Mathieu Larivière

Allele-Specific Chromatin Remodeling in the ZPBP2/GSDMB/ORMDL3 Locus Associated with the Risk of Asthma and Autoimmune Disease

Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes.

Whole-Exome Sequencing Reveals a Rapid Change in the Frequency of Rare Functional Variants in a Founding Population of Humans

Whole-exome or gene targeted resequencing in hundreds to thousands of individuals has shown that the majority of genetic variants are at low frequency in human populations. Rare variants are enriched for functional mutations and are expected to explain an important fraction of the genetic etiology of human disease, therefore having a potential medical interest. In this work, we analyze the whole-exome sequences of French-Canadian individuals, a founder population with a unique demographic history that includes an original population bottleneck less than 20 generations ago, followed by a demographic explosion, and the whole exomes of French individuals sampled from France. We show that in less than 20 generations of genetic isolation from the French population, the genetic pool of French-Canadians shows reduced levels of diversity, higher homozygosity, and an excess of rare variants with low variant sharing with Europeans. Furthermore, the French-Canadian population contains a larger proportion of putatively damaging functional variants, which could partially explain the increased incidence of genetic disease in the province. Our results highlight the impact of population demography on genetic fitness and the contribution of rare variants to the human genetic variation landscape, emphasizing the need for deep cataloguing of genetic variants by resequencing worldwide human populations in order to truly assess disease risk.

Rare allelic forms of PRDM9 associated with childhood leukemogenesis

One of the most rapidly evolving genes in humans, PRDM9, is a key determinant of the distribution of meiotic recombination events. Mutations in this meiotic-specific gene have previously been associated with male infertility in humans and recent studies suggest that PRDM9 may be involved in pathological genomic rearrangements. In studying genomes from families with children affected by B-cell precursor acute lymphoblastic leukemia (B-ALL), we characterized meiotic recombination patterns within a family with two siblings having hyperdiploid childhood B-ALL and observed unusual localization of maternal recombination events. The mother of the family carries a rare PRDM9 allele, potentially explaining the unusual patterns found. From exomes sequenced in 44 additional parents of children affected with B-ALL, we discovered a substantial and significant excess of rare allelic forms of PRDM9. The rare PRDM9 alleles are transmitted to the affected children in half the cases; nonetheless there remains a significant excess of rare alleles among patients relative to controls. We successfully replicated this latter observation in an independent cohort of 50 children with B-ALL, where we found an excess of rare PRDM9 alleles in aneuploid and infant B-ALL patients. PRDM9 variability in humans is thought to influence genomic instability, and these data support a potential role for PRDM9 variation in risk of acquiring aneuploidies or genomic rearrangements associated with childhood leukemogenesis.

Functional impact of sequence variation in the promoter region of TGFB1.

Pathological deregulation of the transforming growth factor, beta 1 (TGFB1) pathway has been implicated in the development of several major diseases, including cancers. Regulatory variation in the TGFB1 gene may lead to altered TGFB1 expression and activity, and thus, modulate an individuals susceptibility to disease. Here, we performed a study of the functional relevance of cis‐acting regulatory variation in the proximal promoter region of the TGFB1 gene. In a previous study, 9 promoter polymorphisms were identified in the 2kb region upstream of the transcription start site and 9 distinct promoter haplotypes were inferred from a panel of individuals from 5 distinct continental population groups. Following experimental validation, we found that the 2 major haplotypes significantly influenced TGFB1 transcriptional activity in an allele‐specific manner and that 3 of the SNPs (−1886G>A, −509C>T and −1550DEL/AGG) altered DNA‐protein complexe formation. Though the biological relevance of these findings remains to be verified, our study suggests that polymorphisms in the TGFB1 promoter could indeed influence gene expression and potentially contribute to the pathogenesis of TGFB1 related diseases.

Identification of functional DNA variants in the constitutive promoter region of MDM2

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (−1494 G > A; indel 40 bp; and −182 C > G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40 bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.

ALG: Automated Genotype Calling of Luminex Assays

Single nucleotide polymorphisms (SNPs) are the most commonly used polymorphic markers in genetics studies. Among the different platforms for SNP genotyping, Luminex is one of the less exploited mainly due to the lack of a robust (semi-automated and replicable) freely available genotype calling software. Here we describe a clustering algorithm that provides automated SNP calls for Luminex genotyping assays. We genotyped 3 SNPs in a cohort of 330 childhood leukemia patients, 200 parents of patient and 325 healthy individuals and used the Automated Luminex Genotyping (ALG) algorithm for SNP calling. ALG genotypes were called twice to test for reproducibility and were compared to sequencing data to test for accuracy. Globally, this analysis demonstrates the accuracy (99.6%) of the method, its reproducibility (99.8%) and the low level of no genotyping calls (3.4%). The high efficiency of the method proves that ALG is a suitable alternative to the current commercial software. ALG is semi-automated, and provides numerical measures of confidence for each SNP called, as well as an effective graphical plot. Moreover ALG can be used either through a graphical user interface, requiring no specific informatics knowledge, or through command line with access to the open source code. The ALG software has been implemented in R and is freely available for non-commercial use either at http://alg.sourceforge.net or by request to mathieu.bourgey@umontreal.ca

