Complex Genomic Architecture Research Articles

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5'UTR Mutations and Copy-Number Variations of NMNAT1.

ABSTRACTLeber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.

Molecular pathogenesis of multiple myeloma.

Multiple myeloma (MM), one of the most intractable malignancies, is characterized by the infiltration and growth of plasma cells, the most differentiated cells in the B-cell lineage, in the bone marrow. Despite the introduction of novel therapeutic agents, including proteasome inhibitors and immunomodulatory drugs, the prognosis of patients with MM is still worse than that of most hematological malignancies. A better understanding of the molecular pathogenesis of the disease is essential to achieve any improvement of treatment outcome of MM patients. All MM cases pass through the phase of asymptomatic expansion of clonal plasma cells, referred to as monoclonal gammopathy of undetermined significance (MGUS). It has long been believed that MM evolves linearly from MGUS to terminal phases, such as extramedullary tumors and plasma cell leukemia via the accumulation of novel mutations. However, recent studies using next-generation sequencing have disclosed the complex genomic architecture of the disease. At each step of progression, the acquisition of novel mutations is accompanied by subclonal evolution from reservoir clones with branching patterns. Each subclone may carry novel mutations and distinct phenotypes, including drug sensitivity. In addition, minor clones already exist at the MGUS stage, which could expand later in the clinical course, resulting in relapse and/or leukemic conversion. The ultimate goal of treatment is to eradicate all clones, including subclonal populations with distinct biological characteristics. This goal could be achieved by further improving treatment strategies that reflect the genomic landscape of the disease.

Dissecting the roles of microRNAs in coronary heart disease via integrative genomic analyses.

The roles of microRNAs (miRNAs) in coronary heart disease (CHD) have not been well characterized. This study sought to systematically characterize the complex genomic architecture of CHD by integrating whole blood miRNA and mRNA expression with genetic variation in 186 CHD cases and 186 controls. At false discovery rate <0.2, 15 miRNAs were differentially expressed between CHD cases and controls. To explore regulatory mechanisms, we integrated miRNA and mRNA expression with genome-wide genotype data to investigate miRNA and mRNA associations and relationships of genetic variation with miRNAs. We identified a large number of correlated miRNA-mRNA pairs and genetic loci that seem to regulate miRNA levels. Subsequently, we explored the relationships of these complex molecular associations with CHD status. We identified a large difference in miRNA-mRNA associations between CHD cases and controls, as demonstrated by a significantly higher proportion of inversely correlated miRNA-mRNA pairs in cases versus controls (80% versus 30%; P<1×10(-16)), suggesting a genome-wide shift in the regulatory structure of the transcriptome in CHD. The differentially coexpressed miRNA-mRNA pairs showed enrichment for CHD risk genetic variants affecting both miRNA and mRNA expression levels, implicating a putatively causal role in CHD. Furthermore, 3 miRNAs (miR-1275, miR-365a-3p, and miR-150-5p) were associated with an mRNA coexpression module that was causally linked to CHD and reflected the dysregulation of B-cell centered immune function. Our results provide novel evidence that miRNAs are important regulators of biological processes involved in CHD via genetic control and via their tight coexpression with mRNAs.

