Expanded Assessments of Germline Genetic Variants Identify Additional Risk Loci for Langerhans Cell Histiocytosis

Introduction: Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by lesions including pathogenic CD207+ dendritic cells among an inflammatory infiltrate. Sequencing studies have identified recurrent, mutually exclusive somatic activating mutations in MAPK pathway genes in ~85% of LCH lesions, including BRAF V600E in 50-65%. Despite advances to elucidate the somatic mutational landscape underlying LCH pathogenesis, germline risk factors remain largely unknown. We previously conducted the first genome-wide association study (GWAS) of LCH and identified and validated a risk variant in SMAD6 (rs12438941, G->A) associated with a 3-fold increase in LCH risk. Our current objective is to further elucidate the role of SMAD6 variation on LCH risk and identify novel LCH susceptibility loci.

Methods: Independent of our discovery GWAS, we sequenced SMAD6 in 466 additional LCH cases and compared them to aggregate-level SMAD6 data from ~15,694 non-cancer controls in gnomAD v2.1.1. Targeted sequencing of SMAD6 was conducted among these cases with ~200x coverage at the Avera Institute of Human Genetics. In parallel, we conducted a GWAS of 319 of those cases and leveraged controlled-access data from 9,921 controls in the ADDHealth study. Genotyping of the 319 cases was also performed at the Avera Institute for Human Genetics on the Illumina Infinium Global Screening Array. We evaluated the role of common variants (minor allele frequency >5%) on LCH risk and applied principal components analysis to account for potential population stratification. A genome-wide threshold of significance was applied at P-value<5.0x10^-8, and threshold of suggestive significance at P-value<1.0x10^-6. Analyses were conducted in Plink and STATAv17.1.

Results: The case-control study of targeted SMAD6 identified novel SMAD6 variants associated with LCH risk (P-values ranging from 4.54x10^-28 to 2.9x10^-4), including our original GWAS locus. All cases with two copies of the alternate alleles in the top 2 SNPs had BRAF V600E⁺ lesions. In terms of the new GWAS, 31 novel loci were associated with LCH at P-value=5x10^-8, including 2 separate loci on Chr 9, with the top hit being intergenic between KCNV2 and VLDLR on Chr 9 and the second being intergenic between OLFM1 and LOC401557. Other identified top hits suggest putative biologic significance in terms of LCH pathogenesis.

Conclusion: We identified additional support that inherited genetic variation impacts LCH risk. We are concurrently assessing the role of ancestry-specific loci on LCH risk and analyzing newly generated SMAD6 targeted sequence and GWAS data in an independent set of case-parent trios (n=185) to integrate with the findings reported here. Next steps include a meta-analysis of our discovery GWAS, the new GWAS reported here, and the recently genotyped new set of 185 case-parent trios. We also aim to determine the role of variants on clinical characteristics (e.g., ancestry, age at onset, BRAF V600E). Our work suggests an important and complex role of germline variants and ancestry in LCH pathogenesis.