Whole-Exome Sequencing in Multiplex Families to Identify Novel AYA Classical Hodgkin Lymphoma Predisposition Genes
Authors: Xu Keren , Wadé Niquelle B , Hwang Amie E. , Conti David V , Salehi Maryam , Mack Thomas M , Cortessis Victoria , Van Den Berg David J , de Smith Adam J. , Cozen Wendy 
Published: November 2021

Abstract
Background:
Hodgkin Lymphoma (HL) is a B-cell malignancy that mainly affects young adults in economically developed countries. Classical HL (cHL) is the most common type, comprising &gt;95% of cases. We demonstrated strong heritability of cHL in adolescents and young adults (AYA) with a high risk to the unaffected co-twins of affected monozygotic (MZ) twins. We and others identified common risk variants by GWAS, and sequencing of familial and sporadic HL patients previously identified rare pathogenic germline variants in genes with varying functions. Few have replicated in other families. A greater understanding of the genes involved in cHL predisposition is needed. We performed germline whole-exome sequencing (WES) for 48 individuals in 14 multiplex families with AYA cHL patients to identify novel cHL predisposition genes.
Methods:
Index cases from multiplex families were ascertained from the USC twin registries (International Twin Registry or California Twin Program, 7 index cases, 2 additional cases, and 7 unaffected relatives) or from the USC Cancer Surveillance Program (7 index cases, 4 additional cases, and 21 unaffected relatives). Among the twin pairs, 5 were concordant MZ pairs, one was a concordant dizygotic pair and one was an MZ discordant pair with a second case occurring in the daughter of the unaffected MZ co-twin. The other 7 families included 4 with two affected siblings, one with a child/father pair, and two with child/uncle pairs; the latter 3 families had specimens available for the index child case only. All index cases were diagnosed under 50 years old, with the median age 27.
DNA samples were extracted from blood or saliva of 48 individuals in 14 multiplex families, with specimens from 20 of 28 reported cases. Germline WES was performed using the Nextera® Rapid Capture Exome kit, with data pre-processing performed based on Genome Analysis Toolkit best practices guidelines, and additional filtering applied using BCFtools. Variants were annotated using ANNOVAR. We filtered out variants with an allele frequency &gt;0.001 in population databases (gnomAD or TOPMed), and variants documented as benign/likely benign in ClinVar. We retained splicing/ncRNA variants and exonic variants with loss-of-function/missense/unknown functional consequences, and with a CADD score &gt;10. We further limited variants to those in 1,073 candidate predisposition genes, including cancer/immunodeficiency/hematological-related genes in the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE) and genes previously identified in sequencing studies or GWAS for cHL. Variants in candidate genes were visually inspected in the Integrative Genomics Viewer and were analyzed for their potential pathogenicity through the PeCanPIE MedalCeremony pipeline. Variants predicted to be tolerated by PeCanPIE were removed. Variants found only in HL patients and not in their sibling controls were considered to be potentially causal.
Results:
In 45 subjects that passed sequencing QC (3 controls were removed due to low mean coverage) and following variant filtering, there were 33 variants in 33 genes found only in cHL patients but not in their sibling controls, among 9 of 14 families. None of the variants appeared in more than one family.
One variant in PGK1 was previously reported as pathogenic/likely pathogenic in ClinVar in patients with phosphoglycerate kinase 1 deficiency associated with hemolytic anemia. Nine variants were reported in ClinVar as variants of uncertain significance, of which 3 were previously reported in patients with immunodeficiency disorders (in genes IL2RA, MALT1, and PRKDC) and 2 were reported in patients with dyskeratosis congenita (TERT) and other anemia-associated disorders (EPB42). Of 22 variants not reported in ClinVar, one was a missense variant in NPAT, a gene previously associated with familial lymphoma; 4 were in genes associated with immune disorders (PLCG2, LIG1, C1QB, and NOD2) and 3 were in genes associated with anemia (SPTB, ALDOA, and GATA1). To our knowledge, none of these genes other than NPAT have previously been implicated in predisposition to HL.
Conclusions:
WES in 20 AYA cHL patients among multiplex families identified putative novel predisposition genes for HL, including genes implicated in anemia and immune function. Assessment of these genes in sequencing studies of independent HL families is required to understand their role in HL predisposition.

Disclosures
Wadé: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company.