CDH1 Missense Variant c.1679C>G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5’ Splice Site

Abstract

Disease-causing germline mutations in CDH1 cause Hereditary Diffuse Gastric Cancer (HDGC). For patients who meet the HDGC screening criteria, the identification and classification of the sequence variants found in CDH1 are critical for risk management of patients. In this report, we describe a germline CDH1 c.1679C>G (p.T560R) variant identified in a 50 year old man who was diagnosed with gastric cancer with a strong family history of gastric cancer (one living brother was diagnosed with gastric cancer at 63 and another brother died of gastric cancer at 45). cDNA analysis, involving fragment analysis and cloning, indicated that the p.T560R mutation created a novel 5’ splice donor site, which led to a novel transcript with a 32 nucleotide deletion in exon 11. This abnormal transcript putatively produces a truncated CDH1 protein (E-cadherin) of 575 amino acids instead of 882. We also demonstrated that the variant completely abolishes normal splicing as the mutant allele does not generate any normal transcript. Furthermore, the CDH1 c.1679C>G (p.T560R) variant segregated with gastric cancer in all three family members affected with gastric cancer in this family. These results support the conclusion that CDH1 c.1679C>G (p.T560R) variant is a pathogenic mutation and contributes to HDGC through disruption of normal splicing.

A study of splicing mutations in disorders of sex development

“…The results so far described here support the conclusion that c.277 + 2 T > G in HSD17B3 and c.544 G > A in SRD5A2 potentially disrupt binding of U1snRNP to the 5′ splice site therefore inhibiting splicing initiation 29 , 30 . These mutations highlight the importance of a conserved 5′ splice site sequence for correct binding of U1 snRNP and spliceosome machinery assembly, and show that disruption of this process may result in human disease 30 , 31 .…”

Clinical and functional characterization of the CDH1 germline variant c.1679C>G in three unrelated families with hereditary diffuse gastric cancer

“…Furthermore, this variant had been reported twice before in the literature-in a young patient with DGC [5] and in a family of Indian origin also with DGC [6]-together with two unpublished cases reported to the Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP; personal communication from R Seruca, 2016). Benusiglio et al did not perform any type of functional analysis [5], while Yelskaya et al described that the c.1679C>G variant disrupts normal splicing, which presumably leads to truncation of the protein [6]. In support of these findings, we also observed that the c.1679C>G change generates an alternative 5′ splice site, leading to the loss of 32 nucleotides in exon 11 or to a premature stop codon at position 576 of the protein.…”

“…The haplotype study indicated that a common ancestry of this variant event for the three families is unlikely. This result was expected for two reasons: (1) a large region of CDH1 (which includes our variant site) shows no linkage disequilibrium (GTEX), which suggests it might be very exposed to recombination; and (2) there are other individuals reported to carry this variant who have very distant geographic origins [5,6].…”

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants

“…Table S1.To avoid double counting of evidence, this PP3 rule cannot be used for canonical splice site variants and should only be applied to splicing variants at locations other than the ±1/2 positions. Note, this code also does not apply to the last nucleotide of exon 3 (c.387G) (Yelskaya, et al, 2016).…”