Identification and validation of novel candidate risk genes in endocytic vesicular trafficking associated with esophageal atresia and tracheoesophageal fistulas

Esophageal atresias/tracheoesophageal fistulas (EA/TEF) are rare congenital anomalies caused by aberrant development of the foregut. Previous studies indicate that rare or de novo genetic variants significantly contribute to EA/TEF risk, and most individuals with EA/TEF do not have pathogenic genetic variants in established risk genes. To identify novel genetic contributions to EA/TEF, we performed whole genome sequencing of 185 trios (probands and parents) with EA/TEF, including 59 isolated and 126 complex cases with additional congenital anomalies and/or neurodevelopmental disorders. There was a significant burden of protein altering de novo coding variants in complex cases (p=3.3e-4), especially in genes that are intolerant of loss of function variants in the population. We performed simulation analysis of pathway enrichment based on background mutation rate and identified a number of pathways related to endocytosis and intracellular trafficking that as a group have a significant burden of protein altering de novo variants. We assessed 18 variants for disease causality using CRISPR-Cas9 mutagenesis in Xenopus and confirmed 13 with tracheoesophageal phenotypes. Our results implicate disruption of endosome-mediated epithelial remodeling as a potential mechanism of foregut developmental defects. This research may have implications for the mechanisms of other rare congenital anomalies.

MEScan: a powerful statistical framework for genome-scale mutual exclusivity analysis of cancer mutations

Motivation Cancer somatic driver mutations associated with genes within a pathway often show a mutually exclusive pattern across a cohort of patients. This mutually exclusive mutational signal has been frequently used to distinguish driver from passenger mutations and to investigate relationships among driver mutations. Current methods for de novo discovery of mutually exclusive mutational patterns are limited because the heterogeneity in background mutation rate can confound mutational patterns, and the presence of highly mutated genes can lead to spurious patterns. In addition, most methods only focus on a limited number of pre-selected genes and are unable to perform genome-wide analysis due to computational inefficiency. Results We introduce a statistical framework, MEScan, for accurate and efficient mutual exclusivity analysis at the genomic scale. Our framework contains a fast and powerful statistical test for mutual exclusivity with adjustment of the background mutation rate and impact of highly mutated genes, and a multi-step procedure for genome-wide screening with the control of false discovery rate. We demonstrate that MEScan more accurately identifies mutually exclusive gene sets than existing methods and is at least two orders of magnitude faster than most methods. By applying MEScan to data from four different cancer types and pan-cancer, we have identified several biologically meaningful mutually exclusive gene sets. Availability and implementation MEScan is available as an R package at https://github.com/MarkeyBBSRF/MEScan. Supplementary information Supplementary data are available at Bioinformatics online.

Clinical significance of TP53, BIRC3, ATM and MAPK-ERK genes in chronic lymphocytic leukaemia: data from the randomised UK LRF CLL4 trial

Despite advances in chronic lymphocytic leukaemia (CLL) treatment, globally chemotherapy remains a central treatment modality, with chemotherapy trials representing an invaluable resource to explore disease-related/genetic features contributing to long-term outcomes. In 499 LRF CLL4 cases, a trial with >12 years follow-up, we employed targeted resequencing of 22 genes, identifying 623 mutations. After background mutation rate correction, 11/22 genes were recurrently mutated at frequencies between 3.6% (NFKBIE) and 24% (SF3B1). Mutations beyond Sanger resolution (<12% VAF) were observed in all genes, with KRAS mutations principally composed of these low VAF variants. Firstly, employing orthogonal approaches to confirm <12% VAF TP53 mutations, we assessed the clinical impact of TP53 clonal architecture. Whilst ≥ 12% VAF TP53mut cases were associated with reduced PFS and OS, we could not demonstrate a difference between <12% VAF TP53 mutations and either wild type or ≥12% VAF TP53mut cases. Secondly, we identified biallelic BIRC3 lesions (mutation and deletion) as an independent marker of inferior PFS and OS. Finally, we observed that mutated MAPK-ERK genes were independent markers of poor OS in multivariate survival analysis. In conclusion, our study supports using targeted resequencing of expanded gene panels to elucidate the prognostic impact of gene mutations.

De novo and recessive forms of congenital heart disease have distinct genetic and phenotypic landscapes

The genetic architecture of sporadic congenital heart disease (CHD) is characterized by enrichment in damaging de novo variants in chromatin-modifying genes. To test the hypothesis that gene pathways contributing to de novo forms of CHD are distinct from those for recessive forms, we analyze 2391 whole-exome trios from the Pediatric Cardiac Genomics Consortium. We deploy a permutation-based gene-burden analysis to identify damaging recessive and compound heterozygous genotypes and disease genes, controlling for confounding effects, such as background mutation rate and ancestry. Cilia-related genes are significantly enriched for damaging rare recessive genotypes, but comparatively depleted for de novo variants. The opposite trend is observed for chromatin-modifying genes. Other cardiac developmental gene classes have less stratification by mode of inheritance than cilia and chromatin-modifying gene classes. Our analyses reveal dominant and recessive CHD are associated with distinct gene functions, with cilia-related genes providing a reservoir of rare segregating variation leading to CHD.

WITER: A powerful method for the estimation of cancer-driver genes using a weighted iterative regression accurately modelling background mutation rate

Genomic identification of driver mutations and genes in cancer cells are critical for precision medicine. Due to difficulty in modeling distribution of background mutations, existing statistical methods are often underpowered to discriminate driver genes from passenger genes. Here we propose a novel statistical approach, weighted iterative zero-truncated negative-binomial regression (WITER), to detect cancer-driver genes showing an excess of somatic mutations. By solving the problem of inaccurately modeling background mutations, this approach works even in small or moderate samples. Compared to alternative methods, it detected more significant and cancer-consensus genes in all tested cancers. Applying this approach, we estimated 178 driver genes in 26 different cancers types. In silico validation confirmed 90.5% of predicted genes as likely known drivers and 7 genes unique for individual cancers as likely new drivers. The technical advances of WITER enable the detection of driver genes in TCGA datasets as small as 30 subjects, rescuing more genes missed by alternative tools.

The “false-positive” conundrum in the NTP 2-year rodent cancer study database

In 1990, Ames and Gold described a conundrum of “too many carcinogens” among chemicals tested in rodent bioassays. Their proposed nongenotoxic carcinogenic mechanism was amplification of the background mutation rate via cytotoxicity induced by high doses of the test chemicals, thereby leading to increases in reparative cellular proliferation rates. Recently, we have statistically and mechanistically analyzed the entire 594-study (470 final reports) NTP 2-year rodent cancer database to better understand the conundrum posed by Ames and Gold. Our analysis provides several lines of evidence that support the contention of Ames and Gold. First, across different routes of administration, relatively phylogenetically similar rats and mice are nonetheless discordant for the development of tumors at similar organ sites. Tumor site concordance across sex within species is higher than tumor site concordance across species. Second, many chemicals negative in the Ames test nonetheless induce tumors in either rats or mice. Third, 11 out of 58 chemicals tested by the inhalation route induce lung tumors in mice and not rats, are negative in the Ames test, and exhibit hyperplasia. In 2017, Tomasetti et al. provided evidence for the clinical relevance in humans of the Ames and Gold mechanism regarding amplification of the background mutation rate by demonstrating that the majority of human tumors result from accumulated mutations due to DNA replication errors.