Digenic inheritance of MSH6 and MUTYH variants in familial colorectal cancer

Abstract We describe a family severely affected by colorectal cancer (CRC) where whole‐exome sequencing identified the coinheritance of the germline variants encoding MSH6 p.Thr1100Met and MUTYH p.Tyr179Cys in, at least, three CRC patients diagnosed before 60 years of age. Digenic inheritance of monoallelic MSH6 variants of uncertain significance and MUTYH variants has been suggested to predispose to Lynch syndrome‐associated cancers; however, cosegregation with disease in the familial setting has not yet been established. The identification of individuals carrying multiple potential cancer risk variants is expected to rise with the increased application of whole‐genome sequencing and large multigene panel testing in clinical genetic counseling of familial cancer patients. Here we demonstrate the coinheritance of monoallelic variants in MSH6 and MUTYH consistent with cosegregation with CRC, further supporting a role for digenic inheritance in cancer predisposition.


| Whole-exome sequencing
Whole-exome sequencing was outsourced to BGI (BGI-Shenzhen, Shenzhen, China); exome libraries were constructed with the BGI capture kit, followed by sequencing on the Complete Genomics' Sequencing Platform (Complete Genomics Inc., San Jose, California). Filtering and variant prioritization was performed as previously described. 10 All variants were selected based on a maximum population frequency <0.01 (in 1000 Genomes phase 3, ExAC 1.0, ESP6500SI-V2 or GoNL release 5).

| Functional MMR assay
In vitro MMR activity assay was performed as previously described. 13

| RESULTS
We performed germline whole-exome sequencing on three CRC patients diagnosed before 60 years of age (III-1, III-7, III-8, Figure 1A) and who belonged to a CRC family comprising of seven cancer patients divided over two generations. Twenty-two rare variants were shared by the three patients (Tables 1 and S1)

| DISCUSSION
Digenic inheritance of monoallelic MSH6 and MUTYH variants has been suggested to predispose to Lynch syndrome-associated cancers.
The involvement of both MSH6 and MUTYH in oxidative DNA damage repair and their physical interaction enhancing MUTYH's repair activity, substantiates the association of variants in these genes. 16 From earlier studies, the inheritance of monoallelic MUTYH variants seemed primarily relevant in patients carrying MSH6 VUSs, which are less strongly associated with MMR deficiency than pathogenic MSH6 variants (Table S2). [4][5][6][7][8][9] Furthermore, a digenic inheritance model was proposed once before for CRC predisposition in a carrier of variants in the oxidative DNA damage repair genes MUTYH and OGG1. 17 Although the functional evidence of combined defects in oxidative DNA damage repair genes is still lacking, the coinheritance of MSH6 and MUTYH variants in at least three, but likely five cancer cases within one family warrants further mechanistic and clinical studies. Next to MSH6 and MUTYH, CUX1 has been described as a cancer-driving gene. 18 CUX1 is implicated in inflammatory bowel disease and various cancer types, although primarily due to loss-offunction somatic mutations. 18,19 This gene codes for several isoforms, including the ubiquitously expressed p200 CUX1, which, among other functions, has been shown to stimulate the repair of oxidized DNA bases by OGG1. 20 The identified CUX1 (NM_001202543: c.1438A > G, p.Ser480Gly) variant, however, was classified as likely benign by the Franklin variant classification tool. 21 Additional gene reportedly linked to tumorigenesis include RYR3, 22 EBNA1BP2, 23 TRIP6, 24 and CAPN9. 25 The RYR3 (NM_001036: c.7812C > G, p.
Asn2604Lys) and EBNA1BP2 (NM_001159936: c.1034A > T, p. Asn345Ile) variants were classified as likely benign and benign, respectively, while the TRIP6 (NM_003302: c.822G > C, p.Glu274Asp) and the CAPN9 (NM_006615: c.55G > T, p.Ala19Ser) variants were classified as VUS. 21 TRIP6 promotes cell migration and invasion through Wnt/β-catenin signaling and was shown to be upregulated in colorectal tumors. 24 Therefore, TRIP6 variants that increase protein stability or expression could potentially stimulate colorectal tumorigenesis. In addition, lost-of-function variants in CAPN9 might promote tumor formation, as Calpain-9 induces cell cycle arrest and apoptosis, and low expression predicts a poorer prognosis in gastric cancer patients. 25 The contribution of the genetic variants, other than MSH6 and MUTYH, to cancer risk cannot be completely excluded. However, none of these variants have been functionally investigated and especially the variants predicted as benign or likely benign are less likely to contribute to an increased cancer risk. Besides, none of these genes have, to date, been associated with a genetic predisposition to any types of cancer.
In conclusion, with the increased application of whole-genome sequencing or large multigene panel testing in clinical genetic counseling, the number of identified individuals carrying multiple potential risk variants is expected to rise. Here, we demonstrate the coinheritance of MSH6 and MUTYH variants consistent with the cosegregation with cancer, further supporting a role for digenic inheritance in CRC predisposition. Our results reiterate that digenic inheritance should be considered as cause of genetic diseases.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy restrictions.