Large-scale data analysis to identify novel disease phenotypes and genes

Diseases can occur due to genetic changes that alter the normal function of genes. These alterations may be either inherited or acquired somatically during lifetime. Aims of this thesis work were to efficiently analyze large quantities of epidemiological and molecular data, and to characterize new susceptibility conditions and genetic causes of human diseases. First, genetic basis of right atrial isomerism (RAI) was studied in a Finnish family with five affected siblings and healthy parents. RAI is a heterotaxy syndrome with disturbances in the left-right axis development resulting in anomalies in heart and other asymmetrical organs. Linkage analysis and candidate-gene approach followed by sequencing revealed two truncating mutations in GDF1 segregating with RAI in an autosomal recessive manner. This finding, supported by the similar phenotype of laterality defects in Gdf1 knockout mice, provides evidence that RAI can be recessively inherited with GDF1 as the causative gene. Second, six patients with severe intellectual disability (ID) of unknown etiology were studied by genetic mapping and whole-genome sequencing (WGS) analysis. Autosomal recessive inheritance of severe ID was confirmed by extensive genealogy, and by linkage analysis showing high statistical significance for a homozygous region at 3p22.1-3p21.1. Three genes, TKT, P4HTM and USP4, with potentially protein damaging sequence changes were identified within the locus. The variants were rare and present only in heterozygous form in population-matched controls. This study facilitates clinical and molecular diagnosis of similar patients and further research on the role of the genes in the development of severe ID. Third, we performed WGS and transcriptome profiling of 38 uterine leiomyomas and corresponding myometrium from 30 women. Uterine leiomyomas are benign tumors that affect approximately three-quarters of women and may cause severe symptoms including abdominal pain and excessive uterine bleeding. Abundant complex chromosomal rearrangement events resembling the recently described chromothripsis phenomenon were detected. The events had created leiomyoma-specific driver changes, and occurred sequentially in some tumors. Four molecular pathways driven by alterations of MED12, FH, HMGA2/HMGA1 or COL4A5/COL4A6 were identified. The clonal origin of multiple separate tumors was also proven. The molecular genetic characterization of uterine leiomyomas will hopefully lead to better understanding of tumor growth and personalized treatment of patients. Fourth, a systematic search for familial aggregation of all tumor types was performed to identify new susceptibility phenotypes. We employed the entire population based data in the Finnish Cancer Registry and clustered 878,593 patients according to family name at birth, municipality of birth and tumor type. The rate of familial occurrence was estimated with a cluster score method. Among known cancer predisposition syndromes, Kaposi sarcoma (KS) with largely unknown genetic background was highlighted. Population records verified majority of the clustered KS patients as true relatives, providing further evidence that the clustering works well in estimating familiality. This study enabled identification of families suitable for a succeeding research on genetic basis of novel tumor predisposition phenotypes.Geneettiset muutokset, jotka muuttavat solujen normaalia toimintaa, vaikuttavat tautien syntyyn. Nämä muutokset voivat olla perittyjä tai elinaikana hankittuja. Tämän väitöskirjan tavoitteena oli (1) analysoida tehokkaasti suuria määriä epidemiologista ja molekyyligeneettistä dataa ja (2) tunnistaa uusia perinnöllisiä tauteja ja geneettisiä muutoksia tautien taustalla. Ensimmäisessä osatyössä tutkittiin oikean isomerismin perinnöllistä taustaa perheessä, jossa oli viisi sairastunutta lasta. Isomerismin taudinkuvaan kuuluu kehityshäiriöitä vasemman ja oikean puoliskon suhteen epäsymmetrisissä elimissä, etenkin sydämessä. Perheen DNA-näytteillä tehtiin kytkentäanalyysi ja taudin kanssa peittyvästi periytyviltä kromosomialueilta etsittiin kandidaattigeenejä kirjallisuutta hyödyntäen. Sairailla lapsilla löydettiin GDF1 geenissä kaksi geenin toiminnan estävää mutaatiota, joista toinen periytyi äidiltä ja toinen isältä. Samankaltainen ilmiasu oli aiemmin nähty hiirissä, joiden perimästä oli poistettu Gdf1, vahvistaen peittyvästi periytyvien GDF1 mutaatioiden kausaalisuutta oikean isomerismin synnyssä. Tulokset mahdollistavat GDF1 geenin tutkimisen, kun taudinkuvaksi epäillään oikeaa isomerismia, ja perinnöllisyysneuvonnan antamisen tarvittaessa. Toisessa osatyössä tutkittiin kuuden vaikeasti kehitysvammaisen lapsen taudin geneettistä syytä kytkentäanalyysin ja koko perimän sekvensoinnin avulla. Sukututkimuksella ja kytkentäanalyysillä todistettiin lapsien vanhempien olevan toisilleen kaukaista sukua ja löydettiin taudin aiheuttava samaperintäinen kromosomialue. Tältä alueelta tunnistettiin geenien toiminnalle mahdollisesti haitallisia perimän virheitä kolmesta eri geenistä (TKT, P4HTM ja USP4). Tutkituilta terveiltä kontrollihenkilöiltä ei koskaan löytynyt samoja geenivirheitä molemmista geenikopioista kuten tutkimuksen kehitysvammaisilla lapsilla. Tulokset mahdollistavat muiden samankaltaisten potilaiden diagnosoimisen ja geenien merkityksen tutkimisen vaikean kehitysvammaisuuden taustalla. Kolmannessa osatyössä käytettiin koko perimän laajuista sekvensointia ja geenien ilmentymistä mittaavaa menetelmää tunnistamaan hyvälaatuisten kohdun kasvainten (leiomyoomien) syntyyn ja kasvuun vaikuttavia geneettisiä muutoksia. Tutkimusaineistona oli 38 leiomyoomaa ja vastaava normaalikudosnäyte 30 potilaalta. Leiomyoomissa havaittiin monimutkaisia kromosomaalisia uudelleenjärjestymiä, jotka muistuttivat aiemmin pahanlaatuisiin syöpiin liittettyä chromothripsis ilmiötä. Monimutkaiset uudelleenjärjestymät aiheuttivat leiomyoomille tyypillisiä geneettisiä muutoksia ja kasvaimet voitiin jakaa neljään ryhmään geenien ilmentymisen perusteella. Joidenkin samassa kohdussa syntyneiden erillisten kasvainten todistettiin olevan klonaalisia. Leiomyoomien syntymekanismien tunteminen avaa toivottavasti mahdollisuuksia kohdennettujen lääkehoitojen kehittämiselle tulevaisuudessa. Neljännessä osatyössä käytettiin Suomen syöpärekisterin väestöpohjaista aineistoa tavoitteena tunnistaa uusia perheittäin esiintyviä syöpiä. Kaikkiaan 878593 syöpätapausta klusteroitiin syntymäsukunimen, -paikkakunnan ja syöpätyypin perusteella, ja perheittäistä esiintymistä arvioitiin klusteroitumisasteen mukaan. Tunnettujen perinnöllisten syöpätyyppien lisäksi muun muassa Kaposin sarkooma osoitti korkeaa klusteroitumisastetta. Sukututkimus vahvisti suuren osan klusteroituneista Kaposin sarkooma potilaista olevan sukulaisia antaen lisäuskottavuutta klusteroinnin toimivuudelle ja mahdollistaen jatkotutkimukset liittyen perheiden syöpäalttiuteen

