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Genomic medicine implementation in healthcare systems can bring us one step closer to making personalised medicine a reality, with major socioeconomic benefits. Citizens and patients canwidely benefit from genomic data analysis for accurate and timely diagnosis, effective treatments with less adverse events, and accurate profiling for disease prevention. Implementation of genomics in healthcare is complex and requires adjustments in the governance, structure and organization of health services, as well as dedicated investments. Implementation is also dependent on the country context. In the context of the 1+Million Genomes (1+MG) initiative, we developed a Maturity Level Model (MLM) for health systems to self-evaluate the maturity of their genomic medicine practices, and define a path to optimization. MLM is a tool for healthcare systems to self-evaluate the level of maturity of their genomic medicine practices according to a common matrix, and to define a path to optimization. A MLM pilot in eight European countries provided important information regarding common strengths, weaknesses and asymmetries across Europe.

IMPORTANCE: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited disease characterized by systemic vasculitis, early-onset stroke, bone marrow failure, and/or immunodeficiency affecting both children and adults. DADA2 is among the more common monogenic autoinflammatory diseases, with an estimate of more than 35 000 cases worldwide, but currently, there are no guidelines for diagnostic evaluation or management. OBJECTIVE: To review the available evidence and develop multidisciplinary consensus statements for the evaluation and management of DADA2. EVIDENCE REVIEW: The DADA2 Consensus Committee developed research questions based on data collected from the International Meetings on DADA2 organized by the DADA2 Foundation in 2016, 2018, and 2020. A comprehensive literature review was performed for articles published prior to 2022. Thirty-two consensus statements were generated using a modified Delphi process, and evidence was graded using the Oxford Center for Evidence-Based Medicine Levels of Evidence. FINDINGS: The DADA2 Consensus Committee, comprising 3 patient representatives and 35 international experts from 18 countries, developed consensus statements for (1) diagnostic testing, (2) screening, (3) clinical and laboratory evaluation, and (4) management of DADA2 based on disease phenotype. Additional consensus statements related to the evaluation and treatment of individuals with DADA2 who are presymptomatic and carriers were generated. Areas with insufficient evidence were identified, and questions for future research were outlined. CONCLUSIONS AND RELEVANCE: DADA2 is a potentially fatal disease that requires early diagnosis and treatment. By summarizing key evidence and expert opinions, these consensus statements provide a framework to facilitate diagnostic evaluation and management of DADA2. ispartof: JAMA Netw Open vol:6 issue:5 pages:e2315894- ispartof: location:United States status: Published online

Purpose Deficiency of adenosine deaminase 2 (DADA2) is an inherited inborn error of immunity, characterized by autoinflammation (recurrent fever), vasculopathy (livedo racemosa, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency, lymphoproliferation, immune cytopenias, and bone marrow failure (BMF). Tumor necrosis factor (TNF-alpha) blockade is the treatment of choice for the vasculopathy, but often fails to reverse refractory cytopenia. We aimed to study the outcome of hematopoietic cell transplantation (HCT) in patients with DADA2. Methods We conducted a retrospective study on the outcome of HCT in patients with DADA2. The primary outcome was overall survival (OS). Results Thirty DADA2 patients from 12 countries received a total of 38 HCTs. The indications for HCT were BMF, immune cytopenia, malignancy, or immunodeficiency. Median age at HCT was 9 years (range: 2-28 years). The conditioning regimens for the final transplants were myeloablative (n = 20), reduced intensity (n = 8), or non-myeloablative (n = 2). Donors were HLA-matched related (n = 4), HLA-matched unrelated (n = 16), HLA-haploidentical (n = 2), or HLA-mismatched unrelated (n = 8). After a median follow-up of 2 years (range: 0.5-16 years), 2-year OS was 97%, and 2-year GvHD-free relapse-free survival was 73%. The hematological and immunological phenotypes resolved, and there were no new vascular events. Plasma ADA2 enzyme activity normalized in 16/17 patients tested. Six patients required more than one HCT. Conclusion HCT was an effective treatment for DADA2, successfully reversing the refractory cytopenia, as well as the vasculopathy and immunodeficiency. Clinical Implications HCT is a definitive cure for DADA2 with > 95% survival.

The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but Ikaros family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2, encoding the transcriptional activator and repressor Helios, which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8⁺ T cell lineage. Protein-protein interaction analysis revealed that the variant abolished heterodimerization of Helios with Ikaros and Aiolos and also prevented Helios binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, and chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis, we show that reduced Helios expression was associated with chronic T cell activation and increased production of proinflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed a profound reduction in circulating MAIT cell numbers. In summary, we show that this previously undescribed loss-of-function variant in Helios leads to an immunodeficiency with signs of immune overactivation.

