Gene Desert Research Articles

Abstract 512: Bcl11b is a Newly Identified Regulator of Vascular Smooth Muscle Function and Stiffness

Background: B-cell leukemia 11b (Bcl11b) is a zinc-finger transcription factor known as master regulator of T lymphocytes and neuronal development during embryogenesis. However, a role for Bcl11b in the cardiovascular system has never been described. Based on human findings from a genome-wide association study (GWAS) that a gene desert region downstream of BCL11B , known to function as BCL11B enhancer, harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness, we sought to examine relations between Bcl11b and arterial function. Methods and Results: We found for the first time that Bcl11b is expressed in the vascular smooth muscle (VSM) of human and murine vasculature and transcriptionally regulates the expression of VSM contractile proteins smooth muscle myosin and smooth muscle α-actin. Lack of Bcl11b in VSM-specific Bcl11b null mice (BSMKO) resulted in an increased expression of Ca ++ -calmodulin-dependent serine/threonine phosphatase calcineurin in BSMKO VSM cells and aortas, which were inversely correlated with levels of phosphorylated VASP S239 , a calcineurin de-phosphorylation target. Decreased pVASP S239 in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio), consistent with pVASP S239 ’s function as regulator of cytoskeletal actin rearrangements, and was normalized by treatment with calcineurin inhibitor cyclosporine A. Functionally, Bcl11b deletion in VSM cells translated in increased aortic force, stress and wall tension, measured ex vivo in BSMKO aortas in organ baths, and increased pulse wave velocity, the in vivo index of arterial stiffness, in BSMKO mice compared to WT littermates. Moreover, Bcl11b and pVASP S239 expression were decreased in aortas of obese and aged mice, two models of arterial stiffness. Bcl11b deletion in VSM had no effect on baseline blood pressure or angiotensin II-induced hypertension, measured in conscious WT and BSMKO mice by radiotelemetry, but dramatically increased the incidence of angII-induced aortic aneurysms in BSMKO mice. Conclusions: Taken together, our results identify VSM Bcl11b as a novel and crucial regulator of VSM cell phenotype and vascular structural and functional integrity

Abstract P531: FOXO3 , a Cardiovascular Protector, is at the Hub of a 46-gene Cell Resilience “Gene Factory” on Human Chromosome 6

The transcription factor FoxO3 regulates multiple genes involved in cell resilience. We have previously implicated variation in non-coding DNA of the FoxO3 gene ( FOXO3 ) with lower blood pressure, reduced inflammation, less hypertension, reduced coronary heart disease mortality, and longevity. The aim of the present study was to determine transcriptional, genetic and genomic mechanisms involving FOXO3 . By DNA sequencing of chromosome 6q21 in lymphoblastoid cell lines of 95 men who had survived to ≥ 95 years of age we identified 110 FOXO3 single nucleotide polymorphisms (SNPs). Thirteen SNPs were at binding sites for 18 transcription factors. Those SNPs appeared to be in physical contact, via RNA polymerase II binding chromatin looping, with sites in the FOXO3 promoter, and likely function together as a cis -regulatory unit. At the chromosome level, FOXO3 was located at the center of a 7.3 Mb 46-gene chromatin domain flanked by gene deserts. We identified distant contact points between FOXO3 and these 46 neighboring genes, through long-range physical contacts via CCCTC-binding factor zinc finger protein (CTCF) binding sites. The genes in this “archipelago” of neighbourhood genes mediate a similar repertoire of functions as FoxO3, including stress resistance, nutrient sensing, cell proliferation, autophagy, apoptosis and stem cell maintenance. The 7.3 Mb gene domain was highly conserved across species, indicating evolutionary importance. We believe that FOXO3 serves as the hub for an “interactome” involved in healthy aging, including cardiovascular disease reduction, in those with favorable FOXO3 genotypes. In support, we found that cellular stress (H 2 O 2 ) could stimulate FOXO3 expression in 20 lymphoblastoid cell lines, being 3-fold stronger for those with a favorable FOXO3 genotype. In FISH experiments, stress-induced activation of FOXO3 caused it to move towards its neighboring genes as suggested by our genomic data. In conclusion, we have shown, for the first time, that FOXO3 is at the central hub of a gene network on chromosome 6 involved in cell protection and healthy aging. The concept of “gene factories” may apply more broadly to genome and genetic mechanisms involved in cardiovascular disease etiology.

Duplication events downstream of IRX1 cause North Carolina macular dystrophy at the MCDR3 locus

Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43 kb was identified in nine families (with evidence for a shared ancestral haplotype), and another one of 45 kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.

