Genome-wide somatic mutation analysis of sinonasal adenocarcinoma with and without wood dust exposure.

BACKGROUND: Sinonasal adenocarcinoma is a rare cancer, encompassing two different entities, the intestinal-type sinonasal adenocarcinoma (ITAC) and the non-intestinal-type sinonasal adenocarcinoma (non-ITAC). Occurrence of ITAC is strongly associated with exposure to hardwood dusts. In countries with predominant exposure to softwood dust the occurrence of sinonasal adenocarcinomas is lower and the relative amount of non-ITACs to ITACs is higher. The molecular mechanisms behind the tumorigenic effects of wood dust remain largely unknown. METHODS: We carried out whole-genome sequencing of formalin-fixed paraffin-embedded (FFPE) samples of sinonasal adenocarcinomas from ten wood dust-exposed and six non-exposed individuals, with partial tobacco exposure data. Sequences were analyzed for the presence of mutational signatures matching COSMIC database signatures. Driver mutations and CN variant regions were characterized. RESULTS: Mutation burden was higher in samples of wood dust-exposed patients (p = 0.016). Reactive oxygen species (ROS) damage-related mutational signatures were almost exclusively identified in ITAC subtype samples (p = 0.00055). Tobacco smoke mutational signatures were observed in samples of patients with tobacco exposure or missing information, but not in samples from non-exposed patients. A tetraploidy copy number (CN) signature was enriched in ITAC subtype (p = 0.042). CN variation included recurrent gains in COSMIC Cancer Gene Census genes TERT, SDHA, RAC1, ETV1, PCM1, and MYC. Pathogenic variants were observed most frequently in TP53, NF1, CHD2, BRAF, APC, and LRP1B. Driver mutations and copy number gains did not segregate by subtype. CONCLUSIONS: Our analysis identified distinct mutational characteristics in ITAC and non-ITAC. Mutational signature analysis may eventually become useful for documentation of occupation-related cancer, while the exact mechanisms behind wood dust-driven carcinogenesis remain elusive. The presence of homologous recombination deficiency signatures implies a novel opportunity for treatment, but further studies are needed.

Selection among critically endangered landlocked salmon (Salmo salar m. sebago) families in survival and growth traits across early life stages and in different environments.

Endangered wild fish populations are commonly supported by hatchery propagation. However, hatchery-reared fish experience very different selective pressures compared to their wild counterparts, potentially causing genotype-by-environment interactions (G × E) in essential fitness traits. We experimentally studied early selection in a critically endangered landlocked Atlantic salmon population, first from fertilization to the swim-up stage in a common hatchery setting, and thereafter until the age of 5 months in two contrasting rearing environments. Swim-up progeny were moved either to standard indoor hatchery tanks involving conventional husbandry or to seminatural outdoor channels providing only natural food. After the first summer, sampled survivors were assigned to their families by genotyping. Early survival until the swim-up stage was mostly determined by maternal effects, but also involved significant variation due to sires and full-sib families (potential genetic effects). High on-growing survival in hatchery tanks (88.7%) maintained a more even distribution among families (relative share 1.5%-4.2%) than the seminatural environment (0.0%-5.4%). This heterogeneity was mostly maternal, whereas no independent paternal effect occurred. Heritability estimates were high for body size traits in both environments (0.62-0.69). Genetic correlations between the environments were significantly positive for body size traits (0.67-0.69), and high body condition in hatchery was also genetically linked to rapid growth in the seminatural environment (0.54). Additive and phenotypic growth variation increased in the seminatural environment, but scaling effects probably played a less significant role for G × E, compared to re-ranking of genotypes. Our results suggest that not only maternal effects, but also genetic effects, direct selection according to the environmental conditions experienced. Consistently high genetic variation in growth implies that, despite its low overall genetic diversity and long history in captive rearing (>50 years), this landlocked Atlantic salmon population still possesses adaptive potential for response to change from hatchery rearing back to more natural conditions.

A pituitary gene network linking vgll3 to regulators of sexual maturation in male Atlantic salmon.

