Carine Bonnard

A Genome-Wide Association Study Identifies Novel and Functionally Related Susceptibility Loci for Kawasaki Disease

Kawasaki disease (KD) is a pediatric vasculitis that damages the coronary arteries in 25% of untreated and approximately 5% of treated children. Epidemiologic data suggest that KD is triggered by unidentified infection(s) in genetically susceptible children. To investigate genetic determinants of KD susceptibility, we performed a genome-wide association study (GWAS) in 119 Caucasian KD cases and 135 matched controls with stringent correction for possible admixture, followed by replication in an independent cohort and subsequent fine-mapping, for a total of 893 KD cases plus population and family controls. Significant associations of 40 SNPs and six haplotypes, identifying 31 genes, were replicated in an independent cohort of 583 predominantly Caucasian KD families, with NAALADL2 (rs17531088, p combined = 1.13×10−6) and ZFHX3 (rs7199343, p combined = 2.37×10−6) most significantly associated. Sixteen associated variants with a minor allele frequency of >0.05 that lay within or close to known genes were fine-mapped with HapMap tagging SNPs in 781 KD cases, including 590 from the discovery and replication stages. Original or tagging SNPs in eight of these genes replicated the original findings, with seven genes having further significant markers in adjacent regions. In four genes (ZFHX3, NAALADL2, PPP1R14C, and TCP1), the neighboring markers were more significantly associated than the originally associated variants. Investigation of functional relationships between the eight fine-mapped genes using Ingenuity Pathway Analysis identified a single functional network (p = 10−13) containing five fine-mapped genes—LNX1, CAMK2D, ZFHX3, CSMD1, and TCP1—with functional relationships potentially related to inflammation, apoptosis, and cardiovascular pathology. Pair-wise blood transcript levels were measured during acute and convalescent KD for all fine-mapped genes, revealing a consistent trend of significantly reduced transcript levels prior to treatment. This is one of the first GWAS in an infectious disease. We have identified novel, plausible, and functionally related variants associated with KD susceptibility that may also be relevant to other cardiovascular diseases.

Genetic Association and Expression Studies Indicate a Role of Toll-Like Receptor 8 in Pulmonary Tuberculosis

Despite high rates of exposure, only 5–10% of people infected with Mycobacterium tuberculosis will develop active tuberculosis (TB) disease, suggesting a significant role for genetic variation in the human immune response to this infection. Here, we studied TB association and expression of 18 genes involved in the Toll-like receptor (TLR) pathways. Initially, we genotyped 149 sequence polymorphisms in 375 pulmonary TB patients and 387 controls from Indonesia. We found that four polymorphisms in the TLR8 gene on chromosome X showed evidence of association with TB susceptibility in males, including a non-synonymous polymorphism rs3764880 (Met1Val; P = 0.007, odds ratio (OR) = 1.8, 95% c.i. = 1.2–2.7). We genotyped these four TLR8 polymorphisms in an independent collection of 1,837 pulmonary TB patients and 1,779 controls from Russia and again found evidence of association in males (for rs3764880 P = 0.03, OR = 1.2, 95% c.i. = 1.02–1.48). Combined evidence for association is P = 1.2×10−3–6×10−4. In addition, a quantitative PCR analysis indicated that TLR8 transcript levels are significantly up-regulated in patients during the acute phase of disease (P = 9.36×10−5), relative to baseline levels following successful chemotherapy. A marked increase in TLR8 protein expression was also observed directly in differentiated macrophages upon infection with M. bovis bacille Calmette-Guérin (BCG). Taken together, our results provide evidence, for the first time, of a role for the TLR8 gene in susceptibility to pulmonary TB across different populations.

New genetic associations detected in a host response study to hepatitis B vaccine

The immune response to hepatitis B vaccination differs greatly among individuals, with 5–10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 × 10−10; OR (95%CI)=0.61 (0.52–0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3′ downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 × 10−6; OR (95%CI)=1.38 (1.2–1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

Multi-variant pathway association analysis reveals the importance of genetic determinants of estrogen metabolism in breast and endometrial cancer susceptibility.

Despite the central role of estrogen exposure in breast and endometrial cancer development and numerous studies of genes in the estrogen metabolic pathway, polymorphisms within the pathway have not been consistently associated with these cancers. We posit that this is due to the complexity of multiple weak genetic effects within the metabolic pathway that can only be effectively detected through multi-variant analysis. We conducted a comprehensive association analysis of the estrogen metabolic pathway by interrogating 239 tagSNPs within 35 genes of the pathway in three tumor samples. The discovery sample consisted of 1,596 breast cancer cases, 719 endometrial cancer cases, and 1,730 controls from Sweden; and the validation sample included 2,245 breast cancer cases and 1,287 controls from Finland. We performed admixture maximum likelihood (AML)–based global tests to evaluate the cumulative effect from multiple SNPs within the whole metabolic pathway and three sub-pathways for androgen synthesis, androgen-to-estrogen conversion, and estrogen removal. In the discovery sample, although no single polymorphism was significant after correction for multiple testing, the pathway-based AML global test suggested association with both breast (p global = 0.034) and endometrial (p global = 0.052) cancers. Further testing revealed the association to be focused on polymorphisms within the androgen-to-estrogen conversion sub-pathway, for both breast (p global = 0.008) and endometrial cancer (p global = 0.014). The sub-pathway association was validated in the Finnish sample of breast cancer (p global = 0.015). Further tumor subtype analysis demonstrated that the association of the androgen-to-estrogen conversion sub-pathway was confined to postmenopausal women with sporadic estrogen receptor positive tumors (p global = 0.0003). Gene-based AML analysis suggested CYP19A1 and UGT2B4 to be the major players within the sub-pathway. Our study indicates that the composite genetic determinants related to the androgen–estrogen conversion are important for the induction of two hormone-associated cancers, particularly for the hormone-driven breast tumour subtypes.