Abstract 1542: Comprehensive genome and transcriptome analyses on small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor of the lung accounting for 15% of all lung cancer cases. SCLC is a highly aggressive disease characterized by early metastasis and a high mortality rate of 95%. The standard of care is chemotherapy, which remains largely ineffective due to quick relapse of chemo-resistant tumors. To date, targeted therapies have not been identified for the treatment of SCLC. In order to comprehensively characterize the genomic alterations in this cancer type, we sought for a worldwide collaboration to collect rare surgical specimen of SCLC patients. Thus, we were able to study copy number alterations in 130 cases, and performed whole genome sequencing on over 78 tumor-normal pairs and transcriptome sequencing of at least 62 primary tumors and 16 SCLC cell lines. In a complementary approach we analyzed tumors derived from a murine SCLC model that was established by the conditional inactivation of Rb1 and Trp53 in lung epithelial cells (Meuwissen et al., 2003). We collected copy number alteration data for 20 mouse SCLC cases and performed exome and genome sequencing of at least 8 tumors derived from SCLC mice which additionally harbored a conditional knockout of Rbl2 (Schaffer et al., 2010). Human SCLC tumors comprise large areas of copy number alterations, revealing focal amplifications of MYC transcription factors. In line with this finding recurrent MYCL1 amplifications were also detected in murine SCLC. Human primary tumors were additionally found to harbor focal homozygous deletions of the CDKN2A locus. Furthermore, low frequent amplifications of oncogenes were identified among which therapeutic tractable FGFR1 amplifications were observed in approximately 6% of the cases. The genomic data allowed for the comprehensive characterization of the TP53 and RB1 locus at higher resolution, aiding in the identification of mutations, hemizygous and homozygous deletions, and larger genomic rearrangements. Consequently, lesions of TP53 and RB1 were found to affect both alleles in almost 100% of the cases analyzed. Other TP53 and RB1 family members were also altered in human tumor specimen thus supporting the mouse models for SCLC. In line with previous findings PTEN inactivation was found in 14% of the cases. Furthermore, recurrent mutations were detected in neuroendocrine specific genes and in chromatin and histone modifiers, among which the inactivation of the histone-acyl-transferases CREBBP and EP300 occurred at a frequency of 24%. In summary, as a continuation of our initial pilot study on 29 SCLC exomes (Peifer et al., 2012) this comprehensive large scale genomic analysis provides further insight into the complex genomic architecture of SCLC tumors and substantially improves the resolution to detect genomic alterations. This larger SCLC tumor cohort allows for the identification of low frequent alterations that might play a decisive role in the development and progression of this disease. Citation Format: Julie George, Martin Peifer, Lynnette Fernandez-Cuesta, Roman Thomas. Comprehensive genome and transcriptome analyses on small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1542. doi:10.1158/1538-7445.AM2014-1542

Unusual genome complexity in Lactobacillus salivarius JCM1046.

BackgroundLactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.ResultsJCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.ConclusionExperimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-771) contains supplementary material, which is available to authorized users.

The shared genomic architecture of human nucleolar organizer regions

The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology.

The Origin of Metazoa: An Algorithmic View of Life

We propose that the sudden emergence of metazoans during the Cambrian was due to the appearance of a complex genome architecture that was capable of computing. In turn, this made defining recursive functions possible. The underlying molecular changes that occurred in tandem were driven by the increased probability of maintaining duplicated DNA fragments in the metazoan genome. In our model, an increase in telomeric units, in conjunction with a telomerase-negative state and consequent telomere shortening, generated a reference point equivalent to a non-reversible counting mechanism.

BiotecVisions 2013, February

BiotecVisions 2013, February

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it.

Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5. A large F(2) population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5. BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence. This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.

The medical genetics of dystrophinopathies: Molecular genetic diagnosis and its impact on clinical practice

A large variety of mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophies, diseases affecting predominantly the striated muscles (skeletal and cardiac). Rare mutations also account for the allelic disorder isolated X-linked dilated cardiomyopathy. Dystrophin protein is encoded by a huge gene located on the X chromosome and the understanding of its complex genomic architecture has unraveled general key functions in gene expression regulation. Dystrophin also exists as a number of other tissue specific isoforms, some exclusively or predominantly expressed in the brain and/or in other tissues. Genotype definition of the dystrophin gene in patients with dystrophinopathies has taught us much about functionally important domains of the protein itself and has also provided insights regarding several regulatory mechanisms governing the gene expression profile. This review focuses on the current understanding of the dystrophin mutations heterogeneity, genotype-phenotype correlations, as well as interpretation of the functional significance of mutations that often require non routine genetic studies. It also explores the impact of genetic diagnosis on clinical definition and on the discovery of biomarkers and personalized therapies. Our aim is to offer an overview of the medical genetic approach on the dystrophin gene and dystrophinopathies with implications for clinical practice and therapeutic perspectives.

Abstract SY34-01: Genome-wide association studies in cancer: A step in the right direction

Abstract SY34-01: Genome-wide association studies in cancer: A step in the right direction

Expression of the SEPT9_i4 isoform confers resistance to microtubule-interacting drugs

The evolutionarily conserved septin family of genes encode GTP binding proteins involved in a variety of cellular functions including cytokinesis, apoptosis, membrane dynamics and vesicle trafficking. Septin proteins can form hetero-oligomeric complexes and interact with other proteins including actin and tubulin. The human SEPT9 gene on chromosome 17q25.3 has a complex genomic architecture with 18 different transcripts that can encode 15 distinct polypeptides. Two distinct transcripts with unique 5' ends (SEPT9_v4 and SEPT9_v4*) encode the same protein. In tumours the ratio of these transcripts changes with elevated levels of SEPT9_v4* mRNA, a transcript that is translated with enhanced efficiency leading to increased SEPT9_i4 protein. We have examined the effect of over-expression of SEPT9_i4 on the dynamics of microtubule polymer mass in cultured cells. We show that the microtubule network in SEPT9_i4 over-expressing cells resists disruption by paclitaxel or cold incubation but also repolymerises tubulin more slowly after microtubule depolymerisation. Finally we show that SEPT9_i4 over-expressing cells have enhanced survival in the presence of clinically relevant microtubule acting drugs but not after treatment with DNAinteracting agents. Given that SEPT9 over-expression is seen in diverse tumours and in particular ovarian and breast cancer, such data indicate that SEPT9_v4 expression may be clinically relevant and contribute to some forms of drug resistance.