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BackgroundSymptomatic primary Epstein-Barr virus infection is a usually self-limiting illness in adolescents. We present a case of an adolescent who had been receiving azathioprine for inflammatory bowel disease for four years and developed a life-threatening primary Epstein-Barr virus infection successfully treated with rituximab.Case presentationAn 11-year-old girl presented with chronic, bloody diarrhea. Endoscopic biopsies confirmed a diagnosis of chronic ulcerative colitis with features of Crohn's disease. Azathioprine was initiated after one year due to active colitis. She responded well and remission was achieved. At the age of 16years she developed a life-threatening Epstein-Barr virus infection including severe multiple organ failure and was critically ill for 4weeks in the intensive care unit. Natural killer cells were virtually absent in the lymphocyte subset analysis. Azathioprine was stopped on admission. She was initially treated with corticosteroids, acyclovir and intravenous immunoglobulin. Approximately 30days after admission, she developed signs of severe hepatitis and pneumonitis and received weekly rituximab infusions for 8weeks. Primary immunodeficiency was excluded by whole exome sequencing in two independent laboratories. Persistent viremia stopped when the natural killer cell count started to rise, approximately 90days after the cessation of azathioprine.ConclusionsWe found 17 comparable cases in the literature. None of the previous cases reported in the literature, who had been treated with azathioprine and developed either a severe or a fatal Epstein-Barr virus infection, underwent full genetic and prospective immunological workup to rule out known primary immunodeficiencies. Recently, azathioprine has been shown to cause rather specific immunosuppression, resulting in natural killer cell depletion. Our case demonstrates that slow recovery from azathioprine-induced natural killer cell depletion, 3months after the stopping of azathioprine, coincided with the clearance of viremia and clinical recovery. Finally, our choice of treating the patient with rituximab, as previously used for patients with a severe immunosuppression and Epstein-Barr virus viremia, appeared to be successful in this case. We suggest testing for Epstein-Barr virus serology before starting azathioprine and measuring natural killer cell counts during the treatment to identify patients at risk of developing an unusually severe primary Epstein-Barr virus infection.

Multiple sclerosis is a complex neurological disease, with ∼20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFNγ biology, and NFκB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS. ispartof: CELL vol:175 issue:6 pages:1679-1695 ispartof: location:United States status: published

International audience; Multiple sclerosis is a complex neurological disease, with ∼20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFNγ biology, and NFκB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

Rheumatoid arthritis (RA) affects millions world-wide. While anti-TNF treatment is widely used to reduce disease progression, treatment fails in ∼one-third of patients. No biomarker currently exists that identifies non-responders before treatment. A rigorous community-based assessment of the utility of SNP data for predicting anti-TNF treatment efficacy in RA patients was performed in the context of a DREAM Challenge (http://www.synapse.org/RA_Challenge). An open challenge framework enabled the comparative evaluation of predictions developed by 73 research groups using the most comprehensive available data and covering a wide range of state-of-the-art modelling methodologies. Despite a significant genetic heritability estimate of treatment non-response trait (h2=0.18, P value=0.02), no significant genetic contribution to prediction accuracy is observed. Results formally confirm the expectations of the rheumatology community that SNP information does not significantly improve predictive performance relative to standard clinical traits, thereby justifying a refocusing of future efforts on collection of other data., Rheumatoid arthritis patients respond differently to anti-TNF treatment. Using community-based challenge, the authors show that currently available data does not reveal meaningful genetic predictors of response to anti-TNF therapy, thus confirming clinical observations.

Multiple sclerosis is a complex neurological disease, with ∼20% of risk heritability attributable to common genetic variants, including >230 identified by genome-wide association studies. Multiple strands of evidence suggest that much of the remaining heritability is also due to additive effects of common variants rather than epistasis between these variants or mutations exclusive to individual families. Here, we show in 68,379 cases and controls that up to 5% of this heritability is explained by low-frequency variation in gene coding sequence. We identify four novel genes driving MS risk independently of common-variant signals, highlighting key pathogenic roles for regulatory T cell homeostasis and regulation, IFNγ biology, and NFκB signaling. As low-frequency variants do not show substantial linkage disequilibrium with other variants, and as coding variants are more interpretable and experimentally tractable than non-coding variation, our discoveries constitute a rich resource for dissecting the pathobiology of MS.

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).

Background Identification of genetic markers of antihypertensive drug responses could assist in individualization of hypertension treatment. Methods and Results We conducted a genome‐wide association study to identify gene loci influencing the responsiveness of 228 male patients to 4 classes of antihypertensive drugs. The Genetics of Drug Responsiveness in Essential Hypertension (GENRES) study is a double‐blind, placebo‐controlled cross‐over study where each subject received amlodipine, bisoprolol, hydrochlorothiazide, and losartan, each as a monotherapy, in a randomized order. Replication analyses were performed in 4 studies with patients of European ancestry (PEAR Study, N=386; GERA I and II Studies, N=196 and N=198; SOPHIA Study, N=372). We identified 3 single‐nucleotide polymorphisms within the ACY3 gene that showed associations with bisoprolol response reaching genome‐wide significance (P−8); however, this could not be replicated in the PEAR Study using atenolol. In addition, 39 single‐nucleotide polymorphisms showed P values of 10−5 to 10−7. The 20 top‐associated single‐nucleotide polymorphisms were different for each antihypertensive drug. None of these top single‐nucleotide polymorphisms co‐localized with the panel of >40 genes identified in genome‐wide association studies of hypertension. Replication analyses of GENRES results provided suggestive evidence for a missense variant (rs3814995) in the NPHS1 (nephrin) gene influencing losartan response, and for 2 variants influencing hydrochlorothiazide response, located within or close to the ALDH1A3 (rs3825926) and CLIC5 (rs321329) genes. Conclusions These data provide some evidence for a link between biology of the glomerular protein nephrin and antihypertensive action of angiotensin receptor antagonists and encourage additional studies on aldehyde dehydrogenase–mediated reactions in antihypertensive drug action.