Analysis of 14 LRRK2 mutations in Parkinson's plus syndromes and late-onset Parkinson's disease

The pleomorphic pathology of postmortem LRRK2‐positive patients and the frequent association with late‐onset Parkinsons disease (LOPD) symptoms suggest that LRRK2 mutations may play a role in Parkinsons Plus disorders and LOPD. Published studies primarily focus on the common G2019S mutation. Analysis of a spectrum of LRRK2 mutations in Parkinsons Plus disorders has yet to be reported. We investigated 14 leucine‐rich repeat kinase 2 (LRRK2) mutations in a cohort of Parkinsons Plus disorders and LOPD. A total of 458 patients with progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal ganglionic degeneration (CBGD), atypical Parkinsonism (AP), and LOPD were screened for 14 mutations that span exons 19 to 41 of the LRRK2 gene. Among the LOPD cases, 1 patient was found to harbor the R1441C mutation. He presented with typical features of PD at age of 58 years old and responded well to levodopa. We did not detect any of the 14 mutations in PSP, MSA, CBGD, and AP patients. We highlight the first case of LRRK2 R1441C mutation in late onset sporadic PD of non‐European ancestry. Furthermore, extensive mutational screen found LRRK2 mutations to be rare among patients who presented with PSP, MSA, CBGD, and AP.

Linkage disequilibrium mapping of CHEK2: Common variation and breast cancer risk

Background Checkpoint kinase 2 (CHEK2) averts cancer development by promoting cell cycle arrest and activating DNA repair in genetically damaged cells. Previous investigation has established a role for the CHEK2 gene in breast cancer aetiology, but studies have largely been limited to the rare 1100delC mutation. Whether common polymorphisms in this gene influence breast cancer risk remains unknown. In this study, we aimed to assess the importance of common CHEK2 variants on population risk for breast cancer by capturing the majority of diversity in the gene using haplotype tagging single nucleotide polymorphisms (tagSNPs). Methods and Findings We analyzed 14 common SNPs spanning 52 kilobases (kb) of the CHEK2 gene in 92 Swedish women. Coverage evaluation indicated that these typed SNPs would efficiently convey association signal also from untyped SNPs in the same region. Six of the 14 SNPs predicted well both the haplotypic and single SNP variations within CHEK2. We genotyped these six tagSNPs in 1,577 postmenopausal breast cancer cases and 1,513 population controls, but found no convincing association between any common CHEK2 haplotype and breast cancer risk. The 1100delC mutation was rare in our Swedish population—0.7% in cases and 0.4% in controls—with a corresponding odds ratio for carriers versus noncarriers of 2.26 (95% confidence interval, 0.99–5.15). Estimates of the population frequency and the odds ratio of 1100delC indicate that our sample is representative of a Northern European population. Conclusions Notwithstanding the involvement of the CHEK2 gene in breast cancer aetiology, we show that common polymorphisms do not influence postmenopausal breast cancer risk.

ESR1 and EGF genetic variation in relation to breast cancer risk and survival

IntroductionOestrogen exposure is a central factor in the development of breast cancer. Oestrogen receptor alpha (ESR1) is the main mediator of oestrogen effect in breast epithelia and has also been shown to be activated by epidermal growth factor (EGF). We sought to determine if common genetic variation in the ESR1 and EGF genes affects breast cancer risk, tumour characteristics or breast cancer survival.MethodsWe genotyped 157 single nucleotide polymorphisms (SNPs) in ESR1 and 54 SNPs in EGF in 92 Swedish controls and selected haplotype tagging SNPs (tagSNPs) that could predict both single SNP and haplotype variation in the genes with an R2 of at least 0.8. The tagSNPs were genotyped in 1,590 breast cancer cases and 1,518 controls, and their association with breast cancer risk, tumour characteristics and survival were assessed using unconditional logistic regression models, Cox proportional hazard models and haplotype analysis.ResultsThe single tagSNP analysis did not reveal association evidence for breast cancer risk, tumour characteristics, or survival. A multi-locus analysis of five adjacent tagSNPs suggested a region in ESR1 (between rs3003925 and rs2144025) for association with breast cancer risk (p = 0.001), but the result did not withstand adjustment for multiple comparisons (p = 0.086). A similar region was also implicated by haplotype analyses, but its significance needs to be verified by follow-up analysis.ConclusionOur results do not support a strong association between common variants in the ESR1 and EGF genes and breast cancer risk, tumour characteristics or survival.

