Genetic Predisposition to Gastric Cancer: CDH1 and Beyond

Gastric cancer is a complex disease influenced by strong genetic and environmental factors. Hereditary gastric cancer syndromes are thought to account for between 1-3% of all cases. The most common hereditary gastric cancer syndrome is Hereditary Diffuse Gastric Cancer (HDGC), an autosomal dominant cancer syndrome that is primarily characterised by an extreme risk of developing diffuse-type gastric cancer. Approximately 40% of families that fit the clinical criteria for HDGC carry a pathogenic variant in germline CDH1. An explanation for the remaining 60% of cases remains largely elusive. While New Zealand as a whole is a country with a low-incidence of gastric cancer, Māori have an age-standardised incidence of gastric cancer more than three times that of non-Māori. Additionally, Māori have an average age of diagnosis approximately 10 years younger than non-Māori, and are one of the few populations worldwide with a higher incidence of the diffuse-type disease. To assess the contribution of HDGC to the high-incidence of diffuse gastric cancer for Māori, we analysed the CDH1 sequence from an unselected cohort of 94 Māori gastric cancer patients and 200 healthy matched controls using next-generation amplicon sequencing, multiplex ligation-dependent probe amplification, and Sanger sequencing. Pathogenic CDH1 variants were identified in 18% of all cases, 34% of diffuse gastric cancers, and 66% of early-onset cases (< 45 years of age). After adjusting for the effect of clinical genetic testing for known Māori HDGC families, we estimate 6% of all Māori gastric cancer patients and 13% of diffuse gastric cancer patients carry pathogenic germline CDH1 variants. Chile is a country with a high-incidence of gastric cancer and no formal genetic screening programme for gastric cancer patients. To explore pathogenic germline CDH1 variants as a cause of gastric cancer in Chile, next-generation amplicon sequencing and Sanger sequencing were used to screen a cohort of 51 Chilean gastric cancer patients that presented with a striking family history or early-onset disease. Overall, one clear pathogenic CDH1 variant was identified, representing 2.0% of all probands and 3.6% of probands who met the clinical criteria for HDGC. Although pathogenic CDH1 variants were rare in this Chilean cohort, we were able to screen the extended family of the one proband with a confirmed mutation and identify five further carriers. These carriers will now benefit from surveillance and early intervention. Finally, whole-exome sequencing was used to examine 14 diffuse gastric cancer patients that fit the clinical criteria for HDGC and did not carry a pathogenic variant in their germline CDH1. Variants in these patients were filtered and prioritised for further evaluation and validation using Sanger sequencing. Single probands were found to carry pathogenic variants in ATM and TP53, genes which are not associated with HDGC, but are known to increase gastric cancer risk. Additional mutations of interest were identified in FARP2, FGD4, and LMO7, genes that are important in the coordination of the actin cytoskeleton and/or cell adhesion, pathways which are dysregulated in diffuse-type gastric tumours. Until now, FARP2, FGD4, and LMO7 were not linked with diffuse gastric cancer risk. It is clear from the current study and other HDGC studies, that there is no other common gene for HDGC, however families may carry private variants in genes rarely associated with disease. Taken together, these studies demonstrate the variable frequency of pathogenic variants in germline CDH1 in different populations, the absence of other commonly mutated genes in familial diffuse gastric cancers, and the importance of genetic screening and targeted interventions for those that carry pathogenic variants

Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

Cancer; Cancer geneticsCàncer; Genètica del càncerCáncer; Genética del cáncerThe contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09–1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers

Germline CDH1 mutations are a significant contributor to the high frequency of early-onset diffuse gastric cancer cases in New Zealand Māori.

New Zealand Māori have a considerably higher incidence of gastric cancer compared to non-Māori, and are one of the few populations worldwide with a higher prevalence of diffuse-type disease. Pathogenic germline CDH1 mutations are causative of hereditary diffuse gastric cancer, a cancer predisposition syndrome primarily characterised by an extreme lifetime risk of developing diffuse gastric cancer. Pathogenic CDH1 mutations are well described in Māori families in New Zealand. However, the contribution of these mutations to the high incidence of gastric cancer is unknown. We have used next-generation sequencing, Sanger sequencing, and Multiplex Ligation-dependent Probe Amplification to examine germline CDH1 in an unselected series of 94 Māori gastric cancer patients and 200 healthy matched controls. Overall, 18% of all cases, 34% of cases diagnosed with diffuse-type gastric cancer, and 67% of cases diagnosed aged less than 45 years carried pathogenic CDH1 mutations. After adjusting for the effect of screening known HDGC families, we estimate that 6% of all advanced gastric cancers and 13% of all advanced diffuse-type gastric cancers would carry germline CDH1 mutations. Our results demonstrate that germline CDH1 mutations are a significant contributor to the high frequency of diffuse gastric cancer in New Zealand Māori

Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

The risk of germline copy number variants (CNVs) in BRCA1 and BRCA2 pathogenic variant carriers in breast cancer is assessed, with CNVs overlapping SULT1A1 decreasing breast cancer risk in BRCA1 carriers.The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.Peer reviewe