Primary sclerosing cholangitis (PSC) is a severe liver disease of unknown etiology leading to fibrotic destruction of the bile ducts and ultimately to the need for liver transplantation. We compared 3,789 PSC cases of European ancestry to 25,079 population controls across 130,422 SNPs genotyped using the Immunochip. We identified 12 genome-wide significant associations outside the human leukocyte antigen (HLA) complex, 9 of which were new, increasing the number of known PSC risk loci to 16. Despite comorbidity with inflammatory bowel disease (IBD) in 72% of the cases, 6 of the 12 loci showed significantly stronger association with PSC than with IBD, suggesting overlapping yet distinct genetic architectures for these two diseases. We incorporated association statistics from 7 diseases clinically occurring with PSC in the analysis and found suggestive evidence for 33 additional pleiotropic PSC risk loci. Together with network analyses, these findings add to the genetic risk map of PSC and expand on the relationship between PSC and other immune-mediated diseases.

Supplementary methods the detailed description of the methods used for the molecular genetics analyses included in this paper. (DOC 41 kb)

Primary sclerosing cholangitis (PSC) is a severe liver disease of unknown etiology leading to fibrotic destruction of the bile ducts and ultimately to the need for liver transplantation. We compared 3,789 PSC cases of European ancestry to 25,079 population controls across 130,422 SNPs genotyped using the Immunochip. We identified 12 genome-wide significant associations outside the human leukocyte antigen (HLA) complex, 9 of which were new, increasing the number of known PSC risk loci to 16. Despite comorbidity with inflammatory bowel disease (IBD) in 72% of the cases, 6 of the 12 loci showed significantly stronger association with PSC than with IBD, suggesting overlapping yet distinct genetic architectures for these two diseases. We incorporated association statistics from 7 diseases clinically occurring with PSC in the analysis and found suggestive evidence for 33 additional pleiotropic PSC risk loci. Together with network analyses, these findings add to the genetic risk map of PSC and expand on the relationship between PSC and other immune-mediated diseases.

Supplementary methods the detailed description of the methods used for the molecular genetics analyses included in this paper. (DOC 41 kb)

Background Diagnostic analysis of patients with developmental disorders has improved over recent years largely due to the use of microarray technology. Array methods that facilitate copy number analysis have enabled the diagnosis of up to 20% more patients with previously normal karyotyping results. A substantial number of patients remain undiagnosed, however. Methods and Results Using the Genome-Wide Human SNP array 6.0, we analyzed 35 patients with a developmental disorder of unknown cause and normal array comparative genomic hybridization (array CGH) results, in order to characterize previously undefined genomic aberrations. We detected no seemingly pathogenic copy number aberrations. Most of the vast amount of data produced by the array was polymorphic and non-informative. Filtering of this data, based on copy number variant (CNV) population frequencies as well as phenotypically relevant genes, enabled pinpointing regions of allelic homozygosity that included candidate genes correlating to the phenotypic features in four patients, but results could not be confirmed. Conclusions In this study, the use of an ultra high-resolution SNP array did not contribute to further diagnose patients with developmental disorders of unknown cause. The statistical power of these results is limited by the small size of the patient cohort, and interpretation of these negative results can only be applied to the patients studied here. We present the results of our study and the recurrence of clustered allelic homozygosity present in this material, as detected by the SNP 6.0 array.