Current status of genome-wide association studies in cancer

Genome-wide association studies in cancer have already identified over 150 regions associated with two dozen specific cancers. Already, a handful of multi-cancer susceptibility regions have been uncovered, providing new insights into perhaps common mechanisms of carcinogenesis. For each new susceptibility allele, investigators now face the arduous task of interrogating each region beginning with fine mapping prior to pursuing the biological basis for the direct association of one or more variants. It appears that there may be a significant number of common alleles that contribute to the heritability of a specific cancer. Since each region confers a small contribution to the risk for cancer, it is daunting to consider any single nucleotide polymorphism (SNP) as a clinical test. Since the complex genomic architecture of each cancer differs, additional genotyping and sequence analysis will be required to comprehensively catalog susceptibility alleles followed by the formidable task of understanding the interactions between genetic regions as well as the environment. It will be critical to assess the applicability of genetic tests in specific clinical settings, such as when to perform screening tests with calculable risks (e.g., biopsies or chemoprevention), before incorporating SNPs into clinical practice. To advance the current genomic observations to the clinical venue, new studies will need to be designed to validate the utility of known genetic variants in assessing risk for cancer as well as its outcomes.

Abstract PL01-02: The heritable component of cancer: Insights from genome-wide association studies and beyond

Abstract PL01-02: The heritable component of cancer: Insights from genome-wide association studies and beyond

Intragenomic diversity of Rhizobium leguminosarum bv. trifolii clover nodule isolates

BackgroundSoil bacteria from the genus Rhizobium are characterized by a complex genomic architecture comprising chromosome and large plasmids. Genes responsible for symbiotic interactions with legumes are usually located on one of the plasmids, named the symbiotic plasmid (pSym). The plasmids have a great impact not only on the metabolic potential of rhizobia but also underlie genome rearrangements and plasticity.ResultsHere, we analyzed the distribution and sequence variability of markers located on chromosomes and extrachromosomal replicons of Rhizobium leguminosarum bv. trifolii strains originating from nodules of clover grown in the same site in cultivated soil. First, on the basis of sequence similarity of repA and repC replication genes to the respective counterparts of chromids reported in R. leguminosarum bv. viciae 3841 and R. etli CFN42, chromid-like replicons were distinguished from the pool of plasmids of the nodule isolates studied. Next, variability of the gene content was analyzed in the different genome compartments, i.e., the chromosome, chromid-like and 'other plasmids'. The stable and unstable chromosomal and plasmid genes were detected on the basis of hybridization data. Displacement of a few unstable genes between the chromosome, chromid-like and 'other plasmids', as well as loss of some markers was observed in the sampled strains. Analyses of chosen gene sequences allowed estimation of the degree of their adaptation to the three genome compartments as well as to the host.ConclusionsOur results showed that differences in distribution and sequence divergence of plasmid and chromosomal genes can be detected even within a small group of clover nodule isolates recovered from clovers grown at the same site. Substantial divergence of genome organization could be detected especially taking into account the content of extrachromosomal DNA. Despite the high variability concerning the number and size of plasmids among the studied strains, conservation of the location as well as dynamic distribution of the individual genes (especially replication genes) of a particular genome compartment were demonstrated. The sequence divergence of particular genes may be affected by their location in the given genome compartment. The 'other plasmid' genes are less adapted to the host genome than the chromosome and chromid-like genes.

The transition from discovery by genome-wide association studies to validation of clinical risk models: Where are we, what will it take, and when can we expect to be there?