CCAT1 and CCAT2 long noncoding RNAs, located within the 8q.24.21 ‘gene desert’, serve as important prognostic biomarkers in colorectal cancer

8q24.21 is a frequently amplified genomic region in colorectal cancer (CRC). This region is often referred to as a 'gene desert' due to lack of any important protein-coding genes, highlighting the potential role of noncoding RNAs, including long noncoding RNAs (lncRNAs) located around the proto-oncogene MYC. In this study, we have firstly evaluated the clinical significance of altered expression of lncRNAs mapped to this genomic locus in CRC. A total of 300 tissues, including 280 CRC and 20 adjacent normal mucosa specimens were evaluated for the expression of 12 lncRNAs using qRT-PCR assays. We analyzed the associations between lncRNA expression and various clinicopathological features, as well as with recurrence free survival (RFS) and overall survival (OS) in two independent cohorts. The expression of CCAT1, CCAT1-L, CCAT2, PVT1, and CASC19 were elevated in cancer tissues (P = 0.039, <0.001, 0.018, <0.001, 0.002, respectively). Among these, high expression of CCAT1 and CCAT2 was significantly associated with poor RFS (P = 0.049 and 0.022, respectively) and OS (P = 0.028 and 0.015, respectively). These results were validated in an independent patient cohort, in which combined expression of CCAT1 and CCAT2 expression was significantly associated with a poor RFS (HR:2.60, 95% confidence interval [CI]: 1.04-6.06, P = 0.042) and a poor OS (HR:8.38, 95%CI: 2.68-37.0, P < 0.001). We established a RFS prediction model which revealed that combined expression of CCAT1, CCAT2, and carcinoembryonic antigen was a significant determinant for efficiently predicting RFS in stage II (P = 0.034) and stage III (P = 0.001) CRC patients. Several lncRNAs located in 8q24.21 locus are highly over-expressed in CRC. High expression of CCAT1 and CCAT2 significantly associates with poor RFS and OS. The expression of these two lncRNAs independently, or in combination, serves as important prognostic biomarkers in CRC.

Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure

BackgroundIn homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation.ResultsWe found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation.ConclusionsOur findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

Mutations of conserved non-coding elements of PITX2 in patients with ocular dysgenesis and developmental glaucoma.

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of this structural variant. Deletion in zebrafish reduced pitx2 expression during development and resulted in shallow anterior chambers. We screened additional patients for copy number variation of the putative regulatory elements and found an overlapping deletion in a second family and in a potentially-ancestrally-related index patient with ASD and glaucoma. These data suggest that mutations affecting conserved non-coding elements of PITX2 may constitute an important class of mutations in patients with ASD for whom the molecular cause of their disease have not yet been identified. Improved functional annotation of the human genome and transition to sequencing of patient genomes instead of exomes will be required before the magnitude of this class of mutations is fully understood.

Deep sequencing reveals a global reprogramming of lncRNA transcriptome during EMT

Several studies have shown that long non-coding RNAs (lncRNAs) may play an essential role in Epithelial-Mesenchymal Transition (EMT), which is an important step in tumor metastasis; however, little is known about the global change of lncRNA transcriptome during EMT. To investigate how lncRNA transcriptome alterations contribute to EMT progression regulation, we deep-sequenced the whole-transcriptome of MCF10A as the cells underwent TGF-β-induced EMT. ResultsDeep-sequencing results showed that the long RNA transcriptome of MCF10A had undergone global changes as early as 8h after treatment with TGF-β. The expression of 3403 known and novel lncRNAs, and 570 known and novel circRNAs were altered during EMT. To identify the key lncRNA-regulator, we constructed the co-expression network and found all junction nodes in the network are lncRNAs. One junction node, RP6-65G23.5, was further verified as a key regulator of EMT. Intriguingly, we identified 216 clusters containing lncRNAs which were located in “gene desert” regions. The expressions of all lncRNAs in these clusters changed concurrently during EMT, strongly suggesting that these clusters might play important roles in EMT. Our study reveals a global reprogramming of lncRNAs transcriptome during EMT and provides clues for the future study of the molecular mechanism of EMT.

Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

Associations between single-nucleotide polymorphisms and inflammatory bowel disease-associated colorectal cancers in inflammatory bowel disease patients: a meta-analysis.

