Genetic and epigenetic contribution to complex traits

Much of the recent advances in functional genomics owe to developments in next-generation sequencing technology, which has contributed to the exponential increase of genomic data available for different human disease and population samples. With functional sequencing assays available to query both the transcriptome and the epigenome, annotation of the non-coding, regulatory genome is steadily improving and providing means to interpret the functional consequences of genetic variants associated with human complex traits. This has highlighted the need to better understand the normal variation in various cellular phenotypes, such as epigenetic modifications, and their transgenerational inheritance. In this review, we discuss different aspects of epigenetic variation in the context of DNA sequence variation and its contribution to complex phenotype

Autismikirjon häiriöiden geneettiset mekanismit

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.Autismi on lapsuusiän kehityksellinen häiriö, jonka tyypillisiä oireita ovat ongelmat vastavuoroisessa sosiaalisessa vuorovaikutuksessa, sanallisessa ja sanattomassa kommunikaatiossa, sekä riippuvuus erilaisista rutiineista ja rituaaleista. Se kuuluu laajempaan autismikirjon häiriöiden ryhmään, jotka kaikki ovat perusoireiltaan samankaltaisia, mutta joiden vakavuusaste vaihtelee huomattavasti. Autismikirjon häiriöitä tavataan maailmanlaajuisesti 0.6-0.7%:lla lapsista, ja ne aiheuttavat inhimillisen kärsimyksen lisäksi merkittäviä kuluja terveydenhuollolle. Vaikka autismikirjon häiriöt ovat vahvasti perinnöllisiä, niiden geneettinen tausta on osoittautunut erittäin heterogeeniseksi, mikä on vaikeuttanut altistavien geneettisten tekijöiden tunnistamista. Viimeaikaisten tutkimusten perusteella kuitenkin tiedetään, että harvinaiset, perhespesifiset geneettiset muutokset selittävät merkittävän osan autismin periytyvyydestä. Tässä väitöskirjassa tutkittiin DISC1-geenin (Disrupted-in-schizophrenia-1) merkitystä autismikirjon häiriöissä. Havaitsimme, että samat geenimerkit ja haplotyypit, joiden on aikaisemmin osoitettu assosioituvan muihin psykiatrisiin fenotyyppeihin, altistavat myös autismikirjon häiriöille. Tunnistimme geenin 3 UTR alueelta neljä polymorfista mikro-RNA:n sitoutumiskohtaa, ja osoitimme, että hsa-miR-559 säätelee DISC1:n ilmentymistä alleelispesifisesti in vitro. Lisäksi tutkimme suurta keskisuomalaista autismisukua, joka polveutuu yhteisistä esivanhemmista 1600-luvulta. Teimme genominlaajuisen kytkentäanalyysin mikrosatelliittimarkkereilla tässä suvussa, jonka kaukaisen sukulaisuuden voidaan olettaa vähentävän geneettistä heterogeenisyyttä. Tunnistimme mahdollisen autismille altistavan lokuksen kromosomissa 19p13.3, ja havaitsimme kaksi jo aiemmin autismiin yhdistettyä lokusta kromosomeissa 1q23 ja 15q11-q13. Tutkiaksemme näitä tuloksia tarkemmin, laajensimme käyttämäämme keskisuomalaistaustaista aineistoa ja teimme genominlaajuisen analyysin käyttämällä tiheästi sijoittuvia SNP-markkereita. Tunnistimme pienen määrän haplotyyppejä, jotka osa suvun autismia sairastavista henkilöistä jakoi keskenään, sekä yhteneviä reaktioreittejä SNP- ja geeniekspressiodatasta, joiden perusteella aksoniohjaukseen osallistuvat molekyylit näyttäisivät liittyvän autismikirjon häiriöiden patogeneesiin. Tutkimuksemme osoittaa, että autismin genettinen tausta on monitekijäinen jopa populaatioisolaateissa ja yksittäisissä perheissä. Saamiemme tulosten perusteella voidaan sanoa, että altistavien genomisten alueiden sekvensointi on välttämätöntä, jos halutaan tunnistaa varsinaiset mutaatiot ja variantit, jotka aiheuttavat autismikirjon häiriöitä. Tunnistimme myös mahdollisen mikro-RNA-välitteisen säätelymekanismin, joka saattaa osittain selittää DISC1-geenin laaja-alaisia neurobiologisia vaikutuksia