Abstract Genome-wide association studies (GWAS) have emerged as an important tool for discovering regions of the genome that harbor genetic variants that confer risk for different types of cancers. The success of GWAS has been a consequence of the convergence of new technologies that can genotype hundreds of thousands of common single nucleotide polymorphism (SNP) markers together with comprehensive annotation of genetic variation. This approach has provided the opportunity to scan across the genome in a sufficiently set of cases and controls without a set of prior hypotheses in search of susceptibility alleles with low effect sizes. So far, susceptibility alleles discovered thus far are common, namely, with a frequency in one or more population of greater than 5% and each allele confers a small contribution to the overall risk for the disease. In prostate cancer, there could be more than 35 distinct regions harboring common susceptibility alleles identified by GWAS whereas in lung cancer, a disease strongly driven by exposure to tobacco products, so far, only three regions have been conclusively established. To date, over 125 regions have been conclusively associated in over a dozen different cancers, yet no more than six regions have been associated with more than one distinct cancer type. GWAS are an important discovery tool that require extensive follow-up to map each region, investigate the biological mechanism underpinning the association and eventually test the optimal markers for assessing risk for a disease or its outcome, such as in pharmacogenomics. The early fruits of success employed by GWAS have opened new horizons for applying genetic variation to the early diagnosis, treatment and perhaps prevention of different types of cancers. At the same time, it has become apparent that the complex genomic architecture of disease susceptibility will require further discovery of uncommon and rare variants using next generation sequence technologies that contribute to different cancers before we can expect to establish robust tests of germline DNA to be clinically useful. To reach this goal, new studies will need to be designed to validate the utility of known genetic variants in assessing risk for cancer.

Abstract PL02-05: Genome-wide association studies in cancer: What have we found and what next

Abstract Cancer genetics has moved into a new era of discovering germ-line genetic variants that influence risk for specific cancers as a consequence of large-scale annotation of common genetic variation across the human genome. With the advent of fixed genotyping platforms and the investment in well-designed molecular epidemiology studies, the focus has shifted away from candidate gene studies towards large scale GWAS. In the process, we have seen clear evidence that substantial replication efforts are needed to conclusively identify new signals associated with cancer etiology. These studies seek to discover markers for disease, but rarely directly identify the variants biologically underlying the observed effect. Consequently, GWAS have primarily identified surrogate markers that necessitate follow-up fine mapping and laboratory analyses. So far, the published literature in cancer has reported approximately 100 novel regions of the genome associated with cancer etiology. GWAS have also demonstrated that the spectrum of cancers could have distinct genomic architectures, which reflects differential contributions of alleles with a range of frequencies and effect sizes. For nearly all regions, the estimated per allele effect sizes are less than 1.3 and the susceptibility alleles are common, with minor allele frequencies greater than 5%. The notable exception is for testicular cancer, which has a very strong tendency towards familial aggregation. In the GWAS of testicular cancer, common genetic markers on chromosome 12q21.32 were found to have per allele estimated risks of greater than 2.5; there is a plausible candidate gene in this region, KITLG. Still, because each common allele appears to confer a small effect, we are beginning to better appreciate the complex nature of common genetic variants that contribute to primary carcinogenesis. Furthermore, it is unlikely that there are many other common alleles with large effects for the common diseases, such as breast, colorectal, lung or prostate cancer because a sufficiently large enough number of cases and controls have been scanned. It is notable that the distribution of the number of regions identified by GWAS varies greatly by cancers, and does not appear to closely mimic hereditary estimates based on twins or family aggregation. For instance, GWAS have identified nearly 30 regions in the genome associated with prostate cancer, all of which are associated with early and aggressive cancers. On the other hand, for lung cancer, only three have been identified and one of them on chromosome 15q24/25 is also strongly associated with smoking behavior phenotypes. These observations raise the likelihood that for some cancers, a substantial environmental effect, such as smoking and lung cancer inhibits the ability to discover low penetrance, common alleles for these diseases. Because GWAS have been conducted in more than a dozen cancers, it is interesting to note that multiple cancers have mapped to two distinct regions, 8q24 and 5p15.33. The former, a ~600MB region centromeric to the MYC gene harbors a series of independent markers associated with breast, colorectal, prostate and urinary bladder cancer as well as chronic lymphocytic leukemia. Similarly, an unexpected spectrum of GWAS have pointed to common variants in a region of chromosome 5p15.33, which harbors the TERT-CLPTM1L locus; these include lung cancer (specifically adenocarcinoma), brain tumors, skin cancer (melanoma and basal cell carcinoma) and pancreatic cancer. This region also harbors less common mutations linked to acute myelogenous leukemia, dyskeratosis congenital (an inherited bone marrow failure syndrome) and pulmonary fibrosis. For each of the regions conclusively identified by GWAS, a series of follow-up studies are needed to fine map the most promising variants, preferably in different populations that can point investigators towards variants for studies designed to investigate plausibility for the association. Subsequently, laboratory investigation will be required to investigate each region that can lead to new insights into pathways and mechanisms underlying the association. To date, the reported GWAS have concentrated on regions associated with cancer etiology and not survival or other clinical outcomes. These suggests that common genetic variation may influence distinct processes important for carcinogenesis as opposed to those associated with disease progression. Though there has been considerable discussion of the promise of common variants as markers for disease risk, it is too early to apply the common variants detected in cancer GWAS to clinical and public health venues. Further studies are needed to map the markers and assess the relationship of multiple markers in sufficiently large studies. In addition, the analytical approach should account for the exposures to environmental and lifestyle choices that interact with germ-line genetic variants. The success of GWAS has opened new horizons for exploration and highlighted the complex genomic architecture of disease susceptibility. Citation Format: Stephen J. Chanock. Genome-wide association studies in cancer: What have we found and what next [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr PL02-05