Age at maturity is a key life history trait and a significant contributor to life history strategy variation. The maturation process is influenced by genetic and environmental factors, but specific causes of variation in maturation timing remain elusive. In many species, the increase in the regulatory gonadotropin-releasing hormone 1 (GnRH1) marks the onset of puberty. Atlantic salmon, however, lacks the gnrh1 gene, suggesting gnrh3 and/or other regulatory factors are involved in the maturation process. Earlier research in Atlantic salmon has found a strong association between alternative alleles of vgll3 and maturation timing. Recently we reported strong induction of gonadotropin genes (fshb and lhb) in the pituitary of Atlantic salmon homozygous for the early maturation allele (E) of vgll3. The induction of gonadotropins was accompanied by increased expression of their direct upstream regulators, c-jun and sf1 (nr5a1b) but the regulatory connection between vgll3 and these regulators has never been investigated in any organism. In this study, we investigated the potential regulatory connection between vgll3 genotypes and these regulators through a stepwise approach of identifying a gene regulatory network (GRN) containing c-jun and sf1, and transcription factor motif enrichment analysis. We found a GRN containing c-jun with predicted upstream regulators, e2f1, egr1, foxj1 and klf4, to be differentially expressed in the pituitary. Finally, we suggest a vgll3 and Hippo pathway -dependent model for transcriptional regulation of c-jun and sf1 in the pituitary, which may have broader implications across vertebrates.

Next-generation sequencing in a large pedigree segregating visceral artery aneurysms suggests potential role of COL4A1/COL4A2 in disease etiology.

BACKGROUND: Visceral artery aneurysms (VAAs) can be fatal if ruptured. Although a relatively rare incident, it holds a contemporary mortality rate of approximately 12%. VAAs have multiple possible causes, one of which is genetic predisposition. Here, we present a striking family with seven individuals affected by VAAs, and one individual affected by a visceral artery pseudoaneurysm. METHODS: We exome sequenced the affected family members and the parents of the proband to find a possible underlying genetic defect. As exome sequencing did not reveal any feasible protein-coding variants, we combined whole-genome sequencing of two individuals with linkage analysis to find a plausible non-coding culprit variant. Variants were ranked by the deep learning framework DeepSEA. RESULTS: Two of seven top-ranking variants, NC_000013.11:g.108154659C>T and NC_000013.11:g.110409638C>T, were found in all VAA-affected individuals, but not in the individual affected by the pseudoaneurysm. The second variant is in a candidate cis-regulatory element in the fourth intron of COL4A2, proximal to COL4A1. CONCLUSIONS: As type IV collagens are essential for the stability and integrity of the vascular basement membrane and involved in vascular disease, we conclude that COL4A1 and COL4A2 are strong candidates for VAA susceptibility genes.

Novel germline variant in the histone demethylase and transcription regulator KDM4C induces a multi-cancer phenotype.

BACKGROUND: Genes involved in epigenetic regulation are central for chromatin structure and gene expression. Specific mutations in these might promote carcinogenesis in several tissue types. METHODS: We used exome, whole-genome and Sanger sequencing to detect rare variants shared by seven affected individuals in a striking early-onset multi-cancer family. The only variant that segregated with malignancy resided in a histone demethylase KDM4C. Consequently, we went on to study the epigenetic landscape of the mutation carriers with ATAC, ChIP (chromatin immunoprecipitation) and RNA-sequencing from lymphoblastoid cell lines to identify possible pathogenic effects. RESULTS: A novel variant in KDM4C, encoding a H3K9me3 histone demethylase and transcription regulator, was found to segregate with malignancy in the family. Based on Roadmap Epigenomics Project data, differentially accessible chromatin regions between the variant carriers and controls enrich to normally H3K9me3-marked chromatin. We could not detect a difference in global H3K9 trimethylation levels. However, carriers of the variant seemed to have more trimethylated H3K9 at transcription start sites. Pathway analyses of ChIP-seq and differential gene expression data suggested that genes regulated through KDM4C interaction partner EZH2 and its interaction partner PLZF are aberrantly expressed in mutation carriers. CONCLUSIONS: The apparent dysregulation of H3K9 trimethylation and KDM4C-associated genes in lymphoblastoid cells supports the hypothesis that the KDM4C variant is causative of the multi-cancer susceptibility in the family. As the variant is ultrarare, located in the conserved catalytic JmjC domain and predicted pathogenic by the majority of available in silico tools, further studies on the role of KDM4C in cancer predisposition are warranted.