Highly accurate quantification of allelic gene expression for population and disease genetics

Publisher Copyright: © 2022 Saukkonen et al.Analysis of allele-specific gene expression (ASE) is a powerful approach for studying gene regulation, particularly when sample sizes are small, such as for rare diseases, or when studying the effects of rare genetic variation. However, detection of ASE events relies on accurate alignment of RNA sequencing reads, where challenges still remain, particularly for reads containing genetic variants or those that align to many different genomic locations. We have developed the Personalised ASE Caller (PAC), a tool that combines multiple steps to improve the quantification of allelic reads, including personalized (i.e., diploid) read alignment with improved allocation of multimapping reads. Using simulated RNA sequencing data, we show that PAC outperforms standard alignment approaches for ASE detection, reducing the number of sites with incorrect biases (>10%) by ∼80% and increasing the number of sites that can be reliably quantified by ∼3%. Applying PAC to real RNA sequencing data from 670 whole-blood samples, we show that genetic regulatory signatures inferred from ASE data more closely match those from population-based methods that are less prone to alignment biases. Finally, we use PAC to characterize cell type–specific ASE events that would be missed by standard alignment approaches, and in doing so identify disease relevant genes that may modulate their effects through the regulation of gene expression. PAC can be applied to the vast quantity of existing RNA sequencing data sets to better understand a wide array of fundamental biological and disease processes.Peer reviewe

Histone lysine methyltransferase-related neurodevelopmental disorders: current knowledge and saRNA future therapies

Neurodevelopmental disorders encompass a group of debilitating diseases presenting with motor and cognitive dysfunction, with variable age of onset and disease severity. Advances in genetic diagnostic tools have facilitated the identification of several monogenic chromatin remodeling diseases that cause Neurodevelopmental disorders. Chromatin remodelers play a key role in the neuro-epigenetic landscape and regulation of brain development; it is therefore not surprising that mutations, leading to loss of protein function, result in aberrant neurodevelopment. Heterozygous, usually de novo mutations in histone lysine methyltransferases have been described in patients leading to haploinsufficiency, dysregulated protein levels and impaired protein function. Studies in animal models and patient-derived cell lines, have highlighted the role of histone lysine methyltransferases in the regulation of cell self-renewal, cell fate specification and apoptosis. To date, in depth studies of histone lysine methyltransferases in oncology have provided strong evidence of histone lysine methyltransferase dysregulation as a determinant of cancer progression and drug resistance. As a result, histone lysine methyltransferases have become an important therapeutic target for the treatment of different cancer forms. Despite recent advances, we still lack knowledge about the role of histone lysine methyltransferases in neuronal development. This has hampered both the study and development of precision therapies for histone lysine methyltransferases-related Neurodevelopmental disorders. In this review, we will discuss the current knowledge of the role of histone lysine methyltransferases in neuronal development and disease progression. We will also discuss how RNA-based technologies using small-activating RNAs could potentially provide a novel therapeutic approach for the future treatment of histone lysine methyltransferase haploinsufficiency in these Neurodevelopmental disorders, and how they could be first tested in state-of-the-art patient-derived neuronal models

Histone lysine methyltransferase-related neurodevelopmental disorders: current knowledge and saRNA future therapies