To assess the correlation between single-nucleotide polymorphisms (SNPs) and inflammatory bowel disease (IBD)-associated colorectal cancer (CRC) in IBD patients. A systematic search of PubMed, EmBase, and Cochrane databases was performed. Five genetic models (allelic, dominant, recessive, heterozygous and homozygous models) were used to analyze the associations, and trial sequential analysis was used to analyze the robustness of the results. We collected and analyzed the results of seven trials including a total of 2287 patients in our meta-analysis. A total of 8 SNPs were tested in IBD patients. For rs1800629 of TNF-α, the allelic model showed that polymorphism at this locus significantly increased the risk of IBD-associated CRC in IBD patients (OR 4.45, 95% CI 3.18-6.21, P<0.001). The results also showed a significant association between rs1800629 and an IBD-associated CRC population (heterozygous model: OR 4.335, 95% CI 2.329-8.069, P<0.001; homozygous model: OR 11.5, 95% CI 2.498-52.592, P=0.002; dominant model: OR 4.986, 95% CI 2.754-9.026, P<0.001; recessive model: OR 7.208, 95% CI 1.588-32.72, P=0.01). Other studies have found that mutation of rs1143627 of IL1B (allelic model: OR 2.97; 95% CI 1.74-5.05, P<0.001) and rs1050152 of OCTN1 (allelic model: OR 1.637, 95% CI 1.078-2.485, P=0.021) increased the proportion of IBD-associated CRC in the population. Moreover, there were significant associations between IBD-associated CRC and ITLN rs2274910, gene desert rs1551398 and rs4871611, FCGR2A rs1801274, and S100-Z rs7712957 in the allelic model. Associations between SNPs and the proportion of IBD-associated CRC in IBD patients were examined, and further investigation of additional SNPs and their association with the risk of morbidity is needed.

Functional Analysis of the Coronary Heart Disease Risk Locus on Chromosome 21q22.

Background. The coronary heart disease (CHD) risk locus on 21q22 (lead SNP rs9982601) lies within a “gene desert.” The aim of this study was to assess if this locus is associated with CHD risk factors and to identify the functional variant(s) and gene(s) involved. Methods. A phenome scan was performed with UCLEB Consortium data. Allele-specific protein binding was studied using electrophoretic mobility shift assays. Dual-reporter luciferase assays were used to assess the impact of genetic variation on expression. Expression quantitative trait analysis was performed with Advanced Study of Aortic Pathology (ASAP) and Genotype-Tissue Expression (GTEx) consortium data. Results. A suggestive association between QT interval and the locus was observed (rs9982601 p = 0.04). One variant at the locus, rs28451064, showed allele-specific protein binding and its minor allele showed 12% higher luciferase expression (p = 4.82 × 10−3) compared to the common allele. The minor allele of rs9982601 was associated with higher expression of the closest upstream genes (SLC5A3 1.30-fold increase p = 3.98 × 10−5; MRPS6 1.15-fold increase p = 9.60 × 10−4) in aortic intima media in ASAP. Both rs9982601 and rs28451064 showed a suggestive association with MRPS6 expression in relevant tissues in the GTEx data. Conclusions. A candidate functional variant, rs28451064, was identified. Future work should focus on identifying the pathway(s) involved.

Pharmacoinformatics, Adaptive Evolution, and Elucidation of Six Novel Compounds for Schizophrenia Treatment by Targeting DAOA (G72) Isoforms.

Studies on Schizophrenia so far reveal a complex picture of neurological malfunctioning reported to be strongly associated with DAOA. Detailed sequence analyses proved DAOA as a primate specific gene having conserved gene desert region on both upstream and downstream region. The analyses of 10 MB chromosomal region of primates, birds, rodents, and reptiles having DAOA evidenced the conserved part in primates and in the rest of species, while DAOA is only present in primates. DAOA has four isoforms having one interaction partner DAO. Protein-protein analyses of four DAOA isoforms with DAO were performed individually and find potential interacting residues computationally. It was observed that molecular docking of approved FDA drugs revealed efficient results but there was no common drug with effective binding to all DAOA isoforms. Library of compounds was constructed by virtual screening of 2D similarity search against recommended SZ drugs in conjunction with their physiochemical properties. Molecular docking resulted in six novel compounds exhibiting maximum binding affinity with selected four DAOA isoforms. However not the entire schizophrenic population responds to the single drug and interestingly in this study six novel compounds having promising results and same binding site to that DAOA that may be used to interact with DAO against four DAOA isoforms were observed.

Control of Hoxd gene transcription in the mammary bud by hijacking a preexisting regulatory landscape

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.

AlphaSim: Software for Breeding Program Simulation.