WNT2 activation through proximal germline deletion predisposes to small intestinal neuroendocrine tumors and intestinal adenocarcinomas.

Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described. We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas. To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture. We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.

Candidate susceptibility variants in angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma with a poor prognosis: the 5-year survival rate is approximately 30%. Somatic driver mutations have been found in TET2, IDH2, DNMT3A, RHOA, FYN, PLCG1, and CD28, whereas germline susceptibility to AITL has to our knowledge not been studied. The homogenous Finnish population is well suited for studies on genetic predisposition. Here, we performed an exome-wide rare variant analysis in 23 AITL patients. No germline mutations were found in the driver genes, implying that they are not frequently involved in genetic AITL predisposition. Potentially pathogenic variants present in at least two patients and showing significant (p < 0.01) enrichment in our sample set were found in ten genes: POLK, PRKCB, ZNF676, PRRC2B, PCDHGB6, GNL3L, TTC36, OTOG, OSGEPL1, and RASSF9. The most significantly enriched variants, causing p.Lys469Ter in a splice variant of POLK and p.Pro588His in PRKCB, are intriguing candidates as Polk deficient mice display a spontaneous mutator phenotype, whereas PRKCB was recently shown to be somatically mutated in 33% of another peripheral T-cell lymphoma, adult T-cell lymphoma. If validated, our findings would provide new insight into the pathogenesis of AITL, as well as tools for early detection in susceptible individuals.

Discovery of potential causative mutations in human coding and noncoding genome with the interactive software BasePlayer.

Next-generation sequencing (NGS) is routinely applied in life sciences and clinical practice, but interpretation of the massive quantities of genomic data produced has become a critical challenge. The genome-wide mutation analyses enabled by NGS have had a revolutionary impact in revealing the predisposing and driving DNA alterations behind a multitude of disorders. The workflow to identify causative mutations from NGS data, for example in cancer and rare diseases, commonly involves phases such as quality filtering, case-control comparison, genome annotation, and visual validation, which require multiple processing steps and usage of various tools and scripts. To this end, we have introduced an interactive and user-friendly multi-platform-compatible software, BasePlayer, which allows scientists, regardless of bioinformatics training, to carry out variant analysis in disease genetics settings. A genome-wide scan of regulatory regions for mutation clusters can be carried out with a desktop computer in ~10 min with a dataset of 3 million somatic variants in 200 whole-genome-sequenced (WGS) cancers.

Germline mutations in young non-smoking women with lung adenocarcinoma.

OBJECTIVES: Although the primary cause of lung cancer is smoking, a considerable proportion of all lung cancers occur in never smokers. Gender influences the risk and characteristics of lung cancer and women are overrepresented among never smokers with the disease. Young age at onset and lack of established environmental risk factors suggest genetic predisposition. In this study, we used population-based sampling of young patients to discover candidate predisposition variants for lung adenocarcinoma in never-smoking women. MATERIALS AND METHODS: We employed archival normal tissue material from 21 never-smoker women who had been diagnosed with lung adenocarcinoma before the age of 45, and exome sequenced their germline DNA. RESULTS AND CONCLUSION: Potentially pathogenic variants were found in eight Cancer Gene Census germline genes: BRCA1, BRCA2, ERCC4, EXT1, HNF1 A, PTCH1, SMARCB1 and TP53. The variants in TP53, BRCA1, and BRCA2 are likely to have contributed to the early onset lung cancer in the respective patients (3/21 or 14%). This supports the notion that lung adenocarcinoma can be a component of certain cancer predisposition syndromes. Fifteen genes displayed potentially pathogenic mutations in at least two patients: ABCC10, ATP7B, CACNA1S, CFTR, CLIP4, COL6A1, COL6A6, GCN1, GJB6, RYR1, SCN7A, SEC24A, SP100, TTN and USH2A. Four patients showed a mutation in COL6A1, three in CLIP4 and two in the rest of the genes. Some of these candidate genes may explain a subset of female lung adenocarcinoma.

Candidate susceptibility variants for esophageal squamous cell carcinoma.