Primary Immunodeficiency (PIDs) categorize a broad and heterogeneous group of inborn immunity errors. Despite being generally quite rare, PIDs collectively account for consistent morbidity and mortality. Currently, more than 350 monogenic PIDs have been recognised to embody clinical phenotypes ranging from life-threatening infections to autoimmune/inflammatory diseases, allergies and/or malignancy. Many PIDs display genetic and allelic heterogeneity with an overlap of symptoms among different syndromes, often making diagnoses challenging. In the past few years, advancements in genomic technologies have revolutionised the world of genetic testing, and currently, next-generation sequencing (NGS)-based approaches are widely applied to routine genetic diagnostics of human disorders. Among the different methods, whole-exome sequencing (WES) proved highly efficient in revealing the genetic variants behind rare disorders. To further depict the genetics of PIDs, a WES-based approach was carried out, targeting the possible disease-causing variants in Finnish subjects lacking a clinical diagnosis. The cohort included patients with a clinical suspicion of immune or/and haematological disorders (n= 212). In the first study, a Finnish founder mutation in the AICDA gene was identified in patients affected by hyper-IgM syndrome type 2 (HIGM2). The disease is a primary antibody deficiency characterised by early-onset recurrent infections, autoimmunity and an absence/low levels of IgG, IgA and IgE but elevated/normal levels of IgM. The retrieved ancestral founder allele is significantly enriched in Finns compared to other European populations (38.56-fold) and has accounted for all HIGM2 cases diagnosed in Finns thus far. In the second study, biallelic ADA2 mutations that cause a deficiency of adenosine deaminase 2 (DADA2) were identified in seven PID patients, all sharing one of the causal variants, which were significantly enriched in Finns (3.31-fold). DADA2 was originally associated with systemic autoinflammation, polyarteritis nodosa-type vasculitis and mild immunodeficiency. Only a fraction of the identified DADA2 patients presented with vasculopathies. In addition, recurrent haematological manifestations are noted, and for the first time, the occurrence of lymphoproliferation is described for some of the patients, expanding the phenotypic spectrum of DADA2. Finally, novel causal variants in telomere biology disorders (TBDs)-associated genes were identified in three families with heterogeneous phenotypes that lacked the classic clinical pathognomonic signs of telomeropathies. The phenotypes ranged from mild signs of Dyskeratosis congenita (DKC) to SCID. The genetic diagnosis was confirmed by an assessment of shortened telomere lengths in patients. In addition, the spectrum of TBD-associated phenotypes was enlarged, showing variable degrees of cytopenia in some patients. This work attests to the validity of clinical WES testing to identify rare disease-causing variants despite the heterogeneous and/or atypical clinical presentations of PIDs. The achievement of a genetic diagnosis allowed for updating the spectrum of reported phenotypes as well as including atypical clinical presentations that might have otherwise remained undiagnosed. In addition, the enrichment of some rare PID-causing mutations in Finland has been depicted, highlighting the correlation of the population history with the distribution of rare deleterious variants of clinical relevance. Primary Immunodeficiency (PIDs) categorize a broad and heterogeneous group of inborn immunity errors. Despite being generally quite rare, PIDs collectively account for consistent morbidity and mortality. Currently, more than 350 monogenic PIDs have been recognised to embody clinical phenotypes ranging from life-threatening infections to autoimmune/inflammatory diseases, allergies and/or malignancy. Many PIDs display genetic and allelic heterogeneity with an overlap of symptoms among different syndromes, often making diagnoses challenging. In the past few years, advancements in genomic technologies have revolutionised the world of genetic testing, and currently, next-generation sequencing (NGS)-based approaches are widely applied to routine genetic diagnostics of human disorders. Among the different methods, whole-exome sequencing (WES) proved highly efficient in revealing the genetic variants behind rare disorders. To further depict the genetics of PIDs, a WES-based approach was carried out, targeting the possible disease-causing variants in Finnish subjects lacking a clinical diagnosis. The cohort included patients with a clinical suspicion of immune or/and haematological disorders (n= 212). In the first study, a Finnish founder mutation in the AICDA gene was identified in patients affected by hyper-IgM syndrome type 2 (HIGM2). The disease is a primary antibody deficiency characterised by early-onset recurrent infections, autoimmunity and an absence/low levels of IgG, IgA and IgE but elevated/normal levels of IgM. The retrieved ancestral founder allele is significantly enriched in Finns compared to other European populations (38.56-fold) and has accounted for all HIGM2 cases diagnosed in Finns thus far. In the second study, biallelic ADA2 mutations that cause a deficiency of adenosine deaminase 2 (DADA2) were identified in seven PID patients, all sharing one of the causal variants, which were significantly enriched in Finns (3.31-fold). DADA2 was originally associated with systemic autoinflammation, polyarteritis nodosa-type vasculitis and mild immunodeficiency. Only a fraction of the identified DADA2 patients presented with vasculopathies. In addition, recurrent haematological manifestations are noted, and for the first time, the occurrence of lymphoproliferation is described for some of the patients, expanding the phenotypic spectrum of DADA2. Finally, novel causal variants in telomere biology disorders (TBDs)-associated genes were identified in three families with heterogeneous phenotypes that lacked the classic clinical pathognomonic signs of telomeropathies. The phenotypes ranged from mild signs of Dyskeratosis congenita (DKC) to SCID. The genetic diagnosis was confirmed by an assessment of shortened telomere lengths in patients. In addition, the spectrum of TBD-associated phenotypes was enlarged, showing variable degrees of cytopenia in some patients. This work attests to the validity of clinical WES testing to identify rare disease-causing variants despite the heterogeneous and/or atypical clinical presentations of PIDs. The achievement of a genetic diagnosis allowed for updating the spectrum of reported phenotypes as well as including atypical clinical presentations that might have otherwise remained undiagnosed. In addition, the enrichment of some rare PID-causing mutations in Finland has been depicted, highlighting the correlation of the population history with the distribution of rare deleterious variants of clinical relevance.

