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 over-represented 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: BRCAI, BRCA2, ERCC4, EXT1, HNF1 A, PTCH1, SMARCB1 and TP53. The variants in TP53, BRCAI, 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, TEN 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.Peer reviewe

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.Peer reviewe

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.</p

Mutation Analysis of Inhibitory Guanine Nucleotide Binding Protein Alpha (GNAI) Loci in Young and Familial Pituitary Adenomas

Peer reviewe

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.Peer reviewe

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.Peer reviewe

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.Peer reviewe

Detecting novel cancer predisposing mutations by utilizing the Finnish Cancer Registry and archival tissue material

Although most cancers are sporadic, a considerable proportion is due to genetic susceptibility. Many cancer types display moderate to high heritability, but predisposition genes have been established for only a few. In this thesis project, we have utilized the data in the Finnish Cancer Registry (FCR) and the Population Information System (PIS) databases to identify cancer patients with a potential genetic predisposition. For most of the patients in the FCR archival tissue material in the form of paraffin-embedded formalin-fixed (FFPE) blocks is available in the pathological department of their local hospital, which makes genetic studies on selected cases possible. The Finnish population is due to its demographic history more homogenous than most populations, and thus optimal for studies on germline predisposition to disease. This thesis includes studies on two cancer types currently lacking well-established susceptibility genes: lung adenocarcinoma (LUAD), and angioimmunoblastic T-cell lymphoma (AITL). In addition, my thesis work examines esophageal squamous cell carcinoma (ESCC) and hereditary diffuse gastric cancer (HDGC) susceptibility. A fraction of these cancers can be attributed to germline mutations in RHBDF2 and CDH1, respectively. We utilized exome sequencing in all studies with the aim to discover novel susceptibility genes for the different phenotypes. Gastric cancer is the second leading cause of cancer mortality. Three distinct hereditary gastric cancer syndromes are recognized: HDGC, familial intestinal gastric cancer, and gastric adenocarcinoma and proximal polyposis of the stomach. Up to one third of HDGC families have heterozygous germline mutations in CDH1, which encodes E-cadherin. Most cases showing familial aggregation do not, however, have a known genetic basis. The aim of the first study was to find the putative cancer-predisposing gene defect in an HDGC family that had previously been tested negative for CDH1 mutations. We identified three candidate variants: p.Glu1313Lys in INSR, p.Arg81Pro in FBXO24 and p.Pro1146Leu in DOT1L. INSR is of special interest as insulin signaling has been shown to modulate E-cadherin glycosylation and stability. ESCC represents the most common subtype of esophageal cancer, a common and often fatal malignancy. Although ESCC shows familial clustering and moderate to high heritability, few studies on moderate to high penetrance predisposition exist. We have previously found familial aggregation of classic Kaposi sarcoma by performing family name at birth and municipality at birth based clustering of all cancer cases in the FCR. In study II, we used the same clustering algorithm to find potential familial cases of ESCC. We managed to find a geographical population subset that showed enrichment of clustered ESCC patients. The finding may indicate that a susceptibility variant is enriched in this population subset. We collected FFPE tissue material and exome sequenced a total of 30 ESCC cases. Six variants passed filtering and analysis criteria, the most frequent being a nonsense mutation in DNAH9 detected in four unrelated patients. One patient's tumor showed loss of the wild type allele of DNAH9, suggesting a tumor-suppressive function. The other candidate genes GKAP1, BAG1, NFX1, FCSK, and DDOST harbored missense variants. A rare variant in EP300, a gene that has previously been implicated in ESCC carcinogenesis, was discovered to segregate in three affected individuals in a single family. Lung cancer is the most common cancer in the world and the leading cause of cancer death in both men and women. It is well established that the primary cause of the malignancy is cigarette smoke, however, other factors are involved as only approximately 15% of smokers develop lung cancer and an estimated 10-25% of all lung cancers occur in never smokers. Gender influences the risk of lung cancer and women are overrepresented among never smokers with the disease. Because of a possible difference in the molecular basis, we chose to study never-smoker women with LUAD in study III. We selected the youngest patients listed in the FCR, who are more likely to harbor a predisposing germline mutation. We managed to collect normal FFPE tissue material from 21 never-smoker women who had been diagnosed with lung adenocarcinoma before the age of 45. 