Rosalie Waller

Discordant Haplotype Sequencing Identifies Functional Variants at the 2q33 Breast Cancer Risk Locus

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance–a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits.

Germline Lysine-Specific Demethylase 1 (LSD1/KDM1A) Mutations Confer Susceptibility to Multiple Myeloma

Given the frequent and largely incurable occurrence of multiple myeloma, identification of germline genetic mutations that predispose cells to multiple myeloma may provide insight into disease etiology and the developmental mechanisms of its cell of origin, the plasma cell (PC). Here, we identified familial and early-onset multiple myeloma kindreds with truncating mutations in lysine-specific demethylase 1 (LSD1/KDM1A), an epigenetic transcriptional repressor that primarily demethylates histone H3 on lysine 4 and regulates hematopoietic stem cell self-renewal. In addition, we found higher rates of germline truncating and predicted deleterious missense KDM1A mutations in patients with multiple myeloma unselected for family history compared with controls. Both monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma cells have significantly lower KDM1A transcript levels compared with normal PCs. Transcriptome analysis of multiple myeloma cells from KDM1A mutation carriers shows enrichment of pathways and MYC target genes previously associated with myeloma pathogenesis. In mice, antigen challenge followed by pharmacologic inhibition of KDM1A promoted PC expansion, enhanced secondary immune response, elicited appearance of serum paraprotein, and mediated upregulation of MYC transcriptional targets. These changes are consistent with the development of MGUS. Collectively, our findings show that KDM1A is the first autosomal-dominant multiple myeloma germline predisposition gene providing new insights into its mechanistic roles as a tumor suppressor during post-germinal center B-cell differentiation.Significance: KDM1A is the first germline autosomal dominant predisposition gene identified in multiple myeloma and provides new insights into multiple myeloma etiology and the mechanistic role of KDM1A as a tumor suppressor during post-germinal center B-cell differentiation. Cancer Res; 78(10); 2747-59. ©2018 AACR.

Trend Displays to Support Critical Care: A Systematic Review

An important aspect of designing information displays to support monitoring and decision-making in critical care is the representation of change of patient data over time. We systematically reviewed articles to identify novel alternatives to tabular and single variable plots of values over time to convey information about change in multiple related variables. Following screening of 5,119 articles, 28 met our inclusion criteria. They described 26 unique displays evaluated in 31 experiments. Methods for representing change varied widely. We classified these methods as enhanced graphical displays (enhanced plots of quantitative data over time), other displays (novel object and metaphoric displays), small multiples displays (multiple co-presented small graphic displays), and simple change indicator displays. Overall, findings support the value of an explicit display of trend information using many different approaches. Few studies directly compared different methods for displaying trend information in ways that would support broader conclusions about which approaches may be preferred for specific applications. The studies suggest that, for displaying patient data trends, it is feasible to develop electronic displays that will outperform both historical paper-based flowcharts and current electronic health record (EHR)-based tabular approaches. There is evidence to suggest that even minor improvements to current approaches such as the automatic presentation of simple line plots of trends on EHRs or the addition of simple graphical indicators of trend direction on patient monitors could lead to clinically meaningful improvements in diagnostic accuracy and efficiency.

Abstract 3267: Targeted DNA and RNA sequencing identifies breast cancer risk variants associated with differential expression of CASP8 and CFLAR/CASP10

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA CASP8 at 2q33 is an established common, low risk susceptibility locus for breast cancer but functional variants are yet to be elucidated. Previously we used haplotype mining of 45 tSNPs in 3778 breast cancer cases and controls to identify a risk haplotype maximizing association evidence. A protective haplotype was also suggested. Here, we perform a focused sequencing study to identify the critical sequence variant/s. To not exclude potential regulatory variants, we designed a custom Agilent SureSelect capture array to target all non-repetitive sequence in the 1 Mb region surrounding CASP8. The resulting 41 libraries for germline DNA (24 breast cancer cases homozygous for the risk haplotype, 17 cancer-free women carrying the protective haplotype) were sequenced on the Life Technologies SOLID system. A total of 1464 sequence variants were prioritized by their propensity to lie on the risk or protective haplotypes, resulting in the selection of 42 variants to follow-up with expression-QTL analysis (MAFs 0.1-0.4). Our RNA sequencing panel comprised 156 tissue samples from 88 women (68 breast tumor, 88 normal breast). Illumina TruSeq stranded mRNA sequencing was performed on an Illumina HiSeq 2000. Genotyping for the 42 selected variants was performed using Illumina BeadXpress. RNAseq eQTL analysis focused on 4 genes: CFLAR, CASP10, CASP8, ALS2CR12 using counts standardized to fragments per kilobase per million bases (FPKM). Differences in FPKM between carriers and non-carriers of the 42 DNA sequence variants were assessed in both normal and tumor tissues. Replication analyses were carried out on available TCGA data and published blood eQTL microarray data. In normal tissue, an intronic variant in ALS2CR12 on the risk haplotype was best associated with CASP8 expression (19% decreased FPKM, p=9x10-5). Eight other variants (all in ALS2CR12 or CASP8) showed similar CASP8 decreases (p<10-3). Increased ALS2CR12 expression was also observed for the same variants, and decreased CASP8 expression was seen in tumor. Despite the difference in technologies, lower coverage and sparser genotyping, the decreased CASP8 and increased ALS2CR12 levels were replicated in both in the TCGA normal tissue (p=3x10-4) and blood eQTL data (p=4x10-14). In tumor, our strongest eQTL results were for decreased expression of CFLAR (p=1x10-4) and CASP10 (p=4x10-5), identified for variants on the protective haplotype (including rs1045485). Examination of TCGA breast tumors did not replicate these findings; however read depths were substantially lower for these genes in the TCGA data. We find evidence for two separate paths to 2q33 breast cancer risk. Reduction of CASP8 expression, which may lead to reduced apoptosis and an environment conducive to tumor development. Reduction of CFLAR-CASP10 expression in tumor may implicate autophagy leading to a challenging environment for tumors, but requires more replication. Citation Format: Nicola J. Camp, Wei-Yu Lin, Alex Bigelow, Marina A. Parry, Tim Mosbruger, George Burghel, Venkatesh Rajamanickam, Sushilaben H. Rigas, Rachel Cosby, Dan Connley, Guoying Wang, Tresa George, Rosalie Waller, Lisa A. Cannon-Albright, Brandt Jones, Rob Sargent, Malcolm W.R. Reed, Angela Cox. Targeted DNA and RNA sequencing identifies breast cancer risk variants associated with differential expression of CASP8 and CFLAR/CASP10. [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 3267. doi:10.1158/1538-7445.AM2014-3267