A region-based gene association study combined with a leave-one-out sensitivity analysis identifies SMG1 as a pancreatic cancer susceptibility gene

Abstract

Pancreatic adenocarcinoma (PC) is a lethal malignancy that is familial or associated with genetic syndromes in 10% of cases. Gene-based surveillance strategies for at-risk individuals may improve clinical outcomes. However, familial PC (FPC) is plagued by genetic heterogeneity and the genetic basis for the majority of FPC remains elusive, hampering the development of gene-based surveillance programs. The study was powered to identify genes with a cumulative pathogenic variant prevalence of at least 3%, which includes the most prevalent PC susceptibility gene, BRCA2. Since the majority of known PC susceptibility genes are involved in DNA repair, we focused on genes implicated in these pathways. We performed a region-based association study using the Mixed-Effects Score Test, followed by leave-one-out characterization of PC-associated gene regions and variants to identify the genes and variants driving risk associations. We evaluated 398 cases from two case series and 987 controls without a personal history of cancer. The first case series consisted of 109 patients with either FPC (n = 101) or PC at ≤50 years of age (n = 8). The second case series was composed of 289 unselected PC cases. We validated this discovery strategy by identifying known pathogenic BRCA2 variants, and also identified SMG1, encoding a serine/threonine protein kinase, to be significantly associated with PC following correction for multiple testing (p = 3.22x10-7). The SMG1 association was validated in a second independent series of 532 FPC cases and 753 controls (p<0.0062, OR = 1.88, 95%CI 1.17-3.03). We showed segregation of the c.4249A>G SMG1 variant in 3 affected relatives in a FPC kindred, and we found c.103G>A to be a recurrent SMG1 variant associating with PC in both the discovery and validation series. These results suggest that SMG1 is a novel PC susceptibility gene, and we identified specific SMG1 gene variants associated with PC risk.

Fig 1. Schematic of gene association study design.

Series A, cases at high risk for hereditary PC. Series B, unselected prospectively collected PC cases. WES, whole-exome sequencing. NGS, next-generation sequencing. SNVs, single nucleotide variants. INDELs, insertions/deletions.

Fig 2. The–log p-value graphs for the LOO-W analysis for BRCA2, BRCA1 and SMG1.

A decrease in the–log p-value is an increase in p-value signifying the window dropped contains variants driving the association with PC risk. Any window with an increase in p-value was analyzed by LOO-V for potential variants of interest. A) LOO-W for BRCA2. B) LOO-W for BRCA1. C) LOO-W for SMG1.

Fig 3. The–log p-value graphs for LOO-V analysis for each significant window for BRCA2, BRCA1, SMG1.

A decrease in the–log p-value corresponds to an increase in p-value, signifying the dropped variant is potentially driving the association with PC risk. The dotted line represents the–log p-value for an increase in p-value of 35% compared with the p-value of the window. This is the threshold for identifying a candidate variant. A) LOO-V for window 1 to window 4 of BRCA2. B) LOO-V for window 1 and window 2 of BRCA1. C) LOO-V for window 1 and window 2 of SMG1.

Fig 4. Pedigrees for two SMG1 variant carriers with segregation opportunities.

A) In family A-78, both the proband and the maternal aunt were included in our case series and were identified to be carriers of the c.4249A>G (p.I1417V) SMG1 variant. *SMGI c.4249A>G carrier status for the maternal uncle with PC was inferred by genotyping his son. B) In family B-105, the maternal aunt was included in our case series and was identified to be a carrier of the c.4952C>G (p.S1651C) SMG1 variant. The proband was found to not be a carrier of this SMG1 mutation.

Fig 5. A lollipop diagram depicting the mutation landscape for SMG1 variants identified in both the discovery and validation series.

SMG1 consists of 4 main functional domains represented by the blue (N-terminal domain—amino acid (aa) 32–140), red (FAT domain—aa 1131–1866), green (PIKK domain—aa 2121–2482), and orange (FATC domain—aa 3629–3661). Variants identified in the discovery series are depicted by the upward lollipops and variants identified in the validation series are depicted by the downward lollipops. The red lollipop located in the N-terminal domain represents the recurrent p.A35T variant identified in both the discovery and validation series. The purple and orange lollipops located in the FAT domain represent the p.I1417V variant, which segregated in kindred A-78, and the p.S1651C variant identified in kindred B-105, respectively. The dark green and black lollipops represent the amino acid position adjacent to the splicing variants identified. The light blue lollipop represents the non-frameshift variant identified.