melanoma (CM), the most fatal skin cancer. The genetic component of BT has only been explored by candidate gene studies with inconsistent results. Our objective was to uncover the genetic factors underlying BT using an hypothesis-free genome-wide approach. Our analysis strategy integrated a genome-wide association study (GWAS) of single nucleotide polymorphisms (SNPs) for BT followed by pathway analysis of GWAS outcomes using the gene-set enrichment analysis (GSEA) method and epistasis analysis within BT-associated pathways. This strategy was applied to two large CM datasets with Hapmap3-imputed SNP data: the French MELARISK study for discovery (966 cases) and the MD Anderson Cancer Center study (1,546 cases) for replication. While no marginal effect of individual SNPs was revealed through GWAS, three pathways, defined by gene ontology (GO) categories were significantly enriched in genes associated with BT (false discovery rate ≤5% in both studies): hormone activity, cytokine activity and myeloid cell differentiation. Epistasis analysis, within each significant GO, identified a statistically significant interaction between CDC42 and SCIN SNPs (pmeta-int =2.2 × 10(-6) , which met the overall multiple-testing corrected threshold of 2.5 × 10(-6) ). These two SNPs (and proxies) are strongly associated with CDC42 and SCIN gene expression levels and map to regulatory elements in skin cells. This interaction has important biological relevance since CDC42 and SCIN proteins have opposite effects in actin cytoskeleton organization and dynamics, a key mechanism underlying melanoma cell migration and invasion.

Integrated pathway and epistasis analysis reveals interactive effect of genetic variants at TERF1 and AFAP1L2 loci on melanoma risk.

Genome-wide association studies (GWASs) have characterized 13 loci associated with melanoma, which only account for a small part of melanoma risk. To identify new genes with too small an effect to be detected individually but which collectively influence melanoma risk and/or show interactive effects, we used a two-step analysis strategy including pathway analysis of genome-wide SNP data, in a first step, and epistasis analysis within significant pathways, in a second step. Pathway analysis, using the gene-set enrichment analysis (GSEA) approach and the gene ontology (GO) database, was applied to the outcomes of MELARISK (3,976 subjects) and MDACC (2,827 subjects) GWASs. Cross-gene SNP-SNP interaction analysis within melanoma-associated GOs was performed using the INTERSNP software. Five GO categories were significantly enriched in genes associated with melanoma (false discovery rate ≤ 5% in both studies): response to light stimulus, regulation of mitotic cell cycle, induction of programmed cell death, cytokine activity and oxidative phosphorylation. Epistasis analysis, within each of the five significant GOs, showed significant evidence for interaction for one SNP pair at TERF1 and AFAP1L2 loci (pmeta-int = 2.0 × 10(-7) , which met both the pathway and overall multiple-testing corrected thresholds that are equal to 9.8 × 10(-7) and 2.0 × 10(-7) , respectively) and suggestive evidence for another pair involving correlated SNPs at the same loci (pmeta-int = 3.6 × 10(-6) ). This interaction has important biological relevance given the key role of TERF1 in telomere biology and the reported physical interaction between TERF1 and AFAP1L2 proteins. This finding brings a novel piece of evidence for the emerging role of telomere dysfunction into melanoma development.

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma.

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole-exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin gene POT1 (chromosome 7, g.124493086C>T; p.Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere lengths and numbers of fragile telomeres, suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two separate Italian families, one variant per family, yielding a frequency for POT1 variants comparable to that for CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in US and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Familial melanoma: clinical factors associated with germline CDKN2A mutations according to the number of patients affected by melanoma in a family.

BACKGROUND: Features associated with an increased frequency of cyclin-dependent kinase inhibitor 2A (CDKN2A) mutations have been identified in families with 3 or more patients with cutaneous melanoma (CM). However, in families with 2 patients with CM, which represent the majority of familial melanoma, these factors have been rarely studied. OBJECTIVE: We investigated association of 3 clinical features with the presence of a CDKN2A mutation in a family by extent of CM family clustering (2 vs ≥3 patients with CM among first-degree relatives in a family). METHODS: We included 483 French families that comprised 387 families with 2 patients with CM (F2 families) and 96 families with 3 or more patients with CM (F3+ families). Three clinical factors were examined individually and in a joint analysis: median age at diagnosis younger than 50 years, and 1 or more patient in a family with multiple primary melanoma or with pancreatic cancer. RESULTS: The frequency of CDKN2A mutations was higher in F3+ families (32%) than in F2 families (13%). Although early age at melanoma diagnosis and occurrence of multiple primary melanoma in 1 or more patient were significantly associated with the risk of a CDKN2A mutation in F2 families, early age at melanoma diagnosis and occurrence of pancreatic cancer in a family were significantly associated with CDKN2A mutations in F3+ families. LIMITATIONS: The study was not population based. CONCLUSIONS: This study shows that factors associated with CDKN2A mutations differ by extent of CM family clustering. It indicates that, in France, families with 2 patients with CM are eligible for genetic testing especially when there is an early age at CM diagnosis and/or 1 or more patients with multiple primary melanoma.

A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma.

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

Characteristics of the coexistence of melanoma and renal cell carcinoma.

BACKGROUND: Patients with melanoma (MM) have an increased risk of kidney cancer, and there is an excess risk of MM among patients with renal cell carcinoma (RCC). The objective of the current study was to analyze a series of 42 patients with both MM and RCC to identify clinical and pathologic features as well as risk factors of this association. METHODS: Clinical and pathologic characteristics of 42 patients who developed both MM and RCC (the MM + RCC series) were compared with 2 published series in each cancer alone: a series of 293 patients with MM (MM series) and a series of 1527 patients with RCC (RCC series). RESULTS: RCC was diagnosed concomitantly or after MM in 83% of patients in the MM + RCC series. Those patients displayed a high proportion of asymptomatic RCC at diagnosis (70%) and a higher frequency of stage I tumors (61%) than patients in the RCC series. Compared with the MM series, patients in the MM + RCC series more often were men, had a higher frequency of blond/red hair, had poor tanning ability, and had a higher number of nevi. In addition, patients in the MM + RCC series had a high aggregation of other malignancies (mainly skin cancers) and a significantly higher frequency of family history of MM (P = .005). Only 2 cyclin-dependent kinase 2A gene (CDKN2A) germline mutations were identified among patients in the MM + RCC series who also were members of MM-prone families. CONCLUSIONS: The high aggregation of cancers among patients in the MM + RCC series and the familial clustering of MM argued for a genetic predisposition that may be partly independent of CDKN2A.

MC1R variant alleles and malignant melanoma risk in Israel.

To evaluate the contribution of MC1R variants to malignant melanoma risk in Israeli Jews, sequencing of the MC1R gene was performed in 132 melanoma patients and 184 ethnically matched controls. Overall, 22 MC1R variants were detected, two were novel (M73I and 496_497insG). Using age and sex-adjusted logistic regression, one specific variant, R151C, conferred significantly increased melanoma risk among Ashkenazim (OR=2.6, 95% CI: 1.3-5.3; p=0.05 after Bonferroni correction). A gene dosage effect was noted, with significantly increased melanoma risk being observed in subjects with at least two variants whether when all variants are pooled (OR=4.8, 95% CI: 2.0-11.2; p=0.002 after Bonferroni correction) or when red hair colour (RHC) variants and non-RHC variants are distinguished (OR=7.6, 95% CI: 2.8-20.3; p=0.0004 after Bonferroni correction). If further studies support these findings, the assessment of MC1R status may be useful in identifying Jewish Israeli individuals at high risk for melanoma.