Neurodevelopmental disorders encompass a group of debilitating diseases presenting with motor and cognitive dysfunction, with variable age of onset and disease severity. Advances in genetic diagnostic tools have facilitated the identification of several monogenic chromatin remodeling diseases that cause Neurodevelopmental disorders. Chromatin remodelers play a key role in the neuro-epigenetic landscape and regulation of brain development; it is therefore not surprising that mutations, leading to loss of protein function, result in aberrant neurodevelopment. Heterozygous, usually de novo mutations in histone lysine methyltransferases have been described in patients leading to haploinsufficiency, dysregulated protein levels and impaired protein function. Studies in animal models and patient-derived cell lines, have highlighted the role of histone lysine methyltransferases in the regulation of cell self-renewal, cell fate specification and apoptosis. To date, in depth studies of histone lysine methyltransferases in oncology have provided strong evidence of histone lysine methyltransferase dysregulation as a determinant of cancer progression and drug resistance. As a result, histone lysine methyltransferases have become an important therapeutic target for the treatment of different cancer forms. Despite recent advances, we still lack knowledge about the role of histone lysine methyltransferases in neuronal development. This has hampered both the study and development of precision therapies for histone lysine methyltransferases-related Neurodevelopmental disorders. In this review, we will discuss the current knowledge of the role of histone lysine methyltransferases in neuronal development and disease progression. We will also discuss how RNA-based technologies using small-activating RNAs could potentially provide a novel therapeutic approach for the future treatment of histone lysine methyltransferase haploinsufficiency in these Neurodevelopmental disorders, and how they could be first tested in state-of-the-art patient-derived neuronal models

Tautimekanismien jäljille iPS-solujen ja biopankkien avulla

Vertaisarvioitu.Indusoidut pluripotentit kantasolut, eli iPS-solut, ovat tavallisista somaattisista soluista tuotettuja kantasoluja, joilla on kyky erilaistua kaikiksi yksilönkehityksen aikana kehittyviksi solutyypeiksi. iPS-solut ovat lyhyessä ajassa mullistaneet useiden sairauksien tutkimusmahdollisuudet, ja niitä käytetään lisääntyvästi sekä tautimallinnuksessa että uusien lääkkeiden ja hoitomuotojen kehittämisessä. Suurimmat läpimurrot on saavutettu monogeenisten sairauksien tutkimuksessa, jossa potilaista tuotetuilla soluilla on keskeinen rooli, mutta edistysaskeleita on otettu myös monitekijäisten sairauksien ymmärtämisessä. Näytemäärien kasvaessa biopankkien merkitys iPS-solulinjojen ja niistä kerätyn tiedon systemoidussa tallentamisessa ja hallinnoinnissa on korostunut. Yhdessä genomitiedon kanssa biopankkitoiminta tarjoaa Suomessa erinomaiset edellytykset hyödyntää iPS-soluja biolääketieteellisessä tutkimuksessa.Peer reviewe

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. Availability: The R package absfilter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

CRISPR activation enables high-fidelity reprogramming into human pluripotent stem cells

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells.Peer reviewe

Differentiation of human induced pluripotent stem cells into cortical neural stem cells

Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/beta-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.Peer reviewe

Assessing the gene regulatory landscape in 1,188 human tumors

Cancer is characterised by somatic genetic variation, but the effect of the majority of non-coding somatic variants and the interface with the germline genome are still unknown. We analysed the whole genome and RNA-seq data from 1,188 human cancer patients as provided by the Pan-cancer Analysis of Whole Genomes (PCAWG) project to map cis expression quantitative trait loci of somatic and germline variation and to uncover the causes of allele-specific expression patterns in human cancers. The availability of the first large-scale dataset with both whole genome and gene expression data enabled us to uncover the effects of the non-coding variation on cancer. In addition to confirming known regulatory effects, we identified novel associations between somatic variation and expression dysregulation, in particular in distal regulatory elements. Finally, we uncovered links between somatic mutational signatures and gene expression changes, including TERT and LMO2, and we explained the inherited risk factors in APOBEC-related mutational processes. This work represents the first large-scale assessment of the effects of both germline and somatic genetic variation on gene expression in cancer and creates a valuable resource cataloguing these effects