Candace D. Middlebrooks

Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors

High rates of APOBEC-signature mutations are found in many tumors, but factors affecting this mutation pattern are not well understood. Here we explored the contribution of two common germline variants in the APOBEC3 region. SNP rs1014971 was associated with bladder cancer risk, increased APOBEC3B expression, and enrichment with APOBEC-signature mutations in bladder tumors. In contrast, a 30-kb deletion that eliminates APOBEC3B and creates an APOBEC3A–APOBEC3B chimera was not important in bladder cancer, whereas it was associated with breast cancer risk and enrichment with APOBEC-signature mutations in breast tumors. In vitro, APOBEC3B expression was predominantly induced by treatment with a DNA-damaging drug in bladder cancer cell lines, and APOBEC3A expression was induced as part of the antiviral interferon-stimulated response in breast cancer cell lines. These findings suggest a tissue-specific role of environmental oncogenic triggers, particularly in individuals with germline APOBEC3 risk variants.

Genome wide association study identified SNP on 15q24 associated with bladder cancer risk in Japanese population

Through genome-wide association analysis and an independent replication study using a total of 1131 bladder cancer cases and 12 558 non-cancer controls of Japanese populations, we identified a susceptibility locus on chromosome 15q24. SNP rs11543198 was associated with bladder cancer risk with odds ratio (OR) of 1.41 and P-value of 4.03 × 10(-9). Subgroup analysis revealed rs11543198 to have a stronger effect in male smokers with OR of 1.66. SNP rs8041357, which is in complete linkage disequilibrium (r(2) = 1) with rs11543198, was also associated with bladder cancer risk in Europeans (P = 0.045 for an additive and P = 0.025 for a recessive model), despite much lower minor allele frequency in Europeans (3.7%) compared with the Japanese (22.2%). Imputational analysis in this region suggested CYP1A2, which metabolizes tobacco-derived carcinogen, as a causative candidate gene. We also confirmed the association of previously reported loci, namely SLC14A1, APOBEC3A, PSCA and MYC, with bladder cancer. Our finding implies the crucial roles of genetic variations on the chemically associated development of bladder cancer.

An Examination of the Relationship between Hotspots and Recombination Associated with Chromosome 21 Nondisjunction

Trisomy 21, resulting in Down Syndrome (DS), is the most common autosomal trisomy among live-born infants and is caused mainly by nondisjunction of chromosome 21 within oocytes. Risk factors for nondisjunction depend on the parental origin and type of meiotic error. For errors in the oocyte, increased maternal age and altered patterns of recombination are highly associated with nondisjunction. Studies of normal meiotic events in humans have shown that recombination clusters in regions referred to as hotspots. In addition, GC content, CpG fraction, Poly(A)/Poly(T) fraction and gene density have been found to be significant predictors of the placement of sex-averaged recombination in the human genome. These observations led us to ask whether the altered patterns of recombination associated with maternal nondisjunction of chromosome 21 could be explained by differences in the relationship between recombination placement and recombination-related genomic features (i.e., GC content, CpG fraction, Poly(A)/Poly(T) fraction or gene density) on 21q or differential hot-spot usage along the nondisjoined chromosome 21. We found several significant associations between our genomic features of interest and recombination, interestingly, these results were not consistent among recombination types (single and double proximal or distal events). We also found statistically significant relationships between the frequency of hotspots and the distribution of recombination along nondisjoined chromosomes. Collectively, these findings suggest that factors that affect the accessibility of a specific chromosome region to recombination may be altered in at least a proportion of oocytes with MI and MII errors.

Evidence for dysregulation of genome-wide recombination in oocytes with nondisjoined chromosomes 21

In oocytes with nondisjoined chromosomes 21 due to a meiosis I (MI) error, recombination is significantly reduced along chromosome 21; several lines of evidence indicate that this contributes to the nondisjunction event. A pilot study found evidence that these oocytes also have reduced recombination genome-wide when compared with controls. This suggests that factors that act globally may be contributing to the reduced recombination on chromosome 21, and hence, the nondisjunction event. To identify the source of these factors, we examined two levels of recombination count regulation in oocytes: (i) regulation at the maternal level that leads to correlation in genome-wide recombination across her oocytes and (ii) regulation at the oocyte level that leads to correlation in recombination count among the chromosomes of an oocyte. We sought to determine whether either of these properties was altered in oocytes with an MI error. As it relates to maternal regulation, we found that both oocytes with an MI error (N = 94) and their siblings (N = 64) had reduced recombination when compared with controls (N = 2723). At the oocyte level, we found that the correlation in recombination count among the chromosomes of an oocyte is reduced in oocytes with MI errors compared with that of their siblings or controls. These results suggest that regulation at the maternal level predisposes MI error oocytes to reduced levels of recombination, but additional oocyte-specific dysregulation contributes to the nondisjunction event.