Comprehensive analysis of the ATM, CHEK2 and ERBB2 genes in relation to breast tumour characteristics and survival: a population-based case-control and follow-up study

BackgroundMutations in the ataxia-telangiectasia mutated (ATM) and checkpoint kinase 2 (CHEK2) genes and amplification of the v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene have been suggested to have an important role in breast cancer aetiology. However, whether common variation in these genes has a role in the development of breast cancer or breast cancer survival in humans is still not clear.MethodsWe performed a comprehensive haplotype analysis of the ATM, CHEK2 and ERBB2 genes in a Swedish population-based study, which included 1,579 breast cancer cases and 1,516 controls. We followed the cases for 8.5 years, on average, and retrieved information on the date and cause of death during that period from the nationwide Swedish causes of death registry. We selected seven haplotype-tagging SNPs (tagSNPs) in the ATM gene, six tagSNPs in the CHEK2 gene and seven tagSNPs in the ERBB2 gene that predicted both haplotypic and single locus variations in the respective genes with R2 values ≥ 0.8. These tagSNPs were genotyped in the complete set of cases and controls. We computed expected haplotype dosages of the tagSNP haplotypes and included the dosages as explanatory variables in Cox proportional hazards or logistic regression models.ResultsWe found no association between any genetic variation in the ATM, CHEK2 or ERBB2 genes and breast cancer survival or the risk of developing tumours with certain characteristics.ConclusionOur results indicate that common variants in the ATM, CHEK2 or ERBB2 genes are not involved in modifying breast cancer survival or the risk of tumour-characteristic-defined breast cancer.

Genetic variation of ESR1 and its co-activator PPARGC1B is synergistic in augmenting the risk of estrogen receptor-positive breast cancer

IntroductionGiven the role of estrogen in breast carcinogenesis and the modification of estrogen receptor (ER) activity by its biochemical cofactors, we hypothesize that genetic variation within ER cofactor genes alters cellular response to estrogen exposure and consequently modifies the risk for ER-positive breast cancer.MethodsWe genotyped 790 tagging SNPs within 60 ER cofactor genes in 1,257 cases and 1,464 controls from Sweden and in 2,215 cases and 1,265 controls from Finland, and tested their associations with either ER-positive or ER-negative breast cancer.ResultsSeven SNPs showed consistent association with ER-positive breast cancer in the two independent samples, and six of them were located within PPARGC1B, encoding an ER co-activator, with the strongest association at rs741581 (odds ratio = 1.41, P = 4.84 × 10-5) that survived Bonferroni correction for multiple testing in the combined ER-positive breast cancer sample (Pcorrected = 0.03). Moreover, we also observed significant synergistic interaction (Pinteraction= 0.008) between the genetic polymorphisms within PPARGC1B and ESR1 in ER-positive breast cancer. By contrast, no consistent association was observed in ER-negative breast cancer. Furthermore, we found that administration of estrogen in the MCF-7 cell line induced PPARGC1B expression and enhanced occupancies of ER and RNA polymerase II within the region of SNP association, suggesting the upregulation of PPARGC1B expression by ESR1 activation.ConclusionsOur study revealed that DNA polymorphisms of PPARGC1B, coding a bona fide ER co-activator, are associated with ER-positive breast cancer risk. The feed-forward transcriptional regulatory loop between PPARGC1B and ESR1 further augments their protein interaction, which provides a plausible mechanistic explanation for the synergistic genetic interaction between PPARGC1B and ESR1 in ER-positive breast cancer. Our study also highlights that biochemically and genomically informed candidate gene studies can enhance the discovery of interactive disease susceptibility genes.

Common genetic variability in ESR1 and EGF in relation to endometrial cancer risk and survival

We investigated common genetic variation in the entire ESR1 and EGF genes in relation to endometrial cancer risk, myometrial invasion and endometrial cancer survival. We genotyped a dense set of single-nucleotide polymorphisms (SNPs) in both genes and selected haplotype tagging SNPs (tagSNPs). The tagSNPs were genotyped in 713 Swedish endometrial cancer cases and 1567 population controls and the results incorporated into logistic regression and Cox proportional hazards models. We found five adjacent tagSNPs covering a region of 15 kb at the 5′ end of ESR1 that decreased the endometrial cancer risk. The ESR1 variants did not, however, seem to affect myometrial invasion or endometrial cancer survival. For the EGF gene, no association emerged between common genetic variants and endometrial cancer risk or myometrial invasion, but we found a five-tagSNP region that covered 51 kb at the 5′ end of the gene where all five tagSNPs seemed to decrease the risk of dying from endometrial cancer. One of the five tagSNPs in this region was in strong linkage disequilibrium (LD) with the untranslated A61G (rs4444903) EGF variant, earlier shown to be associated with risk for other forms of cancer.