Esophageal cancer is common worldwide, and often fatal. The major histological subtype is esophageal squamous cell carcinoma (ESCC). ESCC shows familial aggregation and high heritability. Mutations in RHBDF2 cause tylosis, a very rare disorder characterized by high life-time risk of ESCC, but no other well-established predisposition genes have been identified. To identify candidate susceptibility variants for ESCC we utilized the Population Information System and the Finnish cancer registry to find study materials by clustering ESCC patients by family name at birth and municipality at birth. We collected archival tissue material and exome sequenced a total of 30 ESCC cases. We prioritized shared, deleterious and rare variants that were significantly enriched in our sample set compared to Finnish and population subset specific controls. Six variants passed filtering, the most frequent being a nonsense mutation in DNAH9 (p.Tyr1573Ter) found in four unrelated patients. DNAH9 has been reported to be frequently lost in ESCC tumors. In this study, one patient's tumor showed loss of the wild type allele of DNAH9 suggesting a tumor suppressive function. A missense variant in GKAP1 was shared by three patients, and missense variants in BAG1, NFX1, FUK, and DDOST by two each. EP300 which has previously been implicated in the genesis of ESCC had a missense variant segregating in three affected individuals in a single family. If validated in independent patient sets, these variants could serve as a tool towards prevention and early diagnosis of ESCC.

Exome sequencing reveals three novel candidate predisposition genes for diffuse gastric cancer.

Gastric cancer is the fourth most common cancer worldwide and the second leading cause of cancer mortality. Three hereditary gastric cancer syndromes have been described; hereditary diffuse gastric cancer (HDGC), familial intestinal gastric cancer (FIGC) and gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS). Thirty per cent of HDGC families have heterozygous germline mutations in CDH1, which encodes E-cadherin. A germline truncating mutation in the gene encoding α-E-catenin (CTNNA1) was also recently discovered in a family with HDGC, but no other genes specifically predisposing to gastric cancer have been identified, leaving the majority of cases showing familial aggregation without a known genetic cause. The aim of this study was to find the putative gastric cancer predisposing gene defect in a family with HDGC that had previously been tested negative for mutations in CDH1. In this family, there were six cases of diffuse gastric cancer in two generations. Exome sequencing was applied to two affected family members. The shared variants which were predicted deleterious in silico and could not be found in databases or in a control set of over 4,000 individuals were Sanger sequenced in a third family member. Three candidate variants were identified: p.Glu1313Lys in Insulin receptor (INSR), p.Arg81Pro in F-box protein 24 (FBXO24) and p.Pro1146Leu in DOT1-like histone H3K79 methyltransferase (DOT1L). These variants and adjacent regions were screened for in an additional 26 gastric cancer patients with a confirmed (n = 13) or suspected (n = 13) family history of disease, but no other non-synonymous mutations were identified. This study identifies INSR, FBXO24 and DOT1L as new candidate diffuse gastric cancer susceptibility genes, which should be validated in other populations. Of these genes, INSR is of special interest as insulin signaling was recently shown to affect tumor cell invasion capability by modulating E-cadherin glycosylation.

Whole-Genome Sequencing Identifies STAT4 as a Putative Susceptibility Gene in Classic Kaposi Sarcoma.

BACKGROUND: Classic Kaposi sarcoma (cKS) is an inflammatory tumor caused by human herpesvirus 8 (HHV-8) commonly observed in elderly men of Mediterranean origin. We studied a Finnish family of 5 affected individuals in 2 generations. Except for atypical mycobacterial infection of the index case, the affected individuals did not have notable histories of infection. METHODS: We performed genome and exome sequencing and mapped shared chromosomal regions to identify genetic predisposition in the family. RESULTS: We identified 12 protein-coding candidate variants that segregated in the 3 affected cousins from whom we had samples. The affected mother of the index case was an obligatory carrier. Among the 12 candidates was a rare heterozygous substitution rs141331848 (c.1337C>T, p.Thr446Ile) in the DNA-binding domain of STAT4. The variant was not present in 242 Finnish control genomes or 180 additional regional controls. Activated T-helper cells from the HHV-8-negative variant carriers showed reduced interferon γ production, compared with age and sex matched wild-type individuals. We screened STAT4 in additional 18 familial KS cases and the variant site from 56 sporadic KS cases but detected no pathogenic mutations. CONCLUSIONS: Our data suggest that STAT4 is a potential cKS-predisposition gene, but further functional and genetic validation is needed.

Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15-20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.