Whole-exome sequencing identifies a novel germline variant in PTK7 gene in familial colorectal cancer

Colorectal cancer (CRC) is the third most frequently diagnosed malignancy worldwide. Only 5% of all CRC cases are due to germline mutations in known predisposition genes, and the remaining genetic burden still has to be discovered. In this study, we performed whole-exome sequencing on six members of a Polish family diagnosed with CRC and identified a novel germline variant in the protein tyrosine kinase 7 (inactive) gene (PTK7, ENST00000230419, V354M). Targeted screening of the variant in 1705 familial CRC cases and 1674 healthy elderly individuals identified the variant in an additional familial CRC case. Introduction of this variant in HT-29 cells resulted in increased cell proliferation, migration, and invasion; it also caused down-regulation of CREB, p21 and p53 mRNA and protein levels, and increased AKT phosphorylation. These changes indicated inhibition of apoptosis pathways and activation of AKT signaling. Our study confirmed the oncogenic function of PTK7 and supported its role in genetic predisposition of familial CRC

RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants

Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis

Network analysis for complex neurodegenerative diseases

Purpose of Review Biomedicine is witnessing a paradigm shift in the way complex disorders are investigated. In particular, the need for big data interpretation has led to the development of pipelines that require the cooperation of different fields of expertise, including medicine, functional biology, informatics, mathematics and systems biology. This review sits at the crossroad of different disciplines and surveys the recent developments in the use of graph theory (in the form of network analysis) to interpret large and different datasets in the context of complex neurodegenerative diseases. It aims at a professional audience with different backgrounds. Recent Findings Biomedicine has entered the era of big data, and this is actively changing the way we approach and perform research. The increase in size and power of biomedical studies has led to the establishment of multi-centre, international working groups coordinating open access platforms for data generation, storage and analysis. Particularly, pipelines for data interpretation are under development, and network analysis is gaining momentum since it represents a versatile approach to study complex systems made of interconnected multiple players. Summary We will describe the era of big data in biomedicine and survey the major freely accessible multi-omics datasets. We will then introduce the principles of graph theory and provide examples of network analysis applied to the interpretation of complex neurodegenerative disorders

Genetic risk factors in autoimmune Addison's disease

Autoimmune Addison’s disease is the most common form of primary adrenal insufficiency in the Western world. The low prevalence of the disease has hampered large-scale unbiased genetic studies where the entire genome could be examined at once. By combining the two largest biobanks of DNA from patients with autoimmune Addison’s disease, we identified in Paper I nine independent risk loci with a genome-wide association study of 1223 patients and 4097 geographically matched controls. These results explained up to 41% of the heritability of the disease, which, in a twin study, has been estimated to be as high as 0.97 [95% CI 0.88-0.99]. In Paper II, we derived a polygenic risk score for autoimmune Addison’s disease with the same dataset from the first study. The polygenic risk score enabled an estimation of disease susceptibility at the individual level and the discrimination of other etiologies of primary adrenal insufficiency, uncovering cases previously presumed to have the autoimmune form of Addison’s disease. In Paper III, we explored the use of our polygenic risk score to efficiently triage patients who may benefit most from whole-genome sequencing to achieve the correct diagnosis and appropriate clinical management of the disease. Monogenic forms of primary adrenal insufficiency were found in 5 out of 35 cases with low polygenic risk score for autoimmune Addison’s disease, and we found an additional of three cases with suspected monogenic disease. This study highlights the potential of polygenic risk score as a tool to in the clinical evaluation of primary adrenal insufficiency. In summary, this thesis sheds light on the genetic risk factors behind the development of autoimmune Addison’s disease and their potential utility as diagnostic classifiers. Future studies are warranted to further our understanding of the biological role of the associated genetic risk factors

Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes’ availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy

Mob2 Insufficiency Disrupts Neuronal Migration in the Developing Cortex

Disorders of neuronal mispositioning during brain development are phenotypically heterogeneous and their genetic causes remain largely unknown. Here, we report biallelic variants in a Hippo signaling factor-MOB2-in a patient with one such disorder, periventricular nodular heterotopia (PH). Genetic and cellular analysis of both variants confirmed them to be loss-of-function with enhanced sensitivity to transcript degradation via nonsense mediated decay (NMD) or increased protein turnover via the proteasome. Knockdown of Mob2 within the developing mouse cortex demonstrated its role in neuronal positioning. Cilia positioning and number within migrating neurons was also impaired with comparable defects detected following a reduction in levels of an upstream modulator of Mob2 function, Dchs1, a previously identified locus associated with PH. Moreover, reduced Mob2 expression increased phosphorylation of Filamin A, an actin cross-linking protein frequently mutated in cases of this disorder. These results reveal a key role for Mob2 in correct neuronal positioning within the developing cortex and outline a new candidate locus for PH development

