Genetic and Functional Investigation of Germline JAK2 Alleles That Predispose to Myeloproliferative Neoplasms

Abstract 124

A somatic activating mutation in the pseudokinase domain of JAK2 (JAK2V617F) is found in the majority of patients with myeloproliferative neoplams (MPN). Using a genome-wide approach, we and others identified a germline haplotype in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN. Importantly, this haplotype is associated with in cis acquisition of the somatic JAK2 mutation. An extended linkage disequilibrium block of 300kb is observed at this locus and others have reported an association between single nucleotide polymorphisms (SNPs) within this haplotype and risk of inflammatory bowel disease consistent with increased JAK-STAT signaling in patients who carry this risk haplotype. The mechanism by which this germline locus contributes to MPN pathogenesis has not been delineated. We hypothesized that the identified allele heightens the risk of developing MPN by either a) increasing the mutational rate at the JAK2 locus, or b) imparting a selective advantage on cells that acquire the somatic mutation through increased JAK2 expression.

To address the mutational hypothesis, we performed targeted, high coverage, next-generation sequencing of the entire haplotype and of the entire JAK2 locus in 12 patients homozygous for the risk allele, and in 12 patients without the risk allele. Importantly we did not note an increased rate of somatic mutations (coding or noncoding) in patients homozygous for the risk haplotype. In addition, we expanded our GWAS to include 200 additional cases genotyped using the Illumina 1,000,000 SNP genotyping array. The number of SNPs did not significantly differ between the risk haplotype and non-risk haplotype, further suggesting that there is no increase in mutability attributable to the risk genotype. By constructing a phylogenetic tree, we found that the risk haplotype is ancestral to modern humans and demonstrates evidence of ancestral positive selection, although there was no evidence of recent selection at this locus. Taken together these data suggest that the JAK2 MPN risk hapolotype does not increase the mutational rate at this locus.

We next investigated whether the risk allele affects JAK2 expression in hematopoietic cells. We compared the relative abundance of an exonic SNP within the haplotype using matched genomic DNA and cDNA from 8 MPN patients heterozygous for the risk allele. In each case we found that the risk allele was more highly expressed in cDNA compared to the non-risk allele despite similar allelic ratios in genomic DNA. The results suggest an increase in allele-specific expression of JAK2 associated with the JAK2 risk haplotype. We annotated all germline variants in cis with the JAK2 risk haplotype using next generation sequencing data of the entire JAK2 haplotype from MPN patients and from the 1000 Genomes project. We then used Encode ChIP-seq data and the ConSite web-based transcription factor binding prediction model to identify SNPs within the JAK2 haplotype that affect transcription factor binding. We identified a SNP within the JAK2 promoter region, rs1887428, as a potential causative allele because it is significantly associated with MPN (p=9.11E-11) and c-Fos/c-Jun is predicted to preferentially bind to the risk allele. In order to determine if this preferential transcription factor binding leads to a haplotype-specific increase in expression of JAK2, we performed luciferase assays in cells expressing reporter constructs with the two different alleles at rs1887428. Importantly, this demonstrated increased transcriptional activity in cells containing the risk allele at rs1887428, suggesting that enhanced transcription factor binding at rs1887428 may lead to increased JAK2 expression and confer a selective advantage on cells containing the risk haplotype. The effects of allelic variation at rs1887428 on JAK2 expression in hematopoietic cells will be presented.

Taken together, our data suggests that the JAK2 MPN risk haplotype contributes to MPN pathogenesis through allele-specific transcription factor binding and JAK2 expression, which increases the selective advantage of JAK2 mutations arising on the risk haplotype. This study provides insight into how predisposing loci increase the predisposition to MPN and to other hematopoietic malignancies.