Functional analysis of promoter variants in KU70 and their role in cancer susceptibility

KU70 is involved in the DNA double‐strand break repair pathway, which plays a critical role in maintaining genomic stability and preventing cancer. Genetic variation within the KU70 gene has been shown to be associated with increased risk of several types of cancers including breast cancer. Here, we used gene reporter and gel shift assays combined with site‐directed mutagenesis to characterize genetic variation within the KU70 proximal promoter region and investigate the putative functional role of regulatory variation and altered KU70 expression in modulating an individuals susceptibility to disease. We show that the variant rs2267437C>G significantly influences KU70 transcriptional activity in an allele‐ specific manner and alters DNA–protein complex formation in breast cancer cell lines. Our data provide a possible molecular explanation for the associations observed between the KU70 regulatory variant rs2267437 and breast cancer risk.

Abstract 4335: The genomic landscape of childhood pre-B acute lymphoblastic leukemia

Precursor B-cell acute lymphoblastic leukemia (pre-B ALL) is the most frequent pediatric cancer. Increased understanding of the pathobiology of B-cell ALL has led to risk-targeted treatment regimens and increased survival rates. However, the underlying causes of this pediatric cancer are still unclear. We are using next-generation sequencing technology to better understand the genomic landscape of pre-B ALL and to build a catalogue of variations involved in pediatric ALL onset and/or progression. Using a unique “quartet” design that involves matched tumor (at diagnosis) and normal (remission) samples, as well as DNA from both parents, we will be able to identify somatic mutations driving the leukemic process. Here, we report the deep-sequencing of the whole exomes, and the partial miRNomes, of over 60 childhood B-cell ALL quartets. Using ABI SOLiD technology, we captured over 4.0 Gb of sequence on average per sample with a mean coverage of 40X. Genome-wide genotyping (Illumina9s Omni 2.5 array) was also for quality control and structural variant identification. For each individual, approximately 97% of the targeted region was covered α1X and 80% of the targeted bases passed our thresholds for variant calling (≥ 5X coverage, MQV ≤ 20). According to these criteria, about 25,000 SNPs were found per individual. Using this quartet design we were able to incorporate parental sequence information to reduce sequencing errors and facilitate the identification of true variants within a given family, and of leukemia-specific variants within the ALL cohort. We investigated the somatic mutation profiles of the ALL genomes and identified both recurrent and private leukemia-specific mutations, and highlighted genes/pathways with an increased burden of somatic loss of function variants. Our goal now is to validate potential driver mutations that could play a direct role in leukemogenesis through functional assessment. Ultimately, this work will provide invaluable insights to understand the genetic mechanisms underlying pediatric ALL which could lead to the development of powerful clinical tools to improve detection, diagnosis and treatment of this childhood cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4335. doi:1538-7445.AM2012-4335

Abstract 2484: Whole-exome sequencing of a rare case of familial childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, accounting for approximately 25% of all pediatric cancer cases. However, familial childhood ALL is extremely rare. Few families with multiple non-twinned siblings diagnosed with childhood ALL have been reported, and to date no highly penetrant leukemia susceptibility gene(s) has been identified to explain this uncommon occurrence. We postulated that pure (nonsyndromic) familial childhood ALL could result from the accumulation of disadvantaging rare DNA variants in predisposing genes or biological pathways. To address this hypothesis, we used next-generation sequencing technologies to capture and re-sequence the whole-exomes of a family comprising the mother, father and two male non-twinned affected siblings (sibling A and sibling B). Both brothers were diagnosed with the identical ALL subtype, pre-B hyperdiploid childhood ALL, three years apart. The similar clinical and molecular characteristics of the siblings suggest shared etiologic factors. Using the Agilent SureSelect All Human Exon 38 Mb Kit and the SOLiD 3 Plus system, we captured and sequenced a total of 17.5 Gb of sequence for the entire family, with a mean coverage of 47X. For each individual, approximately 96% or 36.4 Mb of the targeted bases were covered α1X and 70% of the targeted bases or 26.4 Mb passed our thresholds for variant calling. We identified 52,038 positions at which the called allele(s) differed from the reference genome in at least one of the four family members. In total, we identified 19,096 germline variants in sibling A and 28,061 in sibling B, of which 2,355 (12.3%) and 2,125 (7.6%), respectively, were previously undiscovered in dbSNP. We investigated non-synonymous homozygous variant and compound heterozygous positions shared between the siblings, as well as genes/pathways with increased burden of rare non-synonymous variants. Based on several criteria (PolyPhen annotation, known allele frequency, etc.), we identified variants that are strong functional candidates to explain this case of pure familial childhood ALL. In parallel, high-density genotyping was also performed (Illumina Omni 2.5M) for quality control and structural variant detection, allowing the identification of putatively shared copy number variants that may also be involved in leukemogenesis. Though independent validation and functional assessment is required, this is the first study to identify genetic factors involved in pure familial childhood ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2484. doi:1538-7445.AM2012-2484