Muscular dystrophies are a clinically and genetically heterogeneous group of inherited disorders that cause progressive weakness and atrophy of muscles. They are divided into several subgroups based on the clinical and genetic findings. Mutations in dozens of different genes are known to cause the disorders, but unsolved cases exist. Accurate molecular diagnosis is important for genetic counseling and management of the diseases. The aims of this doctoral study were to identify still unknown genetic defects in muscular dystrophy patients using Sanger sequencing and next-generation sequencing (NGS) methods and to develop a targeted NGS gene panel for molecular diagnostics. In the course of this study more than 30 muscular dystrophy families or sporadic patients obtained a molecular diagnosis. A majority of the identified mutations were located in TTN. The gene encodes titin, which is a giant muscle protein having a central role in aspects of development, structure and function of the striated muscle. Due to the huge size of the gene it was not routinely sequenced before the NGS era. Muscle disorders caused by mutations in TTN are called titinopathies. In the first study, missense mutations in TTN exon 343 were identified in 12 families from seven different countries having hereditary myopathy with early respiratory failure (HMERF). The findings considerable expanded the mutational spectrum of HMERF and highlighted the geographically wide occurrence of the disease as well as the role of TTN exon 343 as a mutational hotspot region. In the second study, additional mutations in patients having previously reported tibial muscular dystrophy (TMD) mutation in TTN exon 362 or 363, but atypical phenotypes, were identified. The patients had a frameshift mutation in the other TTN allele causing a more severe TMD phenotype or limb-girdle muscular dystrophy (LGMD). In the last study, recessive distal myopathy patients were identified to have novel mutations in TTN exon 362 or 363 in either homozygous state or in compound heterozygosity with a nonsense or frameshift mutation. The patients had similar more severe TMD phenotype as the patients in the previous study. The findings expanded the list of distal myopathies with a new category: juvenile or early adult onset recessive distal titinopathy. In addition, a few patients having other novel recessive TTN mutations and novel phenotypes were identified in this doctoral study. The targeted NGS gene panel, MyoCap, was designed to capture and sequence 180 myopathy related genes simultaneously. Altogether 61 myopathy patients, negative for previous candidate gene approaches, were screened using the assay and 19 (31%) of them obtained a molecular diagnosis. MyoCap has high sensitivity and specificity and it is easily updated when new discoveries of myopathy causing genes are made. It provides fast and reliable detection of mutations in myopathy patients and it has been implemented in the molecular diagnostics of myopathy patients in Finland. It is now the first method of choice in diagnostics if no clear candidate gene can be determined by conventional clinical examinations. Lihasdystrofiat ovat sekä kliinisesti että geneettisesti heterogeeninen joukko perinnöllisiä sairauksia, jotka aiheuttavat etenevää lihasten heikentymistä ja surkastumista. Taudit on jaettu useisiin alaryhmiin kliinisten ja geneettisten löydösten perusteella. Lihasdystrofioita aiheuttavia geenivirheitä tunnetaan kymmenissä eri geeneissä, mutta selvittämättömiä tapauksia on myös vielä paljon jäljellä. Geenivirheen löytäminen on tärkeää neuvonnan ja taudin hoitamisen kannalta. Tämän väitöskirjatutkimuksen tarkoituksena oli selvittää uusia geenivirheitä lihasdystrofiapotilailta käyttäen sekä perinteistä Sanger- että uuden sukupolven sekvensointimenetelmiä ja lisäksi kehittää geenipaneeli lihastautidiagnostiikkaan. Tutkimuksen aikana yli 30 perhettä tai yksittäistä potilasta saatiin diagnosoitua. Suuri osa löydetyistä uusista mutaatioista sijaitsi TTN-geenissä. Geeni koodaa titiiniä, jättimäistä lihasproteiinia, jolla on keskeinen rooli poikkijuovaisen lihaksen kehityksessä, rakenteessa ja toiminnassa. TTN-geenin suuresta koosta johtuen sitä ei ole aikaisemmin laajasti sekvensoitu. TTN-geenin mutaatioiden aiheuttamia lihastauteja kutsutaan titinopatioiksi. Ensimmäisessä osatutkimuksessa uusia mutaatioita löytyi perinnöllistä HMERF-tautia sairastavilta perheiltä TTN-eksonista 343. Löydökset laajensivat HMERF-mutaatioiden kirjoa ja korostivat eksonin 343 roolia HMERF-mutaatioalueena. Toisessa osatutkimuksessa potilailta, joilla oli tibiaalista lihasdystrofiaa (TMD) aiheuttava mutaatio TTN-eksonissa 362 tai 363, mutta tavallisesta TMD:stä poikkeava fenotyyppi, löytyi toisesta TTN-alleelista frameshift-mutaatio. Tämä johti vakavampaan TMD-tautiin ja osalla potilaista hartia-lantiotyypin lihasrappeumaan (LGMD). Viimeisessä osatutkimuksessa distaalista myopatiaa sairastavilta perheiltä löytyi uusia mutaatioita TTN-eksoneista 362 tai 363 ja lisäksi toisesta TTN-alleelista framshift- tai nonsense-mutaatio. Potilailla oli samanlainen aikaisemmin alkava ja vakavampi TMD-tauti kuin edellisen tutkimuksen potilailla. Nämä löydökset lisäsivät distaalimyopatioihin uuden kategorian: nuoruudessa alkava resessiivinen distaalinen titinopatia. Näiden lisäksi väitöskirjatutkimuksessa löydettiin uusia resessiivisiä TTN-mutaatioita myös muutamalta muulta potilailta, joilla oli uusi aiemmin kuvaamaton fenotyyppi. MyoCap-geenipaneeli suunniteltiin kattamaan 180 lihastautigeenin sekvensointi samanaikaisesti. Tutkimuksessa sitä käytettiin yhteensä 61 potilaan sekvensoimiseen, joilta ei oltu löydetty geenivirhettä aiemmissa tutkimuksissa. Potilaista 19 (31%) saatiin diagnosoitua. MyoCap on tarkka menetelmä ja siihen on helppo lisätä uusia tautigeenejä, kun niitä löydetään. Se tarjoaa nopean ja tehokkaan tavan paikantaa mutaatioita ja se on nykyisin käytössä lihastautipotilaiden diagnostiikassa. Sitä käytetään varsinkin potilailla, joiden tautigeeniä ei pystytä suoraan päättelemään kliinisissä tutkimuksissa.