This paper describes AlphaSim, a software package for simulating plant and animal breeding programs. AlphaSim enables the simulation of multiple aspects of breeding programs with a high degree of flexibility. AlphaSim simulates breeding programs in a series of steps: (i) simulate haplotype sequences and pedigree; (ii) drop haplotypes into the base generation of the pedigree and select single-nucleotide polymorphism (SNP) and quantitative trait nucleotide (QTN); (iii) assign QTN effects, calculate genetic values, and simulate phenotypes; (iv) drop haplotypes into the burn-in generations; and (v) perform selection and simulate new generations. The program is flexible in terms of historical population structure and diversity, recent pedigree structure, trait architecture, and selection strategy. It integrates biotechnologies such as doubled-haploids (DHs) and gene editing and allows the user to simulate multiple traits and multiple environments, specify recombination hot spots and cold spots, specify gene jungles and deserts, perform genomic predictions, and apply optimal contribution selection. AlphaSim also includes restart functionalities, which increase its flexibility by allowing the simulation process to be paused so that the parameters can be changed or to import an externally created pedigree, trial design, or results of an analysis of previously simulated data. By combining the options, a user can simulate simple or complex breeding programs with several generations, variable population structures and variable breeding decisions over time. In conclusion, AlphaSim is a flexible and computationally efficient software package to simulate biotechnology enhanced breeding programs with the aim of performing rapid, low-cost, and objective in silico comparison of breeding technologies.

Heterochromatic histone modifications at transposons in Xenopus tropicalis embryos

Transposable elements are parasitic genomic elements that can be deleterious for host gene function and genome integrity. Heterochromatic histone modifications are involved in the repression of transposons. However, it remains unknown how these histone modifications mark different types of transposons during embryonic development. Here we document the variety of heterochromatic epigenetic signatures at parasitic elements during development in Xenopus tropicalis, using genome-wide ChIP-sequencing data and ChIP-qPCR analysis. We show that specific subsets of transposons in various families and subfamilies are marked by different combinations of the heterochromatic histone modifications H4K20me3, H3K9me2/3 and H3K27me3. Many DNA transposons are marked at the blastula stage already, whereas at retrotransposons the histone modifications generally accumulate at the gastrula stage or later. Furthermore, transposons marked by H3K9me3 and H4K20me3 are more prominent in gene deserts. Using intra-subfamily divergence as a proxy for age, we show that relatively young DNA transposons are preferentially marked by early embryonic H4K20me3 and H3K27me3. In contrast, relatively young retrotransposons are marked by increasing H3K9me3 and H4K20me3 during development, and are also linked to piRNA-sized small non-coding RNAs. Our results implicate distinct repression mechanisms that operate in a transposon-selective and developmental stage-specific fashion.

Genome-Wide Association Mapping for Female Infertility in Inbred Mice.

The genetic factors underlying female infertility in humans are only partially understood. Here, we performed a genome-wide association study of female infertility in 25 inbred mouse strains by using publicly available SNP data. As a result, a total of four SNPs were identified after chromosome-wise multiple test correction. The first SNP rs29972765 is located in a gene desert on chromosome 18, about 72 kb upstream of Skor2 (SKI family transcriptional corepressor 2). The second SNP rs30415957 resides in the intron of Plce1 (phospholipase C epsilon 1). The remaining two SNPs (rs30768258 and rs31216810) are close to each other on chromosome 19, in the vicinity of Sorbs1 (sorbin and SH3 domain containing 1). Using quantitative RT-PCR, we found that Sorbs1 is highly expressed in the mouse uterus during embryo implantation. Knockdown of Sorbs1 by siRNA attenuates the induction of differentiation marker gene Prl8a2 (decidual prolactin-related protein) in an in vitro model of decidualization using mouse endometrial stromal cells, suggesting that Sorbs1 may be a potential candidate gene for female infertility in mice. Our results may represent an opportunity to further understand female infertility in humans.

Brain enhancer activities at the gene-poor 5p14.1 autism-associated locus.

Due to the vast clinical and genetic heterogeneity, identification of causal genetic determinants for autism spectrum disorder (ASD) has proven to be complex. Whereas several dozen ‘rare’ genetic variants for ASD susceptibility have been identified, studies are still underpowered to analyse ‘common’ variants for their subtle effects. A recent application of genome-wide association studies (GWAS) to ASD indicated significant associations with the single nucleotide polymorphisms (SNPs) on chromosome 5p14.1, located in a non-coding region between cadherin10 (CDH10) and cadherin9 (CDH9). Here we apply an in vivo bacterial artificial chromosome (BAC) based enhancer-trapping strategy in mice to scan the gene desert for spatiotemporal cis-regulatory activities. Our results show that the ASD-associated interval harbors the cortical area, striatum, and cerebellum specific enhancers for a long non-coding RNA, moesin pseudogene1 antisense (MSNP1AS) during the brain developing stages. Mouse moesin protein levels are not affected by exogenously expressed human antisense RNAs in our transgenic brains, demonstrating the difficulty in modeling rather smaller effects of common variants. Our first in vivo evidence for the spatiotemporal transcription of MSNP1AS however provides a further support to connect this intergenic variant with the ASD susceptibility.

Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells.

The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10−8) and rs6451692 on chromosome 5 (p = 2.55 x 10−8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L) and EH-domain containing 4 (EHD4). In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6), which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis.

Abstract 2559: Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese

Abstract Colorectal cancer (CRC) is one of the most frequent cancers and leading cause of cancer mortality in the developed world. Twin study has attributed about 35% of the etiology of sporadic CRC to genes. Hitherto, more than 20 single nucleotide polymorphism (SNP) loci from genome-wide association studies (GWAS) were associated with CRC risk. Most of these index SNPs however have small effect sizes and often located in gene deserts. Thus they are unlikely to be causal. Haplotype-based methods may offer a more powerful approach to disease gene mapping. We previously performed a GWAS on 2,000 ethnicity-, age-, and gender-matched Singapore Chinese CRC patients and healthy controls using the Affymetrix SNP 6 platform. Sporadic patients were defined as aged 50 or more at the time of surgery and with no dominant family history of CRC. Haplotype block detection was performed in Golden Helix SVS after this SNP6 dataset was filtered for genotype call rate (&amp;gt;95%), Hardy-Weinberg equilibrium in the controls and principal component analysis. Block detection was performed requiring strong linkage disequilibrium between block pairs, minor allelic frequencies (MAF) of SNPs more than 0.01, the maximum number of markers and length of the block at 30 and 160kb respectively. Genome-wide, 73,333 blocks were computed using 336, 466 markers on 22 autosomes. Haplotype frequencies were estimated using the EM algorithm. Chi-Squared and logistic regression with odds ratio tests of haplotype association were performed using these pre-computed blocks on a per-haplotype basis. Three candidate loci at chromosomes 3, 15 and 20, each with 6 to 9 SNPs and sizes ranging from 4 to 15 kb achieved genome-wide significance and encompass potential novel disease genes. These loci are currently being validated in an independent replication panel using the Fluidgm SNPtype assays and pooled analyses will be performed. Citation Format: Peh Yean Cheah, Lai Fun Thean, Yik Ying Teo, Woon-Puay Koh, Jian-Min Yuan, Min Hoe Chew, Choong Leong Tang. Genome-wide haplotype association tests identified three candidate loci associated with sporadic colorectal cancer risk in Singapore Chinese. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2559.

An intergenic risk locus containing an enhancer deletion in 2q35 modulates breast cancer risk by deregulating IGFBP5 expression.

Breast cancer is the most diagnosed malignancy and the second leading cause of cancer mortality in females. Previous association studies have identified variants on 2q35 associated with the risk of breast cancer. To identify functional susceptibility loci for breast cancer, we interrogated the 2q35 gene desert for chromatin architecture and functional variation correlated with gene expression. We report a novel intergenic breast cancer risk locus containing an enhancer copy number variation (enCNV; deletion) located approximately 400Kb upstream to IGFBP5, which overlaps an intergenic ERα-bound enhancer that loops to the IGFBP5 promoter. The enCNV is correlated with modified ERα binding and monoallelic-repression of IGFBP5 following oestrogen treatment. We investigated the association of enCNV genotype with breast cancer in 1,182 cases and 1,362 controls, and replicate our findings in an independent set of 62,533 cases and 60,966 controls from 41 case control studies and 11 GWAS. We report a dose-dependent inverse association of 2q35 enCNV genotype (percopy OR = 0.68 95%CI 0.55-0.83, P = 0.0002; replication OR = 0.77 95% CI 0.73-0.82, P = 2.1 × 10-19) and identify 13 additional linked variants (r2 > 0.8) in the 20Kb linkage block containing the enCNV (P = 3.2 × 10-15 - 5.6 × 10-17). These associations were independent of previously reported 2q35 variants, rs13387042/rs4442975 and rs16857609, and were stronger for ER-positive than ER-negative disease. Together, these results suggest that 2q35 breast cancer risk loci may be mediating their effect through IGFBP5.

Genetic Analysis of 'PAX6-Negative' Individuals with Aniridia or Gillespie Syndrome.

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3ʹ (telomeric) to PAX6 and one within a gene desert 5ʹ (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.