Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

Redefinition of familial intestinal gastric cancer: clinical and genetic perspectives.

BACKGROUND: Familial intestinal gastric cancer (FIGC) remains genetically unexplained and without testing/clinical criteria. Herein, we characterised the age of onset and disease spectrum of 50 FIGC families and searched for genetic causes potentially underlying a monogenic or an oligogenic/polygenic inheritance pattern. METHODS: Normal and tumour DNA from 50 FIGC probands were sequenced using Illumina custom panels on MiSeq, and their respective germline and somatic landscapes were compared with corresponding landscapes from sporadic intestinal gastric cancer (SIGC) and hereditary diffuse gastric cancer cohorts. RESULTS: The most prevalent phenotype in FIGC families was gastric cancer, detected in 138 of 208 patients (50 intestinal gastric cancer probands and 88 unknown gastric cancer histology relatives), followed by colorectal and breast cancers. After excluding benign and intronic variants lacking impact in splicing, 12 rare high-quality variants were found exclusively in 11 FIGC probands. Only two probands carried potentially deleterious variants, but lacked somatic second-hits, weakly supporting the monogenic hypothesis for FIGC. However, FIGC probands developed gastric cancer at least 10 years earlier and carried more TP53 germline common variants than SIGC (p=4.5E-03); FIGC and SIGC could be distinguished by specific germline and somatic variant profiles; there was an excess of FIGC tumours presenting microsatellite instability (38%); and FIGC tumours displayed significantly more somatic common variants than SIGC tumours (p=4.2E-06). CONCLUSION: This study proposed the first data-driven testing criteria for FIGC families, and supported FIGC as a genetically determined, likely polygenic, gastric cancer-predisposing disease, with earlier onset and distinct from patients with SIGC at the germline and somatic levels.

Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Hereditary Diffuse Gastric Cancer Syndrome: CDH1 Mutations and Beyond.

IMPORTANCE: E-cadherin (CDH1) is a cancer predisposition gene mutated in families meeting clinically defined hereditary diffuse gastric cancer (HDGC). Reliable estimates of cancer risk and spectrum in germline mutation carriers are essential for management. For families without CDH1 mutations, genetic-based risk stratification has not been possible, resulting in limited clinical options. OBJECTIVES: To derive accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and determine if germline mutations in other genes are associated with HDGC. DESIGN, SETTING, AND PARTICIPANTS: Testing for CDH1 germline mutations was performed on 183 index cases meeting clinical criteria for HDGC. Penetrance was derived from 75 mutation-positive families from within this and other cohorts, comprising 3858 probands (353 with gastric cancer and 89 with breast cancer). Germline DNA from 144 HDGC probands lacking CDH1 mutations was screened using multiplexed targeted sequencing for 55 cancer-associated genes. MAIN OUTCOMES AND MEASURES: Accurate estimates of gastric and breast cancer risks in CDH1 mutation carriers and the relative contribution of other cancer predisposition genes in familial gastric cancers. RESULTS: Thirty-one distinct pathogenic CDH1 mutations (14 novel) were identified in 34 of 183 index cases (19%). By the age of 80 years, the cumulative incidence of gastric cancer was 70% (95% CI, 59%-80%) for males and 56% (95% CI, 44%-69%) for females, and the risk of breast cancer for females was 42% (95% CI, 23%-68%). In CDH1 mutation-negative index cases, candidate mutations were identified in 16 of 144 probands (11%), including mutations within genes of high and moderate penetrance: CTNNA1, BRCA2, STK11, SDHB, PRSS1, ATM, MSR1, and PALB2. CONCLUSIONS AND RELEVANCE: This is the largest reported series of CDH1 mutation carriers, providing more precise estimates of age-associated risks of gastric and breast cancer that will improve counseling of unaffected carriers. In HDGC families lacking CDH1 mutations, testing of CTNNA1 and other tumor suppressor genes should be considered. Clinically defined HDGC families can harbor mutations in genes (ie, BRCA2) with different clinical ramifications from CDH1. Therefore, we propose that HDGC syndrome may be best defined by mutations in CDH1 and closely related genes, rather than through clinical criteria that capture families with heterogeneous susceptibility profiles.

Germline mutations in MAP3K6 are associated with familial gastric cancer.

Gastric cancer is among the leading causes of cancer-related deaths worldwide. While heritable forms of gastric cancer are relatively rare, identifying the genes responsible for such cases can inform diagnosis and treatment for both hereditary and sporadic cases of gastric cancer. Mutations in the E-cadherin gene, CDH1, account for 40% of the most common form of familial gastric cancer (FGC), hereditary diffuse gastric cancer (HDGC). The genes responsible for the remaining forms of FGC are currently unknown. Here we examined a large family from Maritime Canada with FGC without CDH1 mutations, and identified a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase kinase 6 (MAP3K6). Based on conservation, predicted pathogenicity and a known role of the gene in cancer predisposition, MAP3K6 was considered a strong candidate and was investigated further. Screening of an additional 115 unrelated individuals with non-CDH1 FGC identified the p.P946L MAP3K6 variant, as well as four additional coding variants in MAP3K6 (p.F849Sfs*142, p.P958T, p.D200Y and p.V207G). A somatic second-hit variant (p.H506Y) was present in DNA obtained from one of the tumor specimens, and evidence of DNA hypermethylation within the MAP3K6 gene was observed in DNA from the tumor of another affected individual. These findings, together with previous evidence from mouse models that MAP3K6 acts as a tumor suppressor, and studies showing the presence of somatic mutations in MAP3K6 in non-hereditary gastric cancers and gastric cancer cell lines, point towards MAP3K6 variants as a predisposing factor for FGC.