Uusien geneettisten epilepsiaoireyhtymien tunnistaminen eksomisekvensoinnilla

Epilepsies are a heterogeneous group of central nervous system diseases characterised by recurrent epileptic seizures. They are one of the most common neurological diseases with a lifetime prevalence of ~4%. Epileptic seizures are also a common comorbidity of various neurobiological disorders where epilepsy is not the primary diagnosis. Most epilepsies have a genetic origin, either monogenic or polygenic, however, the causal genetic variants have remained unknown in a substantial proportion of individuals with epilepsies. Over the past decade, technological advances in DNA sequencing have allowed the characterisation of the genetic basis of human disorders rapidly and efficiently. One of the most widely used methods is whole-exome sequencing (WES) where genetic variants in the protein coding regions of the genome, the exome, are captured. Even though the exome constitutes only ~1.5% of the genome, the majority of disease-causing variants underlying severe, monogenic diseases are located in the protein coding regions. Here, we aimed to decipher the molecular genetic basis of severe epilepsy syndromes by utilising WES to identify disease-causing genetic variants in patients without a genetic diagnosis. We studied patients with progressive myoclonus epilepsy (PME, n=84) or severe infantile-onset epileptic syndromes (n=30), which are one of the most devastating forms of genetic syndromes with epilepsy and characterised by frequent, pharmacoresistant seizures and poor prognosis. Given that the patients had undergone genetic testing to varying extent prior to this study, we specifically aimed to establish novel genes and molecular biological mechanisms underlying these syndromes. We made substantial progress in understanding the genetic architecture and molecular basis of the studied syndromes. For PMEs, we established a new major genetic cause and also expanded the genotypic and phenotypic spectrum of previously established disease genes. For severe infantile-onset epileptic syndromes, we identified one new, definite causal gene and one that requires identification of additional patients to confirm the causal role. The three newly identified disease genes represent three different molecular functions that together give new insight on epileptogenic mechanisms. The new PME subtype is caused by a heterozygous missense variant c.959G>A (p.Arg320His) in KCNC1 that was identified in 11 unrelated patients (13%) in the PME exome sequencing cohort. We have subsequently identified six additional patients. The gene encodes a potassium ion channel KV3.1 that has an important role in generating action potentials in the central nervous system, with the mutation disrupting the ability to transport potassium ions across the cell membrane. This mutation occurs in most families de novo, that is, it is a newly arising mutation. Based on the estimated mutation rate, the recurrent KCNC1 mutation is a worldwide cause of PME with likely hundreds of affected individuals globally. In five families with altogether nine affected siblings, we identified compound heterozygous variants in UBA5 as the cause of an infantile-onset syndrome characterised initially by irritability, followed by epilepsy, dystonic movements, moderate to severe intellectual disability, microcephaly and stagnation of development. The gene encodes an activating enzyme for UFM1, which is a small ubiquitin-like protein that is conjugated to its target proteins. The function of the highly conserved UFM1 conjugation system is still largely unknown. Functional analysis of the UBA5 mutants suggest that the identified variants cause reduced enzymatic