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

Vitamin C boosts DNA demethylation in TET2 mutation carriers

Background Accurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation.Methods In a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members.Results We show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals.Conclusions These results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations.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

Sequence determinants of human gene regulatory elements

Analysis of massively parallel reporter assays measuring the transcriptional activity of DNA sequences indicates that most transcription factor (TF) activity is additive and does not rely on specific TF-TF interactions. Individual TFs can have different gene regulatory activities. DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an similar to 100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.Peer reviewe

Genetic predisposition to uterine leiomyoma is determined by loci for genitourinary development and genome stability

Uterine leiomyomas (ULs) are benign tumors that are a major burden to women's health. A genome-wide association study on 15,453 UL cases and 392,628 controls was performed, followed by replication of the genomic risk in six cohorts. Effects of the risk alleles were evaluated in view of molecular and clinical characteristics. 22 loci displayed a genome-wide significant association. The likely predisposition genes could be grouped to two biological processes. Genes involved in genome stability were represented by TERT, TERC, OBFC1 - highlighting the role of telomere maintenance - TP53 and ATM. Genes involved in genitourinary development, WNT4, WT1, SALL1, MED12, ESR1, GREB1, FOXO1, DMRT1 and uterine stem cell marker antigen CD44, formed another strong subgroup. The combined risk contributed by the 22 loci was associated with MED12 mutation-positive tumors. The findings link genes for uterine development and genetic stability to leiomyomagenesis, and in part explain the more frequent occurrence of UL in women of African origin.Peer reviewe

Nationwide Registry-Based Analysis of Cancer Clustering Detects Strong Familial Occurrence of Kaposi Sarcoma

Many cancer predisposition syndromes are rare or have incomplete penetrance, and traditional epidemiological tools are not well suited for their detection. Here we have used an approach that employs the entire population based data in the Finnish Cancer Registry (FCR) for analyzing familial aggregation of all types of cancer, in order to find evidence for previously unrecognized cancer susceptibility conditions. We performed a systematic clustering of 878,593 patients in FCR based on family name at birth, municipality of birth, and tumor type, diagnosed between years 1952 and 2011. We also estimated the familial occurrence of the tumor types using cluster score that reflects the proportion of patients belonging to the most significant clusters compared to all patients in Finland. The clustering effort identified 25,910 birth name-municipality based clusters representing 183 different tumor types characterized by topography and morphology. We produced information about familial occurrence of hundreds of tumor types, and many of the tumor types with high cluster score represented known cancer syndromes. Unexpectedly, Kaposi sarcoma (KS) also produced a very high score (cluster score 1.91, p-value <0.0001). We verified from population records that many of the KS patients forming the clusters were indeed close relatives, and identified one family with five affected individuals in two generations and several families with two first degree relatives. Our approach is unique in enabling systematic examination of a national epidemiological database to derive evidence of aberrant familial aggregation of all tumor types, both common and rare. It allowed effortless identification of families displaying features of both known as well as potentially novel cancer predisposition conditions, including striking familial aggregation of KS. Further work with high-throughput methods should elucidate the molecular basis of the potentially novel predisposition conditions found in this study.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

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility(1), and are a common cause of hysterectomy(2). They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA2(3). Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex(4), and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice(5). Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.Peer reviewe

Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans

Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis.Peer reviewe