Emma L. Ivansson

Genome-wide Association Study of Susceptibility Loci for Cervical Cancer

BACKGROUND Cervical carcinoma has a heritable genetic component, but the genetic basis of cervical cancer is still not well understood. METHODS We performed a genome-wide association study of 731 422 single nucleotide polymorphisms (SNPs) in 1075 cervical cancer case subjects and 4014 control subjects and replicated it in 1140 case subjects and 1058 control subjects. The association between top SNPs and cervical cancer was estimated by odds ratios (ORs) and 95% confidence intervals (CIs) with unconditional logistic regression. All statistical tests were two-sided. RESULTS Three independent loci in the major histocompatibility complex (MHC) region at 6p21.3 were associated with cervical cancer: the first is adjacent to the MHC class I polypeptide-related sequence A gene (MICA) (rs2516448; OR = 1.42, 95% CI = 1.31 to 1.54; P = 1.6×10(-18)); the second is between HLA-DRB1 and HLA-DQA1 (rs9272143; OR = 0.67, 95% CI = 0.62 to 0.72; P = 9.3×10(-24)); and the third is at HLA-DPB2 (rs3117027; OR=1.25, 95% CI = 1.15 to 1.35; P = 4.9×10(-8)). We also confirmed previously reported associations of B*0702 and DRB1*1501-DQB1*0602 with susceptibility to and DRB1*1301-DQA1*0103-DQB1*0603 with protection against cervical cancer. The three new loci are statistically independent of these specific human leukocyte antigen alleles/haplotypes. MICA encodes a membrane-bound protein that acts as a ligand for NKG2D to activate antitumor effects. The risk allele of rs2516448 is in perfect linkage disequilibrium with a frameshift mutation (A5.1) of MICA, which results in a truncated protein. Functional analysis shows that women carrying this mutation have lower levels of membrane-bound MICA. CONCLUSIONS Three novel loci in the MHC may affect susceptibility to cervical cancer in situ, including the MICA-A5.1 allele that may cause impaired immune activation and increased risk of tumor development.

Variants of chemokine receptor 2 and interleukin 4 receptor, but not interleukin 10 or Fas ligand, increase risk of cervical cancer

Cervical cancer is caused by persistent infection of oncogenic human papillomavirus (HPV). Most infected women clear the virus without developing cervical lesions and it is likely that immunological host factors affect susceptibility to cervical cancer. The impact of the human leukocyte antigen (HLA) locus on the risk of cervical cancer is established and several other genes involved in immunological pathways have been suggested as biologically plausible candidates. The aim of this study was to examine the potential role of polymorphisms in 4 candidate genes by analysis of 1,306 familial cervical cancer cases and 288 controls. The following genes and polymorphisms were studied: Chemokine receptor 2 (CCR‐2) V64I; Interleukin 4 receptor α (IL‐4R) I75V, S503P and Q576R; Interleukin 10 (IL‐10) −592; and Fas ligand (FasL) −844. The CCR‐2 64I variant was associated with decreased risk of cervical cancer; homozygote carriers of the 64I variant had an odds ratio of 0.31 (0.12–0.77). This association was detected in both carriers and noncarriers of the HLA DQB1*0602 cervical cancer risk allele. The IL‐4R 75V variant was associated with increased risk of cervical tumors, cases homozygote for 75V had an odds ratio of 1.91 (1.27–2.86) with a tendency that the association was stronger in noncarriers of the DQB1*0602 allele. We did not find any association for IL‐10 −592, or FasL −844, previously reported to be associated with cervical cancer in the Dutch and Chinese populations, respectively.

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

BackgroundCanine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible.ResultsThrough genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors.ConclusionsMapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease.

Interaction of immunological genes on chromosome 2q33 and IFNG in susceptibility to cervical cancer.

OBJECTIVE Cervical cancer is caused by persistent infection with human papillomavirus and genetic susceptibility factors may augment disease risk. The immune response consists of complex interactions and it was recently proposed that the association of combinations of genotypes at several genes should be examined. In support of this the combination CD28+17(TT)/IFNG+874(AA) was shown to increase cervical cancer risk in a Brazilian population (VB Guzman et al. New approach reveals CD28 and IFNG gene interaction in the susceptibility to cervical cancer. Hum Mol Genet 2008;17:1838-44) and our aim was to replicate this finding. METHODS We re-examined the proposed associations by analysis of polymorphisms at CD28, IFNG, TNF, PDCD1, ICOS and CTLA4 in 1306 Swedish cases and 811 controls. RESULTS Logistic regression analysis detected association at single SNP level for CD28+17 (p=0.01), IFNG+874 (p=0.02), and PDCD1+7785 (p=0.04). The two locus combination CD28+17(TT)/IFNG+874(AA) (OR=0.76 (0.60-0.96, empirical p=0.03) and the three-locus combination CD28+17(TT)/IFNG+874(AA)/ICOS+1564(TT) (OR=0.65(0.49-0.87), empirical p=0.006) were associated with decreased risk. The strongest association was detected for the combination CTLA4-319 (CC)/IFNG (AA) (OR=0.67(0.53-0.84), empirical p=0.0007). CONCLUSION The observation that these combinations of loci are associated in different populations supports their importance in cervical cancer development although the opposite directions of the effect call for clarification. The polymorphisms studied might not be the functional variants per se, but linked to those exerting a functional effect. The opposite associations in the two populations could then be explained by differences in linkage disequilibrium and population structure.