A large part of the genomic arrangement regulating the immune system is located in the Major Histocompatibility Complex (MHC) region on chromosome 6p21.31. Human leucocyte antigen (HLA) genes, which encode the antigen presenting molecules, are located in MHC class I and II, while the MHC class III region contains non-HLA genes, e.g. butyrophilin-like protein 2 (BTNL2), tumor necrosis factor (TNF) and complement C4. Most genes in the MHC region play an important role in susceptibility to autoimmune and infectious diseases. Sarcoidosis is a complex granulomatous disorder with varying a clinical presentation. A multitude of studies have reported strong associations between genes in the MHC and sarcoidosis. The structure of the MHC is characterized by a high degree of polymorphism, due to population-specific and highly conserved extended haplotypes, leading to a complicated pattern of linkage disequilibrium (LD). This makes dissecting disease associations within individual MHC genes difficult. The primary aims of this Study were to study the overall polymorphism of the MHC region in Finnish subjects, to investigate MHC as a susceptibility locus for sarcoidosis and to find predictive markers for the prognosis of the disease. In Study I, extended MHC haplotypes covering genes within MHC class I (HLA-A, -B), MHC class II (HLA-DRB1, -DQB1 and DPB1) and MHC class III (e.g. TNF and BTNL2) were constructed and LD between the genes studied in a Finnish population sample. We discovered that the extended MHC haplotypes could be grouped, based on a similar functional variant (e.g. truncating protein) or a similar structure of the peptide binding site. In Study II, we replicated a previously reported HLA-DRB1*03:01 association with a favourable prognosis of sarcoidosis in Finland (P=0.007, OR=2.7). Using the extended MHC haplotype approach, we detected novel and independent variants associated with sarcoidosis (HLA-DPB1*04:02; P=0.003, OR=0.48) or the disease course of sarcoidosis (HLA-DRB1*04:01-DPB1*04:01; P=0.02, OR=3.07). In Study IV, we explored four genes in the MHC class III region (LTA, TNF, AGER, BTNL2) and HLA-DRA, and performed a replication and meta-analysis in four European populations. After adjusting for sex, population stratification and HLA-DRB1 alleles, four SNPs in the HLA- DRA/BTNL2 region were associated with non-Löfgren sarcoidosis; the strongest signal association was detected with rs3177928 (1.79x10-7, OR=1.90). In this Study, we also addressed guidelines for disease association studies dealing with immunogenetic data. The results of the extended MHC haplotype analysis discovered a unique haplotype admixture in the Finnish population, which may have implications to MHC-related disease associations. Our sarcoidosis study provided new insights to predicting the disease prognosis, with different distributions of MHC variants associating different patterns of progression, further promoting the importance of MHC region in sarcoidosis predisposition. Especially, the region covering the genes BTNL2 and HLA-DRA warrants further studies in larger samples and in different ethnic groups. To conclude, this Study showed the importance of studying extended MHC haplotypes in well-characterized samples, in order to understand the complex MHC structure and to distinguish variants associations with the trait. Sarkoidoosi on useita elimiä samanaikaisesti vaurioittava tulehdussairaus, jonka aiheuttajaa ei tunneta. Sarkoidoosissa eri elimiin muodostuu pieniä tulehdussolukertymiä eli granuloomia, varsinkin keuhkoihin ja imusolmukkeisiin. Suomessa sarkoidoosiin sairastuu vuosittain noin 500 henkilöä. Sarkoidoosin taudinkuva on vaihteleva, yleisoireena on tyypillisesti yskää, kohtalaista kuumeilua ja väsymystä. Suurin osa tautiin sairastuneista ei tarvitse hoitoa, koska lieväoireinen tauti paranee monesti itsestään. 