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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


CDH1 c.1679 C>G (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.(A) Sequence adjacent to wild type allele, CDH1 c.1679 C, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, CDH1 c.1679 G, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.
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pone.0165654.g004: CDH1 c.1679 C>G (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.(A) Sequence adjacent to wild type allele, CDH1 c.1679 C, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, CDH1 c.1679 G, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.

Mentions: To determine whether the CDH1 c.1679 G mutant allele completely disrupts normal splicing, i.e., whether the mutant allele is able to generate any CDH1 exon 11 full length transcripts with the T560R missense mutation, we cloned the RT-PCR products into the TOPO sequencing vector and then sequenced 21 colonies. All 21 clones from the patient and his brother contained the normal “C” allele in the full length transcript, indicating that the mutant allele was unable to generate any normal transcript (Fig 4A). However, all clones with the 32 nt deletion contained the mutant “G” allele (Fig 4B). These results indicate that the mutant “G” allele completely abolishes normal splicing through activation of a cryptic splice site within exon 11 of CDH1. We also confirmed the exon 11 deletion by Sanger sequencing (data not shown).


CDH1 Missense Variant c . 1679C > G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5 ’ Splice Site
CDH1 c.1679 C>G (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.(A) Sequence adjacent to wild type allele, CDH1 c.1679 C, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, CDH1 c.1679 G, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.
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Related In: Results  -  Collection

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pone.0165654.g004: CDH1 c.1679 C>G (p.T560R) mutant allele creates a novel splice site that results in a 32 nucleotide deletion of the 3’ end of exon 11.(A) Sequence adjacent to wild type allele, CDH1 c.1679 C, indicating normal splice site. Electropherogram data of normally spliced cDNA with wild type allele highlighted in blue. (B) Sequence adjacent to mutant allele, CDH1 c.1679 G, indicating aberrant splice site produced by mutant allele (solid lines) and location of normal splice site (dashed lines). Electropherogram data of aberrantly spliced cDNA with the mutant allele highlighted in light blue.
Mentions: To determine whether the CDH1 c.1679 G mutant allele completely disrupts normal splicing, i.e., whether the mutant allele is able to generate any CDH1 exon 11 full length transcripts with the T560R missense mutation, we cloned the RT-PCR products into the TOPO sequencing vector and then sequenced 21 colonies. All 21 clones from the patient and his brother contained the normal “C” allele in the full length transcript, indicating that the mutant allele was unable to generate any normal transcript (Fig 4A). However, all clones with the 32 nt deletion contained the mutant “G” allele (Fig 4B). These results indicate that the mutant “G” allele completely abolishes normal splicing through activation of a cryptic splice site within exon 11 of CDH1. We also confirmed the exon 11 deletion by Sanger sequencing (data not shown).

View Article: PubMed Central - PubMed

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.

No MeSH data available.