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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.


Semi-quantitative fragment analysis of CDH1 RNA transcripts.(A) RT-PCR fragments generated from CDH1 spanning exons 10–12 cDNAs from patient, patient’s brother, and eight controls (results from one representative fragment analysis run were shown) were analyzed by capillary electrophoresis (3730 Genetic Analyzer). The three peaks observed (from left to right) are: del Exon 11, del 32nt, and Full length. (B) Quantification of the three transcripts in controls and affected patients (the proband and his brother). The peak heights for the three transcripts in the patient and patient’s brother were averaged because the ratios are similar. The peak heights for the eight control samples were also averaged. Blue, red and green bars represent percentages of fragments with exon 11deletion, a deletion of 32 nucleotides within exon 11, and wild type fragments. Error bars represent standard error in comparing controls and patients for each fragment. Asterisks represent statistical significance between controls and patients.
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pone.0165654.g003: Semi-quantitative fragment analysis of CDH1 RNA transcripts.(A) RT-PCR fragments generated from CDH1 spanning exons 10–12 cDNAs from patient, patient’s brother, and eight controls (results from one representative fragment analysis run were shown) were analyzed by capillary electrophoresis (3730 Genetic Analyzer). The three peaks observed (from left to right) are: del Exon 11, del 32nt, and Full length. (B) Quantification of the three transcripts in controls and affected patients (the proband and his brother). The peak heights for the three transcripts in the patient and patient’s brother were averaged because the ratios are similar. The peak heights for the eight control samples were also averaged. Blue, red and green bars represent percentages of fragments with exon 11deletion, a deletion of 32 nucleotides within exon 11, and wild type fragments. Error bars represent standard error in comparing controls and patients for each fragment. Asterisks represent statistical significance between controls and patients.

Mentions: Based on the in silico prediction results and patient’s strong personal and family history, we decided to evaluate the effect of the CDH1 c.1679 C>G on mRNA splicing by amplifying regions of CDH1 using cDNA derived from the patient, patient’s living affected brother (who also carries the same variant), and individuals who do not carry this variant. The PCR was designed to generate fragments containing exons 10–12 of CDH1, which are the exons most likely to be affected by the mutation. Fragment analysis of RT-PCR products spanning CDH1 exons 10–12 resulted in three transcripts. One transcript of predicted size of RT-PCR product, “Wild type”, was observed in the controls as well as in the patient and his brother. It is the fragment generated by the wild type allele(s). Another transcript observed in the controls, patient and his brother, was shorter by 146 base pairs than the full-length fragment, “del Exon 11.” This fragment is consistent with alternative CDH1 transcript that skips exon 11. Interestingly, we observed a novel transcript that is unique in the patient and his living brother: 32 nucleotides shorter than the full-length fragment, “del 32nt” (Fig 3A).


CDH1 Missense Variant c . 1679C > G (p.T560R) Completely Disrupts Normal Splicing through Creation of a Novel 5 ’ Splice Site
Semi-quantitative fragment analysis of CDH1 RNA transcripts.(A) RT-PCR fragments generated from CDH1 spanning exons 10–12 cDNAs from patient, patient’s brother, and eight controls (results from one representative fragment analysis run were shown) were analyzed by capillary electrophoresis (3730 Genetic Analyzer). The three peaks observed (from left to right) are: del Exon 11, del 32nt, and Full length. (B) Quantification of the three transcripts in controls and affected patients (the proband and his brother). The peak heights for the three transcripts in the patient and patient’s brother were averaged because the ratios are similar. The peak heights for the eight control samples were also averaged. Blue, red and green bars represent percentages of fragments with exon 11deletion, a deletion of 32 nucleotides within exon 11, and wild type fragments. Error bars represent standard error in comparing controls and patients for each fragment. Asterisks represent statistical significance between controls and patients.
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Related In: Results  -  Collection

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pone.0165654.g003: Semi-quantitative fragment analysis of CDH1 RNA transcripts.(A) RT-PCR fragments generated from CDH1 spanning exons 10–12 cDNAs from patient, patient’s brother, and eight controls (results from one representative fragment analysis run were shown) were analyzed by capillary electrophoresis (3730 Genetic Analyzer). The three peaks observed (from left to right) are: del Exon 11, del 32nt, and Full length. (B) Quantification of the three transcripts in controls and affected patients (the proband and his brother). The peak heights for the three transcripts in the patient and patient’s brother were averaged because the ratios are similar. The peak heights for the eight control samples were also averaged. Blue, red and green bars represent percentages of fragments with exon 11deletion, a deletion of 32 nucleotides within exon 11, and wild type fragments. Error bars represent standard error in comparing controls and patients for each fragment. Asterisks represent statistical significance between controls and patients.
Mentions: Based on the in silico prediction results and patient’s strong personal and family history, we decided to evaluate the effect of the CDH1 c.1679 C>G on mRNA splicing by amplifying regions of CDH1 using cDNA derived from the patient, patient’s living affected brother (who also carries the same variant), and individuals who do not carry this variant. The PCR was designed to generate fragments containing exons 10–12 of CDH1, which are the exons most likely to be affected by the mutation. Fragment analysis of RT-PCR products spanning CDH1 exons 10–12 resulted in three transcripts. One transcript of predicted size of RT-PCR product, “Wild type”, was observed in the controls as well as in the patient and his brother. It is the fragment generated by the wild type allele(s). Another transcript observed in the controls, patient and his brother, was shorter by 146 base pairs than the full-length fragment, “del Exon 11.” This fragment is consistent with alternative CDH1 transcript that skips exon 11. Interestingly, we observed a novel transcript that is unique in the patient and his living brother: 32 nucleotides shorter than the full-length fragment, “del 32nt” (Fig 3A).

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.