Variation in the Zinc Finger of PRDM9 is Associated with the Absence of Recombination along Nondisjoined Chromosomes 21 of Maternal Origin

Variation in the zinc finger-binding domain (ZFBD) of the protein PR Domain-Containing Protein 9 (PRDM9) is associated with altered placement of recombination in the human genome. As both the absence and altered placement of recombination are observed among chromosomes 21 that nondisjoin, we genotyped the PRDM9 ZFBD among mothers of children with Trisomy 21 in efforts to determine if variation within this region is associated with the recombination-related risk for chromosome 21 nondisjunction (NDJ). In our approach, PCR was used to amplify the ZFBD of PRDM9 and products were then subjected to bi-directional Sanger sequencing. DNA sequencing reads were aligned and compared to the sequence of the PRDM9 alleles previously identified. Chi-Square analysis was used to compare allele frequencies between cases (N=235, mothers of children with maternally-derived Trisomy 21) and controls (N=48, fathers of children with maternally-derived Trisomy 21). Results of our analysis showed that the frequency of PRDM9 ZF minor alleles is significantly increased among women displaying NDJ of chromosome 21 and no recombination on 21q (p=0.02). Even more, when compared to those for the PRDM9 major A-allele, these minor alleles displayed fewer predicted binding sites on 21q. These findings suggest that allelic variation in the ZF of PRDM9 may play a role in the risk for chromosome 21 NDJ by leading to reduced recombination on 21q.

Abstract 2562: Bladder cancer GWAS signal at 4p16.3 affects response of TMEM129 to chemically-induced endoplasmic reticulum stress

Background: SNP rs798766 within the 4p16.3 region was associated with bladder cancer risk in several GWAS. This SNP is located within a linkage disequilibrium block that includes TMEM129, TACC3, FGFR3 and SLBP. We aimed to explore molecular mechanisms of this GWAS signal. Methods: Expression analysis of all isoforms of TMEM129, TACC3, FGFR3 and SLBP was performed in the Cancer Genome Atlas (TCGA) bladder cancer dataset (N = 412). Expression was analyzed by linear regression in relation to rs798766 genotypes adjusting for age, sex, race, DNA CpG methylation status and copy number variation (CNV). Expression in additional bladder tumors (N = 42) was tested with TaqMan expression assays and with splicing form-specific in situ hybridization (ISH) assays in a tissue microarray that included 7 normal-tumor bladder tissue pairs. Allele-specific TMEM129 exontrap assays in 5 bladder cancer cell lines were used to evaluate alternative splicing. TMEM129 isoforms were cloned and overexpressed in cell lines for functional characterization by confocal microscopy, reporter assays, RNA-seq and qRT-PCR arrays to explore their effects on cell signaling and cell viability. Endoplasmic reticulum (ER) stress was induced by treating bladder cancer cell lines with chemicals dithiothreitol (DTT), tunicamycin and MG132. Results: Of all the isoforms of four genes tested the strongest association with rs798766 genotypes was observed for TMEM129-b transcript (p adj = 1.65E-06); expression was decreased with bladder cancer risk allele. Exontrap analysis revealed that allele G of a SNP rs2236786 located within exon 3 of TMEM129 (r 2 = 1.0 with GWAS SNP rs798766) is responsible for alternative splicing of TMEM129. Allele-specific alternative splicing also results in lower expression of TMEM129-b and creation of a new TMEM129-a2 transcript. TMEM129-a2 is predicted to undergo nonsense mediated decay (NMD) except when under ER stress. TMEM129 is an E3 ligase and its protein isoforms are predicted to be functionally different. Confocal imaging showed that all isoforms are located in ER and thus can compete with each other. Overexpression of TMEM129 isoforms resulted in differential activation of genes from the main ER stress response pathways - IRE1, PERK and ATF6. Chemically-induced ER stress increased expression of all TMEM129 isoforms, although the induction was lower in cell lines with risk G allele of rs2236786. Conclusions: The risk of bladder cancer is known to be increased by environmental exposures. Our results suggest that bladder cancer risk could be modulated by a genetic variant that affects the function of TMEM129, an E3 ligase. This mechanism could be important for response to environmentally-induced ER stress and subsequent development of bladder cancer. Citation Format: A. Rouf Banday, Eniko Kiss, Wusheng Yan, Candace Middlebrooks, Ludmila Prokunina-Olsson. Bladder cancer GWAS signal at 4p16.3 affects response of TMEM129 to chemically-induced endoplasmic reticulum stress. [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 2562.