activity of UBA5. Symptoms of the UBA5 patients and our findings in the central nervous system specific knockout mice for Ufm1 together indicate that UFM1-cascade is essential for normal development and function of the central nervous system. Finally, we identified compound heterozygous variants in ADAM22 as the likely cause of the disease in a patient with an infantile-onset rapidly progressing encephalopathy with epilepsy and cortical atrophy. The gene encodes a postsynaptic protein that functions as a receptor for LGI1, and we show that the identified variants abolish the ability of ADAM22 to bind to LGI1. The LGI1-ADAM22 complex is an antiepileptogenic factor regulating synaptic transmission throughout life. Highlighting the important role of this complex, knockout of Adam22 and Lgi1 in mice causes lethal epilepsy. Autosomal dominant LGI1 variants also cause epilepsy in humans. Identification of a patient with loss-of-function variants in ADAM22 suggest that also this gene is linked to epilepsy in humans. This connection should be confirmed through identification of additional affected individuals with ADAM22 variants. Altogether, this thesis demonstrates the power of WES in identification of causal genetic variants even in phenotypically heterogeneous patient cohorts subjected to prior genetic screenings. The findings improve diagnostics of these syndromes, increase knowledge of the underlying molecular mechanisms and potentially aid in developing new therapeutic interventions. Finally, for these families, establishment of the genetic diagnosis ends years of uncertainty and frustration of not knowing the cause of the disease and prevents need for unnecessary diagnostic testing.Epilepsiat ovat heterogeeninen joukko keskushermostosairauksia, jotka ilmenevät toistuvina epileptisinä kohtauksina. Niiden elämänaikainen esiintyvyys on noin 4 % eli ne ovat yksiä yleisimmistä neurologisista sairauksista. Epileptisiä kohtauksia esiintyy myös osana muita keskushermoston sairauksia, joissa epilepsia ei ole päädiagnoosi. Useimmat epilepsiat ovat geneettisiä joko mono- tai polygeenisiä mutta tautia aiheuttavat geneettiset variantit jäävät tunnistamatta merkittävällä osalla epilepsiaa sairastavista. Viimeisen vuosikymmenen aikana teknologinen kehitys DNA-sekvensoinnissa on mahdollistanut ihmisen sairauksien geneettisen taustan selvittämisen nopeasti ja tehokkaasti. Yksi käytetyimmistä menetelmistä on eksomisekvensointi, jossa geneettiset variantit koko perimän proteiinia koodaavilla alueilla eli eksomissa pystytään tunnistamaan. Vaikka eksomi on vain noin 1,5 % koko perimästä, suurin osa vakavia monogeenisiä sairauksia aiheuttavista muutoksista sijaitsee proteiinia koodaavilla alueilla. Tässä väitöstutkimuksessa tavoitteenamme oli selvittää vakavien epileptisten oireyhtymien molekyyligeneettistä taustaa hyödyntämällä eksomisekvensointia tautia aiheuttavien varianttien tunnistamisessa potilailla, joilla ei ole geneettistä diagnoosia. Tutkimusaineistomme koostui potilaista, joilla on joko progressiivinen myoklonusepilepsia (PME, n=84) tai vakava imeväisikäisenä alkava epileptinen oireyhtymä (n=30). Nämä oireyhtymät kuuluvat vakavimpien epileptisten oireyhtymien joukkoon ja niihin liittyy toistuvia, lääkeresistenttejä kohtauksia ja niillä on huono ennuste. Koska aineiston potilaille oli tehty geneettisiä diagnostisia testejä ennen tutkimukseen osallistumista, ensisijaisena tavoitteenamme oli tunnistaa uusia tautigeenejä ja molekyylimekanismeja näiden oireyhtymien taustalla. Edistimme tutkimuksen avulla merkittävästi ymmärrystämme näiden oireyhtymien molekyyligeneettisestä taustasta. Tunnistimme uuden PME-alatyypin ja kaksi uutta autosomaalisesti peittyvästi periytyvää