Genetics Journal Club Sumeet A. Khetarpal 10 December 2015

A common overall problem…

A common overall problem… Phenotype/Disease Unbiased genomic discovery Causal variant? Mechanism? Variants Gene Pathway Actionable deliverable

Published Genome-Wide Associations through 2013 ~12,000 SNP-trait associations NHGRI GWA Catalog

Maris lab at CHOP: Heritability of Neuroblastoma Bosse & Maris. Cancer. 2015

Neuroblastoma is a Pediatric Cancer Tumor of sympathetic nervous system most frequently presents in adrenal gland 650 cases/year in USA 10% of all pediatric cancers and 15% of all pediatric cancer deaths <50% of high-risk cancers contain recurrent somatic mutations (Pugh et al. 2013) Germline genetic variation may substantially impact heritability Germline mutations in the ALK/PHOX2B genes are implicated in rare familial neuroblastoma cases J Nucl Med, 2004

Neuroblastoma susceptibility loci from GWAS DUSP12 PLoS Genet 2011 HSD17B12 DDX4/IL31RA Low-Risk BARD1 Nat Genet 2009 LINC00340 NEJM 2008 LMO1 Nature 2011 High-Risk HACE1/LIN28B Nat Genet, 2012 TP53 JNCI, 2014

LIM Domain Only 1 (LMO1) Transcriptional co-regulator Part of a family of related proteins (LMO1-4) with oncogenic activity in T-ALL, breast cancer, neuroblastoma Matthews et al. Nat Rev Cancer. 2013

(Protective Genotype) (Protective Genotype) LMO1 is a neuroblastoma oncogene implicated in both tumor initiation and maintenance LMO1 Knockdown (Risk Genotype) LMO1 Knockdown (Protective Genotype) LMO1 Overexpression (Protective Genotype) Wang et al. Nature, 2011.

Hypothesis GWAS associated noncoding SNPs at LMO1 perturb LMO1 gene expression Goal Determine the causal variant and mechanism underlying association of LMO1 noncoding SNPs with neuroblastoma risk

Approach Fine mapping of the LMO1 locus Characterization of SNPs using publicly available datasets Impact of lead SNP on gene expression Impact of lead SNP on gene transcription

Fine Mapping of the LMO1 Locus Neuroblastoma GWAS ~2800 cases vs. 7500 controls Germline SNP Imputation from 1000 Genomes 2100 4200

Fine Mapping of the LMO1 Locus 27 SNPs with MAF>0.01 and P<10-5 Fig. 1a

Direct genotyping and meta-analysis of rs2168101 association 3200 Cases vs. 8500 Controls Common in Europeans + East Asians, rare/absent in Africans Direct genotyping of 146 / 2101 cases from GWAS  86% imputation accuracy Table 1

Conditional analysis of other LMO1 SNPs after adjusting for rs2168101 Ext. Fig. 1a

Conditional analysis of rs2168101 after adjusting for other LMO1 SNPs rs2168101 is the lead SNP of the lone signal at the LMO1 locus Ext. Fig. 1b

ENCODE ChIP Seq overlap of LMO1 SNPs in SKNSH neuroblastoma cell line Fig. 1b

Does the LMO1 lead SNP affect transcription factor binding? JASPAR Database Consensus TF binding motifs based on experimental ChIP-Seq data 202 nonredundant profiles in vertebrates http://jaspar.genereg.net

Does the LMO1 lead SNP affect transcription factor binding? Lead LMO1 SNP falls in conserved enhancer and GATA binding motif Fig. 1b

Impact of lead SNP on LMO1 expression LMO1 RNA reads in 127 high-risk tumors separated by genotype No T/T genotypes found in high-risk neuroblastoma tumors  risk alleles ‘predispose’ to the high-risk subset Fig. 2a

Impact of lead SNP on LMO1 expression Allelic imbalance of LMO1 expression in tumors from hets Fig. 2b – 12 tumors were heterozygous. Fig. 2a-b

Impact of lead SNP on LMO1 expression Allelic imbalance of nascent LMO1 expression in heterozygous cell lines Ancestral GATA allele of lead SNP  increased LMO1 gene expression Cis regulation? Fig. 2a-c

G(O)TA find the GATA GATA2 and GATA3 are highly expressed GATA factors in neuroblastoma tumors Neuroblastoma cell lines overexpressing GATA3 show increased LMO1 expression if G allele present Ext. Fig. 5a

GATA3 binding at LMO1 by ChIP GATA3 ChIP-Seq in neuroblastoma cell lines with differing rs2168101 genotype Fig. 4a

GATA3 binding at LMO1 by ChIP Allele-specific GATA3 ChIP-Seq at LMO1 lead SNP in cell lines ~Zero GATA3 binding to rs2168101 T allele! Fig. 4b

LMO1 lead SNP lies in active enhancer Fig. 4c, e

Summary Fine mapping validates LMO1 locus-neuroblastoma association and identifies single signal Lead SNP lies in conserved GATA TF binding motif Protective allele disrupts GATA3 binding, enhancer activity, gene expression Protective allele associated with reduced cell growth & increased survival in high-risk disease

Lingering Questions Busted enhancer, broken TF binding, both, or one-in-the-same? Molecular mechanism of disruption? Why is this particular GATA binding site so important for this region? Other long-range interactions disrupted? (Is it really all just LMO1?) Is locus really a ‘super-enhancer’?

Can this locus be defined as a super-enhancer? Proposed model for impact of lead SNP on GATA3-mediated LMO1 transcription Risk “G” allele of rs2168101: Exon 1 GATA3 AGATAA Exon 1 Exon 2 Exon 3 Exon 4 LMO1 Gene Can this locus be defined as a super-enhancer? Protective “T” allele of rs2168101: GATA3 Exon 2 Exon 3 Exon 4 Exon 1 ATATAA LMO1 Gene

Enhancers and Super-enhancers DNA element that activates transcription from a distance Enrichment of H3K4me1, H3K27ac, DNase I hypersensitivity Few 100 bp Super-enhancers Unusually strong enrichment for coactivators (e.g. Med1) Enrichment of Pol II, eRNA, p300, CBP, H3K27ac, H3K4me1/2, DNase I hypersensitivity Few 1000 bp High controversy over distinction from enhancers Poor agreement over definition Pott & Lieb. Nat Genet. 2015

‘Super-enhancers’ are controversial

Back to our common problem…

Back to our common problem… Phenotype/Disease How can we find causal variants and their mechanisms? Unbiased genomic discovery Variants Gene Pathway Actionable deliverable

There are some tools to help… Spain & Barrett. Hum Mol Genet. 2015

…but it’s still a hot mess Spain & Barrett. Hum Mol Genet. 2015

...and only a few have ‘succeeded’ so far Spain & Barrett. Hum Mol Genet. 2015

Germline genetic variation and pediatric cancer risk Whole-genome or whole-exome sequencing in ~1100 pediatric participants with cancer vs. ~960 cancer-free participants from 1000 genomes 8.5% of cancer patients had deleterious germline mutation Underestimate of total germline heritability

Thanks for coming!