Abstract ED3-1: Genome-wide association studies in cancer: Insights in cancer etiology

Abstract Genome-wide association studies (GWAS) in cancer have already discovered over 100 novel regions of the genome associated with cancer risk. For nearly all regions conclusively identified by GWAS, the estimated per allele effect sizes are less than 1.3 and the susceptibility alleles are common, with minor allele frequencies greater than 5%. Each common allele appears to confer a small effect overall, underscoring the complex nature of common genetic variants that contribute to primary carcinogenesis. Interestingly, none of the variant markers associated with etiology have been strongly associated with outcomes, such as metastatic disease or survival, which suggests that common genetic variation may influence distinct processes important for carcinogenesis as opposed to those associated with disease progression. GWAS have also demonstrated that the spectrum of cancers could have distinct genomic architectures (e.g., differential contributions of alleles with a range of frequencies and effect sizes). GWAS have identified perhaps as many as 30 regions in the genome associated with prostate cancer, whereas for lung cancer, only three so far, and one of them on chromosome 15q24/25 is also strongly associated with smoking behavior phenotypes. This observation could be related to the strong environmental effect of smoking on lung cancer. It is also notable that GWAS have mapped multiple cancers to two distinct regions, 8q24 and 5p15.33. Consequently, follow-up studies are needed to map the most promising variants, preferably in different populations that can point investigators towards the optimal variants for studies designed to investigate plausibility for the association. Consequently, parallel laboratory investigation of each region can lead to new insights into novel pathways and mechanisms associated with carcinogenesis. Though there has been considerable discussion of the promise of common variants as markers for disease risk, it is too early to apply the common variants to clinical and public health venues. The success of GWAS has opened new horizons for exploration and highlighted the complex genomic architecture of disease susceptibility. Citation Information: Clin Cancer Res 2010;16(7 Suppl):ED3-1

Genome-wide association studies in cancer--current and future directions

Genome-wide association studies (GWAS) have emerged as an important tool for discovering regions of the genome that harbor genetic variants that confer risk for different types of cancers. The success of GWAS in the last 3 years is due to the convergence of new technologies that can genotype hundreds of thousands of single-nucleotide polymorphism markers together with comprehensive annotation of genetic variation. This approach has provided the opportunity to scan across the genome in a sufficiently large set of cases and controls without a set of prior hypotheses in search of susceptibility alleles with low effect sizes. Generally, the susceptibility alleles discovered thus far are common, namely, with a frequency in one or more population of >10% and each allele confers a small contribution to the overall risk for the disease. For nearly all regions conclusively identified by GWAS, the per allele effect sizes estimated are <1.3. Consequently, the findings of GWAS underscore the complex nature of cancer and have focused attention on a subset of the genetic variants that comprise the genomic architecture of each type of cancer, which already can differ substantially by the number of regions associated with specific types of cancer. For instance, in prostate cancer, there could be >30 distinct regions harboring common susceptibility alleles identified by GWAS, whereas in lung cancer, a disease strongly driven by exposure to tobacco products, so far, only three regions have been conclusively established. To date, >85 regions have been conclusively associated in over a dozen different cancers, yet no more than five regions have been associated with more than one distinct cancer type. GWAS are an important discovery tool that require extensive follow-up to map each region, investigate the biological mechanism underpinning the association and eventually test the optimal markers for assessing risk for a disease or its outcome, such as in pharmacogenomics, the study of the effect of genetic variation on pharmacological interventions. The success of GWAS has opened new horizons for exploration and highlighted the complex genomic architecture of disease susceptibility.

Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.