imeväisikäisten vakavaa enkefalopatiaa, joista toisen kohdalla vaaditaan vielä lisäpotilaiden tunnistamista varmistuaksemme havaitsemiemme varianttien patogeenisyydestä. Lisäksi laajensimme aiemmin tunnistettujen tautigeenien genotyyppi- ja fenotyyppikirjoa. Nämä kolme uutta tautigeeniä edustavat kolmea eri molekulaarista mekanismia, jotka yhdessä lisäävät tietoa epilepsioiden taustalla olevista tekijöistä. Väitöskirjan ensimmäisessä osatyössä tunnistamamme uusi PME-alatyyppi aiheutuu heterotsygoottisesta missense-variantista c.959G>A (p.Arg320His) geenissä KCNC1. Tämä muutos on 11 potilaalla (13 %; eivät toisilleen sukua) PME-eksomisekvensointiaineistossa. Alkuperäisen löydöksen jälkeen olemme tunnistaneet kuusi lisäpotilasta. KCNC1 koodaa kaliumionikanavaa KV3.1, jolla on keskushermostossa tärkeä tehtävä aktiopotentiaalien muodostamisessa. Tunnistamamme mutaatio tässä geenissä vahingoittaa kanavan kykyä siirtää kaliumioneita solukalvon läpi. Mutaatio on niin kutsuttu de novo mutaatio eli se on uusi muutos, joka ei ole periytynyt vanhemmilta. Arvioimamme mutaatiotaajuuden perusteella tämä mutaatio KCNC1-geenissä aiheuttaa PME:n sadoilla potilailla maailmanlaajuisesti. Toisessa osatyössä tunnistimme viidessä perheessä yhteensä yhdeksällä potilaalla yhdistelmäheterotsygootit UBA5-geenin variantit, jotka aiheuttavat imeväisikäisenä ilmenevän oireyhtymän. Tämä oireyhtymä ilmenee aluksi ärtyvyytenä, ja muita myöhemmin esiintyviä oireita ovat epileptiset kohtaukset, dystoniset liikkeet, älyllinen kehitysvamma ja pienipäisyys. UBA5 on UFM1-proteiinia aktivoiva entsyymi. UFM1 on pieni ubikitiinin kaltainen proteiini, joka kiinnitetään sen kohdeproteiineihin entsyymien katalysoimien reaktioiden kautta. UFM1-kaskadin tehtävä soluissa on suurelta osin tuntematon. Funktionaaliset kokeemme viittaavat siihen, että tunnistamamme UBA5-variantit vähentävät UBA5:n entsymaattista aktiivisuutta. UBA5-potilaiden oireet ja löydökset tutkimallamme Ufm1-hiirimallilla osoittavat, että UFM1-kaskadi on välttämätön keskushermoston kehittymiselle ja toiminnalle. Kolmannessa osatyössä tunnistimme yhdistelmäheterotsygoottiset variantit ADAM22-geenissä todennäköisenä aiheuttajana potilaan imeväisikäisenä alkaneelle etenevälle enkefalopatialle, johon liittyy epileptisiä kohtauksia ja aivokuoren atrofiaa. ADAM22-proteiini toimii LGI1:n reseptorina ja osoitimme tutkimuksessa, että tunnistamamme variantit estävät ADAM22-proteiinin ja LGI1:n välisen interaktion. LGI1-ADAM22 kompleksi on antiepileptinen tekijä, joka säätelee synaptista transmissiota läpi elämän. Näiden proteiinien tärkeää tehtävää korostaa se, että Lgi1- ja Adam22-poistogeeniset hiiret kärsivät epileptisistä kohtauksista ja kuolevat 2-3 viikkoa syntymänsä jälkeen. Autosomaalisesti vallitsevasti periytyvät variantit LGI1-geenissä aiheuttavat epilepsiaa ihmisellä. Vakavasta epileptisestä oireyhtymästä kärsivällä potilaalla tunnistamamme proteiinin toiminnan estävät, peittyvästi periytyvät variantit ADAM22-geenissä viittaavat siihen, että myös ADAM22 kytkeytyy epilepsiaan ihmisellä. Tämän kytköksen vahvistaminen edellyttää lisäpotilaiden tunnistamista. Kokonaisuudessaan tämä väitöstutkimus osoittaa eksomisekvensoinnin tehokkuuden tautia aiheuttavien varianttien tunnistamisessa heterogeenisissä potilaskohorteissa, joille on tehty aiempia diagnostisia testejä. Löydöksemme edistävät näiden epileptisten oireyhtymien diagnostiikkaa, lisäävät tietoa niiden taustalla olevista molekyylimekanismeista ja mahdollisesti auttavat kehittämään uusia hoitokeinoja. Perheille diagnoosin saaminen merkitsee myös sairauden aiheuttajaan liittyneen epätietoisuuden ja turhautuneisuuden päättymistä ja tarpeettomien diagnostisten testien loppumista