Abstract 2563: Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region

Background: Genome-wide association studies (GWAS) of bladder cancer risk recently identified several signals on chromosome 20p12.2 marked by single nucleotide polymorphisms (SNPs) rs6104690, rs62185668 and rs6108803 (Figueroa et al, HMG, 2015). Interestingly, associations of SNPs rs62185668 and rs6108803 were significantly stronger for the risk of muscle-invasive (stages T2-T4) compared to non-muscle-invasive (stages Ta and T1) bladder cancer, suggesting that detailed analysis of these signals could further our understanding of aggressive bladder cancer. Methods: We used data from The Cancer Genome Atlas (TCGA) bladder cancer set (n = 412) and an independent RNA-seq dataset for 12 bladder tissues. qRT-PCR analysis was performed with custom TaqMan assays in an additional set of 40 tumor and 40 adjacent normal bladder tissue samples. RNAscope in situ hybridization analysis with custom probes was used to evaluate mRNA expression in bladder tissue microarrays that included 7 pairs of normal-tumor bladder tissues. Results: At a distance of more than 300 and 800 Kb, JAG1 and BTD3 are the closest genes to the GWAS signals in the 20p12.2 region. Analysis of TCGA data did not show significant association of JAG1 and BTD3 expression with genotypes of 20p12.2 GWAS markers analyzed through TCGA-genotyped proxies SNPs rs6040291 and rs6074214. Using RNA-seq data in an independent set of 12 bladder tissue samples, we discovered a novel putative gene in the 20p12.2 region. Cloning of this gene from bladder tumor tissue and annotation by several methods showed two alternative splicing forms, designated as major and minor transcripts. Expression of the major transcript in bladder tumors was significantly increased with the rs6104690-A allele (P = 0.014), which is associated with increased risk of bladder cancer. SNPs with stronger association with muscle-invasive bladder cancer (rs62185668 and rs6108803) showed weaker or non-significant associations with expression of this novel transcript (P = 0.037 and P = 0.42, respectively). This transcript might be protein-coding as it is predicted to have several open reading frames for short peptides, or it may be a long non-coding RNA (lncRNA). RNAscope analysis in bladder tumors showed nuclear expression of the major transcript. Functional analysis of this novel gene in relation to bladder cancer is underway. Conclusions: A novel gene might be related to the GWAS signals within the 20p12.2 region and the risk of bladder cancer. Citation Format: Rouf Banday, Wusheng Yan, Candace D. Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. Analysis of a novel gene in relation to bladder cancer GWAS signals within the 20p12.2 region. [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 2563.

Abstract 4617: An alternatively spliced isoform of TMEM129 shows association with bladder cancer GWAS marker rs798766

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Genome-wide association studies (GWAS) have identified a number of single nucleotide polymorphisms (SNPs) associated with bladder cancer risk. Here, we explored the bladder cancer GWAS signal SNP rs798766 (C/T), which is located in the intron of TACC3 upstream of FGFR3 at 4p16.3 locus. This region represents a high linkage disequilibrium (LD) block and includes two more genes, TMEM129 and SLBP. Several SNPs in r2 = 1 with rs798766 are located within coding and non-coding regions of these genes; however, the coding SNPs are synonymous. Previously, genetic association of this GWAS marker has been shown by eQTL analysis with FGFR3 and TACC3 mRNA expression in adipose tissue. However, genetic association for all four genes and their several alternatively spliced isoforms remains largely unexplored in primary bladder normal and tumor tissue. We used genotype and RNA-seq data from The Cancer Genome Atlas (TCGA), bladder cancer data set (n = 242). Within the 1000 Kb region surrounding rs798766 we imputed genotypes of 13,529 SNPs in the TCGA data set based on the 1000 Genomes reference panel. Next, we performed eQTL analysis of FGFR3, TACC3, SLBP and TMEM129 both at gene level and at individual isoform level with genotypes of GWAS and several other markers. For validation, we used an independent panel of 42 bladder tumor and 42 adjacent normal tissue samples, using TaqMan expression assays. Expression was analyzed by general linear regression based on an additive genetic model, adjusting for age, sex and race. At the gene level in the TCGA data, we found significant association with TACC3 expression (p = 1.9 × 10−2unadj and p = 3.5 × 10−2 adj). The association was stronger for expression of an alternatively spliced isoform of TMEM129, designated TMEM129β, (p = 4.57×10−4unadj and p = 3.5 × 10−3adj). Computational annotation revealed that SNP rs2236786, r2 = 1 with GWAS marker, is located within a cassette exon that is an intron for isoform TMEM129β. Further analysis revealed that rs2236786 alleles affect a binding motif for alternative splicing factors SF2ASF, hnRNPF and CUG-BP. The functional exploration of this finding is underway. In conclusion, we suggest that the molecular phenotype of the 4p16.3 bladder cancer GWAS signal could be associated with differential expression of more than one gene (FGFR3, TACC3, SLBP and TMEM129) and their alternatively spliced isoforms. We present the first evidence for possible association of alternative splicing of TMEM129 and bladder cancer GWAS signal. Note: This abstract was not presented at the meeting. Citation Format: A. Rouf Banday, Ashley Paquin, Candace Middlebrooks, Eniko Kiss, Ludmila Prokunina-Olsson. An alternatively spliced isoform of TMEM129 shows association with bladder cancer GWAS marker rs798766. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2015-4617