Genetic Predisposition to Gastric Cancer: CDH1 and Beyond

Gastric cancer is a complex disease influenced by strong genetic and environmental factors. Hereditary gastric cancer syndromes are thought to account for between 1-3% of all cases. The most common hereditary gastric cancer syndrome is Hereditary Diffuse Gastric Cancer (HDGC), an autosomal dominant cancer syndrome that is primarily characterised by an extreme risk of developing diffuse-type gastric cancer. Approximately 40% of families that fit the clinical criteria for HDGC carry a pathogenic variant in germline CDH1. An explanation for the remaining 60% of cases remains largely elusive. While New Zealand as a whole is a country with a low-incidence of gastric cancer, Māori have an age-standardised incidence of gastric cancer more than three times that of non-Māori. Additionally, Māori have an average age of diagnosis approximately 10 years younger than non-Māori, and are one of the few populations worldwide with a higher incidence of the diffuse-type disease. To assess the contribution of HDGC to the high-incidence of diffuse gastric cancer for Māori, we analysed the CDH1 sequence from an unselected cohort of 94 Māori gastric cancer patients and 200 healthy matched controls using next-generation amplicon sequencing, multiplex ligation-dependent probe amplification, and Sanger sequencing. Pathogenic CDH1 variants were identified in 18% of all cases, 34% of diffuse gastric cancers, and 66% of early-onset cases (< 45 years of age). After adjusting for the effect of clinical genetic testing for known Māori HDGC families, we estimate 6% of all Māori gastric cancer patients and 13% of diffuse gastric cancer patients carry pathogenic germline CDH1 variants. Chile is a country with a high-incidence of gastric cancer and no formal genetic screening programme for gastric cancer patients. To explore pathogenic germline CDH1 variants as a cause of gastric cancer in Chile, next-generation amplicon sequencing and Sanger sequencing were used to screen a cohort of 51 Chilean gastric cancer patients that presented with a striking family history or early-onset disease. Overall, one clear pathogenic CDH1 variant was identified, representing 2.0% of all probands and 3.6% of probands who met the clinical criteria for HDGC. Although pathogenic CDH1 variants were rare in this Chilean cohort, we were able to screen the extended family of the one proband with a confirmed mutation and identify five further carriers. These carriers will now benefit from surveillance and early intervention. Finally, whole-exome sequencing was used to examine 14 diffuse gastric cancer patients that fit the clinical criteria for HDGC and did not carry a pathogenic variant in their germline CDH1. Variants in these patients were filtered and prioritised for further evaluation and validation using Sanger sequencing. Single probands were found to carry pathogenic variants in ATM and TP53, genes which are not associated with HDGC, but are known to increase gastric cancer risk. Additional mutations of interest were identified in FARP2, FGD4, and LMO7, genes that are important in the coordination of the actin cytoskeleton and/or cell adhesion, pathways which are dysregulated in diffuse-type gastric tumours. Until now, FARP2, FGD4, and LMO7 were not linked with diffuse gastric cancer risk. It is clear from the current study and other HDGC studies, that there is no other common gene for HDGC, however families may carry private variants in genes rarely associated with disease. Taken together, these studies demonstrate the variable frequency of pathogenic variants in germline CDH1 in different populations, the absence of other commonly mutated genes in familial diffuse gastric cancers, and the importance of genetic screening and targeted interventions for those that carry pathogenic variants

CDH1 gene mutation in early-onset, colorectal signet-ring cell carcinoma

Aim: Colorectal signet-ring cell carcinomas (SRCC) are highly malignant tumours with poor prognosis that disproportionately affect younger patients. There is growing evidence of a unique set of molecular features that separate SRCC from conventional colorectal adenocarcinoma. Identification of these distinct features may have diagnostic and prognostic significance for patients and families. CDH1, which encodes E-cadherin, a cell adhesion protein, is commonly mutated in gastric SRCC and our study aimed to identify whether CDH1 mutation was also a common phenomenon in colorectal SRCC.Methods: DNA was extracted from formalin-fixed paraffin embedded tumour tissue, the CDH1 gene was analysed by next generation sequencing and the pathogenicity of mutations assessed in silico. Sections cut from the same blocks were immunostained to identify the presence of the E-cadherin protein.Results: We found 8 CDH1 mutations that meet our inclusion criteria in seven of 11 samples. Of these, five (from four patients), were likely to be germline mutations. E-cadherin staining was absent or markedly reduced in all of the seven samples with CDH1 mutation.Conclusion: Our finding of CDH1 mutations in a proportion of signet-ring cell carcinomas and associated reduction in E-cadherin in these tumours supports previous findings of a role for mutation of this gene in the development of this disease. In addition, the finding of likely germline mutations suggests that a subset of these tumours may be familial. Loss of E-cadherin staining in the absence of CDH1 mutations however also suggests a role for environmental factors in a subset of these tumours

Comprehensive Assessment of BARD1 Messenger Ribonucleic Acid Splicing With Implications for Variant Classification

Introduction: Case–control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/−1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate

Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers