(P 414) GENOMIC ANALYSIS OF EWING SARCOMA AND HERNIA IDENTIFIES A POSITIVE GENETIC CORRELATION AND SUGGESTS BOTH ARE LINKED TO AN HMGA2 VARIANT

Objective: The literature on the causes of Ewing sarcoma (EWS) is exceedingly limited, but a few clues have emerged with surprising consistency. In particular, a 2005 meta-analysis by Valery et al (PMID: 15992697) examined five case-control studies of EWS and found significant associations between this rare cancer and umbilical [odds ratio (OR) = 3.3; 95% confidence interval (CI): 1.3-8.0] and any hernia [OR = 3.2; 95% CI: 1.3-8.0]. Hernias are heterogeneous defects but all are characterized by the intrusion of a tissue or organ into an adjacent, inappropriate space; many are also considered developmental defects. While associations with hernias could expand understanding of the etiology of EWS, few epidemiologic studies of EWS have further examined the association. Recently, the genetic epidemiology of EWS has begun to be characterized, most recently in a genome-wide association study (GWAS) by Machiela et al (PMID: 30093639) who identified six germline susceptibility loci with genome-wide significance and strong per-allele ORs. The genetic epidemiology of hernias has also come into focus, with Fadista et al (PMID: 35680855) describing 81 independent loci for five types of hernia, many of which overlap. With these two datasets we aimed to conduct a comprehensive analysis of the genetic relationship between different types of hernia and EWS.

Methods: We used summary statistics from a GWAS of 733 EWS cases and 1346 controls conducted by Machiela et al and the summary statistics derived from the GWAS study of 28707, 31193, 6402, 1049, and 4644 inguinal, diaphragmatic, umbilical, femoral and ventral hernia cases, respectively, and 65,492 controls in the UK Biobank conducted by Fadista et al. All datasets focused on Europeans. We analyzed SNPs with minor allele frequency > 1% in all datasets. We evaluated the globalized and localized relationship between EWS and each hernia type, using Bonferonni adjustment for multiple testing. On the global level, we estimated the pairwise genetic correlation by linkage disequilibrium score (LDSC) regression and the casual relationship by Mendelian Randomization analysis (MR), In the MR analysis, we examined the model assumptions by checking for directional pleiotropy, strength of the instrumental variables (IV), heterogeneity, and filtered SNPs which failed Steiger’s directional test. We used a random-effect, inverse-variance weighted (IVW) method as the primary analysis whereas MR Egger, weighted mean, simple mode, and weighted mode MR were performed as sensitivity analyses.

On the regional level, we used the Multi-Trait Analysis of GWAS (MTAG) algorithm that leverages the genetic correlation among different hernias to improve the GWAS estimates for each hernia type except femoral hernia, based on which we constructed the COLOC algorithm on the regions 10,000 base pair upstream and downstream from the six genome-wide significant EWS variants (rs11366169, rs7742053, rs10822056, rs2412476, rs6047482, rs6106336) and the genome-wide significant lead SNPs for hernias identified from the MTAG estimates. The resulting test for colocalization establishes whether the same variant is associated with both phenotypes. We further estimated the local genetic covariance for the LD blocks around these regions using the SUPERGNOVA algorithm.

Lastly, we examined colocalized SNPs for evidence of being tumor expression quantitative trait loci (eQTL) using tumor RNA-seq and germline array data generated by Crompton et al (PMID: 25186949) and downloaded from dbGap (phs000804).

Results: The five hernia types were significantly correlated with each other on a global level, except for a few marginally significant pairs (Figure 1). None of the hernia had a statistically significant genetic correlation with EWS. However, all estimates suggested a positive global relationship, among which ventral hernia had the strongest global correlation with EWS (genetic correlation = 0.26) and inguinal hernia had the weakest association (genetic correlation = 0.02).

In the MR analyses, the odds of EWS was 2.26 higher among those with diaphragmatic hernia compared to those without and was statistically significant [95% confidence interval (CI) = 1.30,3.95, p-value = 0.004]. Additionally, we observed a marginally significant causal relationship between inguinal hernia and EWS with an OR = 1.27, 95% CI = (1.01, 1.59), and p-value = 0.041. There was no indication of violation of model assumptions and no concern for having weak IV. The MR analysis on single SNPs suggests that the significance was mainly driven by two SNPs, one of them was rs12810758.

Among six SNPs associated with EWS and 120 SNPs associated with any of the hernia in MTAG, we identified one region surrounding rs12810758 (chr12: 66318027 – 66338027, GRCh37) which had posterior probability of sharing the same SNP between EWS and inguinal hernia as high as 0.80. 23 SNPs included in the analysis were located within the DNA helicase G (HMGA2) gene. An additional sensitivity analysis limiting to 0.5kb around rs12810758 showed a posterior probability of 0.81 with 3 SNPs. The top SNP rs12810758 was positively associated with inguinal hernia (p-value = 8.9x10-11, MTAG p-value = 4.5x10-11) and positively associated with EWS (p-value = 0.00089). We observed that having the alternative allele T for rs12810758 suggested lower mean values in the expression levels of HMGA2 in EWS tumors, although this difference was not significant (Figure 2).

Conclusion: We present the first genetic investigation of five hernia types (diaphragmatic, inguinal, umbilical, femoral and ventral) with EWS using two independent datasets - UKBiobank data (>60,000 individuals) and a EWS GWAS data (733 EWS cases and 1346 controls). We demonstrated, through genetic epidemiologic methods, evidence of causal positive relationships between EWS and diaphragmatic and inguinal hernias. There was no evidence of a relationship between EWS and the other three hernia types. Interestingly, we observed a locally mild but cumulatively strong relationship between diaphragmatic hernia and EWS, while we found several local genetic loci which indicated shared pathways between inguinal hernia and EWS. This analysis provides additional genetic evidence for the observed association between hernia with EWS, and also demonstrates the ability of genetic analysis to aid in unraveling the etiology of rare sarcomas.