Boveri at 100: Theodor Boveri and genetic predisposition to cancer.

One hundred years have passed since the publication of Theodore Boveri's Zur Frage der Entstehung maligner Tumouren [Concerning the Origin of Malignant Tumours]. This prescient publication created the foundations for much of our understanding of the origins of cancer and in particular the genetic basis of some cancers. In his work, Boveri suggested that loss of key cellular attributes, now known as tumour suppressor genes, are a key driver event in the development of cancer and inheritance could play a role in cancer susceptibility. He also predicted that chromosomal (genomic) instability as a key hallmark of cancer. Whilst these key insights that still inform the practice of cancer genetics, they were not the main theme of Boveri's text, which was to describe the role of chromosomal abnormalities in the development of cancer. In making his case he also suggested that genetic information could be contained in distinct packages (genes) that are linearly arranged along chromosomes and that cancers arise from single cells. These remarkably accurate hypotheses add weight to the need to celebrate this landmark publication for its accurate prediction of so much that we take for granted. Here we focus on Boveri's contributions to our understanding of hereditary cancers, which, alongside the astute clinical observations of Paul Broca and Aldred Scott Warthin, were published decades before the field became respectable, yet could still inform anyone studying hereditary cancers.

A recurrent germline PAX5 mutation confers susceptibility to pre-B cell acute lymphoblastic leukemia.

Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.

An α-E-catenin (CTNNA1) mutation in hereditary diffuse gastric cancer.

Diffuse gastric cancers typically present as late-stage tumours and, as a result, the 5 year survival rate is poor. Some gastric cancers are hereditary and these tend to be of the diffuse type; 30-40% of hereditary diffuse gastric cancers (HDGCs) can be explained by defective germline alleles of E-cadherin (CDH1), but for the remaining families the factors driving susceptibility remain unknown. We had access to a large HDGC pedigree with no obvious mutation in CDH1, and applied exome sequencing to identify new genes involved in gastric cancer. We identified a germline truncating allele of α-E-catenin (CTNNA1) that was present in two family members with invasive diffuse gastric cancer and four in which intramucosal signet ring cells were detected as part of endoscopic surveillance. The remaining CTNNA1 allele was silenced in the two diffuse gastric cancers from the family that were available for screening, and this was also true for signet ring cells identified in endoscopic biopsies. Since α-E-catenin functions in the same complex as E-cadherin, our results call attention to the broader signalling network surrounding these proteins in HDGC. We also detected somatic mutations in one tumour and found substantial overlap with genes mutated in sporadic gastric cancer, including PIK3CA, ARID1A, MED12 and MED23.

Germline BRCA1 and BRCA2 mutations in ovarian cancer: utility of a histology-based referral strategy.

OBJECTIVE: To estimate the frequency of BRCA1 and BRCA2 germline mutations in women with nonmucinous epithelial ovarian carcinoma unselected for a family history of breast or ovarian cancer. METHODS: From 2004 to 2009, women undergoing surgical staging for nonmucinous epithelial ovarian carcinoma, including fallopian tube and primary peritoneal carcinoma, were invited to participate in tumor banking and genetic counseling for BRCA1 and BRCA2 mutations. Pathology and family history obtained by the gynecologic oncology surgeon and genetic counselors were reviewed. RESULTS: Of 131 women fulfilling entry criteria, germline BRCA1 and BRCA2 mutations were found in 20% (26/131) and were exclusively associated with high-grade serous histology (26/103 [25%]). Restricting BRCA1 and BRCA2 testing to women with family histories of hereditary breast and ovarian cancer, as ascertained by the surgeon, missed 14 mutation carriers, lowering detection rates to 9% (12/131) or 11.6% (12/103) if only considering the patients with high-grade serous histology. This improved to 16% (21/131) or 20.4% (21/103) when ascertained by the genetic counselor; however, 5 of 26 (19%) mutation carriers did not have a family history of hereditary breast or ovarian cancer. CONCLUSION: Germline BRCA1 and BRCA2 mutations in ovarian (pelvic) cancer are associated with high-grade serous histology. The high incidence (25%) of germline BRCA1 and BRCA2 mutations specific to the high-grade serous subtype suggests that genetic assessment of all women diagnosed with high-grade serous ovarian (pelvic) carcinoma will improve detection rates and capture mutation carriers otherwise missed by referral based on family history alone. LEVEL OF EVIDENCE: II.