20%:lla tauti kuitenkin kroonistuu jolloin keuhkofibroosin ohella voi löytyä muutoksia ihossa, silmissä, maksassa/pernassa, luustossa, sydänlihaksessa, keskushermostossa jne. Nykykäsityksen mukaan sarkoidoosin syntymisen taustalla ovat sekä ympäristötekijät että geenit, joista tärkeimpinä pidetään MHC-alueen geenejä, kuten HLA-DRB1 ja BTNL2. MHC-alue kromosomissa 6 (6p21.3, Major Histocompatibility Complex) sisältää joukon geenejä, joista suurin osa liittyy ihmisen immuunivasteen muodostumiseen ja säätelyyn. MHC-alue jakaantuu kolmeen luokkaan. Elinsiirtojen kudossopeutuvuutta määrittelevät klassiset transplantaatiogeenit, HLA-A, -B, -DRB1, -DQB1 ja -DPB1, kuuluvat joko luokkaan I tai II. Näiden HLA-geenien päätehtävä on esitellä peptidejä (antigeenejä) immuunijärjestelmälle. Luokan III alueella on geenejä, kuten esimerkiksi C4 komplementtigeenit ja BTNL2, jotka liittyvät mm. tulehdusreaktion syntyyn sekä solujen väliseen viestintään. Tämän väitöskirjatyön tarkoituksena oli kartoittaa, miten suomalaisten MHC-alueen geenit jakaantuvat ja esiintyvät yhdessä, ja käyttää näitä tietoja hyväksi tutkiessa MHC geenien merkitystä sarkoidoosin alttiuteen ja ennusteeseen. I. osajulkaisussa tutkimme erilaisten MHC-geenikombinaatioiden (haplotyyppien) jakaumia ja geenien välistä kytkeytymistä (kytkentäepätasapainoa). Totesimme, että MHC-haplotyyppejä voi ryhmitellä sen mukaan miten ne sisältävät samoja toiminnallisia geenimuotoja (esim. C4 geenin puutos) tai samankaltaisia antigeenin sitoutumiskohtia. Tutkimustuloksemme osoittivat, että usean MHC-geenin samanaikainen analysointi ja haplotyyppirakenteiden tunteminen voi auttaa ehdokasgeenien paikantamisessa. Osajulkaisuissa II ja III toistimme aiemmin muista väestöistä löydettyjen geenien, HLA-DRB1*03:01 and BTNL2, yhteyden sarkoidoosin ennusteen myös suomalaisessa väestössä. Tutkimuksissamme löysimme myös uusia sarkoidoosille altistavia (HLA-DRB1*04:01-DPB1*04:01) ja siltä suojaavia (HLA-DPB1*04:02) geenejä. IV osajulkaisussa monikansallinen otos vahvisti sarkoidoosiin liittyvien MHC luokan II ja III ehdokasgeenien ja niissä löytyneiden geenimuotojen merkityksen taudin alttiudelle. Sarkoidoosille altistavan geenin osoittaminen on MHC-alueella haastavaa, koska alueen geenit ovat tiukasti kytkeytyneet toisiinsa, ja ehdokasgeenit voivat vaihdella eri väestöjen välillä. Tässä väitöskirjatyössä osoitimme, että MHC-alueen kokonaisvaltaisesta tutkimisesta on etua alttiusgeenien paikantamisessa. Hyödyntäen tätä lähestymistapaa - tutkimalla useita MHC-geenejä samanaikaisesti- löysimme MHC-geenialueelta uusia sarkoidoosiin ja sarkoidoosin ennusteeseen liittyviä geenimuotoja. Ne auttavat ymmärtämään sarkoidoosin monimuotoista geneettistä taustaa ja osoittavat MHC-geenien merkityksen sarkoidoosin alttiudelle. Jotta MHC-alueen geenejä voidaan käyttää ennustamaan suomalaisten sarkoidoosipotilaiden taudinkuvaa, tulee tulokset toistaa isommassa otoksessa.