Breed Distribution of SOD1 Alleles Previously Associated with Canine Degenerative Myelopathy

Background Previous reports associated 2 mutant SOD1 alleles (SOD1:c.118A and SOD1:c.52T) with degenerative myelopathy in 6 canine breeds. The distribution of these alleles in other breeds has not been reported. Objective To describe the distribution of SOD1:c.118A and SOD1:c.52T in 222 breeds. Animals DNA from 33,747 dogs was genotyped at SOD1:c.118,SOD1:c.52, or both. Spinal cord sections from 249 of these dogs were examined. Methods Retrospective analysis of 35,359 previously determined genotypes at SOD1:c.118G>A or SOD1:c.52A>T and prospective survey to update the clinical status of a subset of dogs from which samples were obtained with a relatively low ascertainment bias. Results The SOD1:c.118A allele was found in cross‐bred dogs and in 124 different canine breeds whereas the SOD1:c.52T allele was only found in Bernese Mountain Dogs. Most of the dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A homozygotes, but 8 dogs with histopathologically confirmed degenerative myelopathy were SOD1:c.118A/G heterozygotes and had no other sequence variants in their SOD1 amino acid coding regions. The updated clinical conditions of dogs from which samples were obtained with a relatively low ascertainment bias suggest that SOD1:c.118A homozygotes are at a much higher risk of developing degenerative myelopathy than are SOD1:c.118A/G heterozygotes. Conclusions and Clinical Importance We conclude that the SOD1:c.118A allele is widespread and common among privately owned dogs whereas the SOD1:c.52T allele is rare and appears to be limited to Bernese Mountain Dogs. We also conclude that breeding to avoid the production of SOD1:c.118A homozygotes is a rational strategy.

MHC loci affecting cervical cancer risk: distinguishing the effects of HLA-DQB1 and non-HLA genes TNF, LTA, TAP1 and TAP2.

Cervical cancer has been associated with specific human leukocyte antigen (HLA) haplotypes/alleles and with polymorphisms at the nearby non-HLA loci TNF, LTA, TAP1 and TAP2. Distinguishing effects of individual loci in the major histocompatibility complex (MHC) region are difficult due to the complex linkage disequilibrium (LD) pattern characterized by high LD, punctuated by recombination hot spots. We have evaluated the association of polymorphism at HLA class II DQB1 and the TNF, LTA, TAP1 and TAP2 genes with cervical cancer risk, using 1306 familial cases and 288 controls. DQB1 was strongly associated; alleles *0301, *0402 and *0602 increased cancer susceptibility, whereas *0501 and *0603 decreased susceptibility. Among the non-HLA loci, association was only detected for the TAP2 665 polymorphism, and interallelic disequilibrium analysis indicated that this could be due to LD with DQB1. As the TAP2 665 association was seen predominantly in non-carriers of DQB1 susceptibility alleles, we hypothesized that TAP2 665 may have an effect not attributable to LD with DQB1. However, a logistic regression analysis suggested that TAP2 665 was strongly influenced by LD with DQB1. Our results emphasize the importance of large sample sizes and underscore the necessity of examining both HLA and non-HLA loci in the MHC to assign association to the correct locus.

Pathway-based analysis of genetic susceptibility to cervical cancer in situ: HLA-DPB1 affects risk in Swedish women.

We have conducted a pathway-based analysis of genome-wide single-nucleotide polymorphism (SNP) data in order to identify genetic susceptibility factors for cervical cancer in situ. Genotypes derived from Affymetrix 500k or 5.0 arrays for 1076 cases and 1426 controls were analyzed for association, and pathways with enriched signals were identified using the SNP ratio test. The most strongly associated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were Asthma (empirical P=0.03), Folate biosynthesis (empirical P=0.04) and Graft-versus-host disease (empirical P=0.05). Among the 11 top-ranking pathways were 6 related to the immune response with the common denominator being genes in the major histocompatibility complex (MHC) region on chromosome 6. Further investigation of the MHC revealed a clear effect of HLA-DPB1 polymorphism on disease susceptibility. At a functional level, DPB1 alleles associated with risk and protection differ in key amino-acid residues affecting peptide-binding motifs in the extracellular domains. The results illustrate the value of pathway-based analysis to mine genome-wide data, and point to the importance of the MHC region and specifically the HLA-DPB1 locus for susceptibility to cervical cancer.