Exome sequencing in rare neurological disorders

PhD ThesisNeurological disorders are complex traits, manifesting as a range of diverse phenotypes. The current diagnostic approach involves either stepwise testing, which is expensive and time consuming, or targeted next generation sequencing with a limited portfolio of genes. Both of these approaches have a lower diagnostic yield. Whole exome sequencing may be a more advantageous and faster method to discover disease causing gene mutations. This study evaluates the use of whole exome sequencing for diagnostic purposes in neurological disorders. Whole exome sequencing was performed in a heterogeneous cohort of patients with suspected inherited ataxia as an example of a neurological disorder, with the aim to identify candidate gene mutations. The study cohort consisted of 35 affected individuals from 22 randomly selected families of white European descent with no known consanguinity. All common sporadic, inherited and metabolic causes were excluded on routine clinical investigations prior to inclusion in this study. Whole exome sequencing was performed on 30 affected individuals. In-house bioinformatic analysis was based on previously published tools. A variant filtering algorithm excluded synonymous variants and focused on protein altering variants, nonsense mutations, exonic insertions/deletions and splice site variants. Minor allele frequency (MAF) was set at 1% in dbSNP137, 1000 genomes (April 2012 data release) and NHLBI-ESP6500 databases as well as in 286 unrelated in-house controls. Selection of the remaining variants was based on mode of inheritance. The variants were prioritized for brain and nerve cell expression and defined using carefully selected criteria. Genetic analysis was supported further by molecular genetic approaches (Sanger sequencing, reverse transcription PCR, quantitative pyrosequencing, cloning for allelic cis-trans study) and proteomics (Western blotting, immunohistochemistry). Confirmed pathogenic variants were found in 9/22 probands (41%) implicating 6 genes (KCNC3, SPG7, TUBB4A, SLC1A3, SACS and NPC1). Likely de novo dominant TUBB4A mutations were found in two families. In one family quantitative pyrosequencing revealed varying degrees of mosaicism in the mildly affected mother and heterozygosity in the severely affected offspring. In silico analysis further supported pathogenicity of the mutation and revealed that it could potentially disrupt ! ! ii! polymerizations of αβ-tubulin heterodimers. Possible pathogenic variants were identified in 5/22 probands (23%) implicating 5 genes (ZFYVE26, ZFYVE27, WFS1, WNK1 and FASTKD2). A predicted splice site mutation was detected in three members of an autosomal dominant pedigree in the previously described gene ZFYVE27. The ZFYVE27 protein (protrudin) levels were increased approximately 2.5 fold in the cerebellum but not in the frontal cortex of the affected individual. Protrudin is an endoplasmic reticulum (ER) protein and its anomalies have previously been shown to cause ER stress. In this study levels of the master regulator of ER stress, BiP/GRP78, were significantly increased in the patient’s cerebellum, which may indicate the ER pathology. In one family with possible pathogenic compound heterozygous FASTKD2 mutations, the in silico splice-site prediction was validated by sequencing analysis of cDNA clones. Likely de novo compound heterozygous mutations in ZFYVE26 (SPG15) in one family was validated with sequencing of cloned alleles and the result confirmed occurrence of the mutations in trans, therefore supporting their autosomal recessive inheritance. In conclusion, the likely molecular diagnosis in 14 out of 22 families (64%) was defined; a total of 11 genes were implicated. Disease genes previously described in isolated families were validated and the clinical phenotypes of known disease genes was broadened. This study has also demonstrated genetic heterogeneity of hereditary ataxias but shows the impact of exome sequencing in a group of patients difficult to diagnose genetically

Clinical and molecular investigation of rare genetic overgrowth disorders

Genetic overgrowth disorders are a group of rare conditions characterised by generalised and/or regional overgrowth. They are associated with a wide spectrum of clinical features including intellectual disability, developmental disorders, congenital anomalies, and other medical problems. In recent years several novel overgrowth genes have been identified but the clinical phenotypes and natural history of these emerging conditions are not yet fully understood. The Phenotyping of Overgrowth Disorders (POD) study was established to investigate the clinical and molecular features of rare genetic overgrowth disorders. Comprehensive clinical phenotyping data was collected from 100 participants and entered in an electronic data capture system. Genomic testing was performed on a custom targeted next generation sequencing panel of overgrowth genes. Additional molecular investigation with whole exome sequencing was performed in selected participants and trios. This work identified a molecular genetic diagnosis in over 40% of the study cohort, confirmed the genetic heterogeneity of overgrowth disorders, and identified phenotypic overlap between overgrowth disorders and other rare genetic disorders. Knowledge of the clinical phenotypes of rare genetic overgrowth disorders has been expanded, including the clinically significant discovery of vascular complications in PDGFRB-related disorders that may be amenable to targeted molecular therapy