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 very likely to have contributed to the early-onset lung cancer in the respective patients. This supports the idea that lung adenocarcinoma can be a component of certain cancer predisposition syndromes. In addition, 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. Some of these candidate genes may explain a subset of female lung adenocarcinoma. AITL is a subtype of peripheral T-cell lymphoma with a bleak prognosis. 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. In study IV we performed an exome-wide rare variant analysis in 23 AITL patients. No germline mutations were found in the driver genes, indicating that they are not frequently involved in genetic AITL predisposition. Potentially pathogenic variants present in at least two patients and showing significant 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 were p.Lys469Ter in a splice variant of POLK and p.Pro588His in PRKCB. These two are intriguing candidates, as Polk deficient mice display a spontaneous mutator phenotype and PRKCB has been found somatically mutated in 33% in adult T-cell lymphoma, another form of peripheral T-cell lymphoma. These studies show that rarely performed next-generation sequencing of DNA extracted from FFPE tissue samples is well suited for research and should be utilized to a greater extent. We found plausible candidate genes for the phenotypes under study. However, due to relatively small sample sets, our findings need to be genetically validated in independent sample series and through functional studies to prove causality.Syövän syntyyn vaikuttavat ympäristötekijät, sattuma sekä perimä. Valtaosa syövistä on sporadisia, eli ne kehittyvät pääosin ympäristötekijöiden (joihin lukeutuvat elintavat) ja sattuman seurauksena. Osa kuitenkin aiheutuu ensisijaisesti geneettisestä alttiudesta. Geneettinen alttius muodostuu riskimutaatioista ituradassa, ja nämä mutaatiot sekä niiden aiheuttamat riskit periytyvät vanhemmilta lapsille. Useita alttiusgeenejä ja -mutaatioita on jo tunnistettu ja karakterisoitu, mutta on todennäköistä, että huomattava määrä on yhä löytämättä. Tämän väitöskirjan osatöissä tutkimme eksomisekvensoinnin avulla perinnöllistä alttiutta neljälle eri syövälle: diffuusille mahasyövälle, ruokatorven levyepiteelisyövälle, keuhkon adenokarsinoomalle sekä angioimmunoblastiselle T-solulymfoomalle. Tavoitteenamme oli löytää uusia alttiusgeenejä näille syöpätyypeille ja sitä kautta lisätä ymmärrystä syövän syntymekanismeista sekä edistää kyseisten syöpien ennaltaehkäisyä ja hoitoa. Suomen väestö on geneettisesti yhtenäinen, eli siinä on vähemmän vaihtelua kuin väestöissä yleensä, mikä helpottaa tauteja aiheuttavien geenimuutosten löytämistä. Hyödynsimme geneettiseen tutkimukseen hyvin soveltuvan väestön lisäksi Suomen Syöpärekisteriä sekä väestötietojärjestelmää. Nämä rekisterit ovat kattavia ja korkeatasoisia, ja niissä olevan tiedon avulla on mahdollista löytää potilaita, jotka ovat korkealla todennäköisyydellä geneettisesti alttiita jollekin syöpätyypille. Tällaiseen alttiuteen voi viitata esimerkiksi nuori sairastumisikä, useat primäärikasvaimet ja syöpätyypin rikastuminen potilaan suvussa. Syöpäpotilaiden diagnosoinnissa käytettyä kudosmateriaalia arkistoidaan yleisesti formaliinifiksoimalla ja parafiiniin upottamalla. Näistä FFPE-kudosnäytteistä voidaan eristää DNA:ta geneettisiä analyysejä varten, mutta niitä käytetään kuitenkin melko harvoin lähtömateriaalina eksomisekvensoinnissa. Tämä johtuu siitä, että fiksointi ja säilöminen vahingoittavat DNA:ta, ja vaikeuttavat siten sen sekvensointia sekä analyysejä. Osatöissä II-IV käytimme eksomisekvensoinnin lähtömateriaalina FFPE-näytteitä. Väitöskirjatyöni osoittaa, että FFPE-kudosnäytteistä eristetty DNA soveltuu varsin hyvin eksomin laajuisiin sekvensointiprojekteihin. Tunnistimme potentiaalisia alttiusvariantteja tutkituille syöpätyypeille. Osatyön II tulokset tukevat ajatusta, että keuhkosyöpä on osa tiettyjen syöpäoireyhtymien syöpäkirjoa, sillä osa potilaista kantoi variantteja BRCA1-, BRCA2- ja TP53-geeneissä. Osatyössä havaitut variantit ovat väestössä hyvin harvinaisia ja ominaisuuksiltaan sellaisia, että ne ovat suurella todennäköisyydellä patogeenisiä

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 (pPeer reviewe

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 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.Peer reviewe