Contribution of TMC6 and TMC8 (EVER1 and EVER2) variants to cervical cancer susceptibility

Cervical cancer (CxCa) is caused by persistent human papillomavirus (HPV) infection; genetic predisposition is also suspected to play a role. Our study is a targeted candidate gene follow‐up based on: (i) strong clinical evidence demonstrating that mutations in the TMC6 and TMC8 (EVER1 and EVER2) genes associate with the HPV‐associated disease epidermodysplasia verruciformis (EV) and (ii) recent epidemiological data suggesting a genetic susceptibility conferred by polymorphisms in such genes for skin and CxCa. Clarifying the association of the TMC6/8 genes with risk of CxCa will help in understanding why some HPV‐infected women develop persistent infection, cervical lesions and eventually cancer while others do not. Twenty‐two single nucleotide polymorphisms (SNPs) harboring the TMC6/8 genes were genotyped in 2,989 cases with cervical intraepithelial neoplasia grade III or invasive CxCa and 2,281 controls from the Swedish population. Association was evaluated in logistic regression models. Two SNPs displayed association with cervical disease: rs2290907 [odds ratio (OR)GGvsAA = 0.6, 95% confidence interval (95% CI): 0.3–0.9, p = 0.02)] and rs16970849 (ORAGvsGG = 0.8, 95% CI: 0.66–0.98, p = 0.03). The present data support the involvement of the TMC6/8 region in CxCa susceptibility but further analyses are needed to replicate our findings, fully characterize the region and understand the function of the genetic variants involved.

Temporal trends over 3 decades and intrafamilial clustering of HPV types in Swedish patients with cervical cancer in situ

Information on HPV type distribution in cervical cancer in situ in different populations is needed for evaluation of prophylactic vaccination programs targeting HPV 16 and 18. In our study, the HPV type prevalence in 1,079 Swedish women from multicase families diagnosed with cervical cancer in situ 1965–1993 was investigated using real‐time PCR and archival tissue material. HPV type information was obtained for 974 samples. Among these, HPV 16 (61%) was the dominant type followed by HPV 33/52/58 (24%), HPV 31 (13%) and HPV 18/45 (12%). The detected prevalence of HPV 16 among cancer in situ decreased by 13% over the study period while the group of low frequency high‐risk types increased. Related women were not prone to infection by the same type. These data suggest that the prevalence of individual HPV types has changed over time in Swedish patients with cervical cancer in situ. Large‐scale studies of pathology biobank materials will enable further insight into the temporal changes of individual HPV types, as baseline information to properly evaluate the effect of vaccine programs. The findings also indicate that genetic susceptibility to cervical cancer operates through general and not type specific susceptibility to HPV infection.

TGFβ Receptor 1: An Immune Susceptibility Gene in HPV-Associated Cancer

Only a minority of those exposed to human papillomavirus (HPV) develop HPV-related cervical and oropharyngeal cancer. Because host immunity affects infection and progression to cancer, we tested the hypothesis that genetic variation in immune-related genes is a determinant of susceptibility to oropharyngeal cancer and other HPV-associated cancers by performing a multitier integrative computational analysis with oropharyngeal cancer data from a head and neck cancer genome-wide association study (GWAS). Independent analyses, including single-gene, gene-interconnectivity, protein-protein interaction, gene expression, and pathway analysis, identified immune genes and pathways significantly associated with oropharyngeal cancer. TGFβR1, which intersected all tiers of analysis and thus selected for validation, replicated significantly in the head and neck cancer GWAS limited to HPV-seropositive cases and an independent cervical cancer GWAS. The TGFβR1 containing p38-MAPK pathway was significantly associated with oropharyngeal cancer and cervical cancer, and TGFβR1 was overexpressed in oropharyngeal cancer, cervical cancer, and HPV(+) head and neck cancer tumors. These concordant analyses implicate TGFβR1 signaling as a process dysregulated across HPV-related cancers. This study demonstrates that genetic variation in immune-related genes is associated with susceptibility to oropharyngeal cancer and implicates TGFβR1/TGFβ signaling in the development of both oropharyngeal cancer and cervical cancer. Better understanding of the immunogenetic basis of susceptibility to HPV-associated cancers may provide insight into host/virus interactions and immune processes dysregulated in the minority of HPV-exposed individuals who progress to cancer.