Cancer Res Treat.  2024 Oct;56(4):1126-1135. 10.4143/crt.2024.328.

Identification of New Pathogenic Variants of Hereditary Diffuse Gastric Cancer

• Oh SY
• Jang G
• Kim J
• Jeong KY
• Kim HM
• Kwak YJ
• Kong SH
• Park DJ
• Lee HJ
• Cho SY
• Kim JI
• Yang HK

• KMID: 2560247
• DOI: http://doi.org/10.4143/crt.2024.328

Abstract

Purpose
Hereditary diffuse gastric cancer (HDGC) presents a significant genetic predisposition, notably linked to mutations in the CDH1 and CTNNA1. However, the genetic basis for over half of HDGC cases remains unidentified. The aim of this study is to identify novel pathogenic variants in HDGC and evaluate their protein expression.
Materials and Methods
Among 20 qualifying families, two were selected based on available pedigree and DNA. Whole genome sequencing (WGS) on DNA extracted from blood and whole exome sequencing on DNA from formalin-fixed paraffin-embedded tissues were performed to find potential pathogenic variants in HDGC. After selection of a candidate variant, functional validation, and enrichment analysis were performed.
Results
As a result of WGS, three candidate germline mutations (EPHA5, MCOA2, and RHOA) were identified in one family. After literature review and in-silico analyses, the RHOA mutation (R129W) was selected as a candidate. This mutation was found in two gastric cancer patients within the family. In functional validation, it showed RhoA overexpression and a higher GTP-bound state in the Rhoa^R129W mutant. Decreased phosphorylation at Ser127/397 suggested altered YAP1 regulation in the Rho-ROCK pathway. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses linked Rhoa^R129W overexpression to changed migration/adhesion in MKN1 cell line. However, this RHOA mutation (R129W) was not found in index patients in other families.
Conclusion
The RHOA mutation (R129W) emerges as a potential causative gene for HDGC, but only in one family, indicating a need for further studies to understand its role in HDGC pathogenesis fully.

Keyword

Familial gastric cancer; Hereditary diffuse gastric cancer; Germ-line mutation; Pathogenic variant; RHOA

Figure

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  Fig. 1. Flowchart showing the family selection for whole genome sequencing in this study.

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  Fig. 2. (A, B) Pedigrees of two families with hereditary diffuse gastric cancer who underwent whole genome sequencing (WGS) to identify new candidate variants of hereditary diffuse gastric cancer. Where known, the individual’s age is presented to the right of the symbols. The age is underlined if DNA from blood sample was available. Not all relatives consented to undergo genetic screening. A dotted circle is drawn around the symbol of the individual for whom WGS was performed and red dotted circle means individuals with RHOA R129W germline mutation (square, male; circle, female; oblique line, deceased; black, disease; *, index patient; number’, year of birth; unknown, year of birth unknown; numberY, age at diagnose; ?, all unknown).

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  Fig. 3. RhoA expression level in each gastric cancer cell line. RhoA expression level was the lowest in the MKN1 gastric cancer cell line.

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  Fig. 4. Functional validation of RHOA mutation (R129W). (A) Levels of exogenous Myc-tagged wild-type RhoA (myc-RhoA) and Myc-tagged mutant RhoA (myc-RhoAR129W). (B) Overexpression of myc-RhoA and myc-RhoAR129W exhibited a similar inhibitory effect on cell growth. (C) RhoAR129W has a higher potential to exist in a GTP-bound state than the wild-type RhoA. (D) Expression levels of ROCK1 and p-MLC2 showed no difference between cells overexpressing myc-RhoA- and myc-RhoAR129W. (E) Phosphorylation of Ser127/397 was lower in the myc-RhoAR129W–overexpressing cells than in the myc-RhoA–overexpressing cells. (F) Although the expression levels of RhoA and the mRNA were similar in wild-type RhoA and RhoAR129W overexpressing cells, up- or down-regulated genes were differed. (G) Genes related cell migration and cytoskeleton reorganization were enriched in RhoAR129W overexpressing cells. CPM, counts per million; DAVID, Database for Annotation, Visualization and Integrated Discovery; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type.

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  Fig. 5. ANKRD1 (A) and AXL (B) level in wild-type RhoA and RhoAR129W overexpressing cells. Among ANKRD1 and AXL, only ANKRD1 is upregulated in RhoAR129W-overexpressing cells. CPM, counts per million.

Reference

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