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system. Current available treatments can slow down the disease progression in the majority of patients, but can neither stop the progression nor cure the patients. MS is a debilitating neurological disease of young adults and it affects 0.1% of the populations of Northern European descent. The prevalence of MS varies globally and locally, and there are several isolated high-risk populations. One such high-risk population is in the Southern Ostrobothnia region of Finland, where the incidence and prevalence are approximately twice those of the surrounding regions. Both environmental and genetic factors are thought to contribute to disease pathogenesis. Twin, half-sib and adoption studies all point towards underlying genetic factors that predispose to MS. In recent years, genome-wide association studies have revealed over fifty genes that are associated with MS. At the start of this thesis project, only one MS locus, HLA-DRB1, was known, whilst shortly after the start two more loci, IL2RA and IL7R, were reported. The aim of this thesis was to find new associated loci, confirm newly found loci and assess the relevance of these known loci within the Southern Ostrobothnian high-risk isolate, and in the Finnish population overall. The association analyses were performed using genome-wide association analysis methods, haplotype analysis and meta-analysis methods. In the first study, 68 Southern Ostrobothnian cases and 136 identity-by-state matched controls were analyzed in a genome-wide association analysis, where an association signal from the SNP rs744166 in the STAT3 gene was discovered. The association signal was replicated in an international sample set of 4487 MS cases and 9778 controls (OR 1.18, 95% CI 1.12-1.24, p=2.75×10-10). In order to further understand the association signal, a haplotype analysis was performed, revealing that the association signal was on a common haplotype. Further, in a genome-wide homozygosity analysis, excess homozygosity was found in the 68 cases compared to the 136 controls in three genomic loci, 1q42.12, 2q24.3 and 12q24.33. The importance of these loci requires further study. The second study aimed to replicate loci that had previously been observed to associate with MS in an international genome-wide meta-analysis. Three SNPs in three loci, IRF8, TNFRSF1A and CD6, were genotyped in 608 trios, 8439 MS cases, and 9280 controls from 11 populations of European origin, replicating the associations in all three loci. The SNP in IRF8 was strongly associated with MS, while the SNPs in TNTRSF1A and CD6 indicated slightly weaker association. The odds ratios for all the three loci were similarly modest: IRF8 OR 1.11, TNFRSF1A OR 1.12, and CD6 OR 1.11. Since the prevalence and incidence of MS is two-fold in the Southern Ostrobothnian region and there is evidence for founder effect, the aim of the last study was to assess the accumulation of recently identified genome-wide significant MS-associated common risk alleles in the Southern Ostrobothnian isolate MS cases and in familial samples. However, we could not detect accumulation of common variants in the isolate region or in the families compared to general Finnish samples or non-familial samples. Multippeliskleroosi, eli MS-tauti, on krooninen monitekijäinen keskushermoston autoimmuunisairaus, jonka syntyyn vaikuttavat monet ympäristö- ja perintötekijät. Tässä väitöskirjatyössä on etsitty ja tutkittu perintötekijöitä, jotka voivat osaltaan altistaa kantajansa MS-taudille. Tutkimuksissamme havaittiin, että STAT3-geeni on yksi monesta MS taudin riskigeenistä. Lisäksi vahvistimme aiemmin tunnistettujen TNFRSF1A, IRF8 ja CD6 geenien yhteyden MS-tautiriskiin. Näiden geenien uskotaan vaikuttavan elimistön immuunipuolustuksen valkosolujen toimintaan ja kehitykseen. MS-taudissa elimistön oma immuunipuolustus tuhoaa paikallisesti aivojen ja selkäytimen hermosolujen viejähaarakkeita peittävää myeliiniä, ja aiheuttaa samalla hermosolujen viejähaarakkeiden tuhoutumista. Sairauden oirekirjo on laaja, mutta tauti ilmenee usein aaltomaisesti tai tasaisesti etenevänä vammautumisena. Taudin syntysyy ja mekanismi tunnetaan puutteellisesti, eikä tautiin ole löydetty parantavaa tai etenemisen pysäyttävää hoitoa. Tautimekanismin parempi tuntemus onkin perusedellytys parempien hoitomuotojen kehittämiselle MS-taudissa. Viimeaikaiset geneettiset ja solubiologiset tutkimukset ovat tunnistaneet perimässämme useita taudille altistavia geenimuotoja, jotka ovat valottaneet taudin syntyyn vaikuttavia mekanismeja. Vaikka ympäristötekijöillä on merkittävä rooli taudin synnyssä, niitä tunnetaan edelleen vähän. Suomessa noin yksi ihminen tuhannesta sairastaa MS-tautia, mutta taudin esiintyvyys vaihtelee sekä Suomen sisällä, että maailmanlaajuisesti. Etelä-Pohjanmaalla MS-taudin esiintyvyys on kaksinkertainen verrattuna muuhun Suomeen, ja alueen MS-potilaiden perheissä on usein muitakin MS-potilaita. On oletettavaa, että alueelle on kertynyt perinnöllisiä tekijöitä, jotka altistavat MS-taudille ja ovat mahdollisesti harvinaisia muualla maailmassa. Olemme tutkimuksissamme keskittyneet erityisesti Etelä-Pohjanmaan MS-potilaisiin, joiden avulla löysimme yhteyden MS-taudin ja STAT3-geenin tietyn muodon välillä. Löytämämme geenimuodon vaikutus yksittäisenä tekijänä on pieni, kuten muidenkin MS taudille altistavien geenimuotojen. Yhdessä muun tiedeyhteisön tutkimuksen kanssa ne kuitenkin lisäävät ja tarkentavat käsitystämme MS-taudin perusmekanismeista.

Osteoarthritis (OA) is a complex disease characterized by the destruction of the articular cartilage of the joints. The main symptoms are pain and disability, caused most probably by secondary synovitis. Secondary manifestations of OA are considered to be bone changes, osteophytes, subchondral sclerosis, and bone cysts. The suspected risk factors for OA include genetic predisposition, gender (greater in females), old age, obesity (also in non-weight-bearing joints), previous injury and various physical loading conditions. Prevalence of the disease varies between approximately 8 % and 60 %, dependent on the joint, but also largely on the age of study subjects, the population and OA definition. The disease affects the cartilage, bone, and synovium, but the etiology is still mainly unknown. Even the initiating events and tissue are unknown, but mechanical wear and tear is considered as the prime candidate for the root cause of the disease. The role of the genetic component in common skeletal diseases is well established but remains poorly understood. Heritability of hand and knee OA in women has been shown to range from 39 % to 65 %. Previous association studies have provided some evidence for genes affecting bone density, inflammation, composition and break down of the extracellular matrix. However, these results have been inconsistent. The objective of the present study was to identify predisposing genes for OA using single nucleotide polymorphism (SNP) markers. The study consisted of hand, knee and hip OA cases (n = 1466) and controls (n = 4475). A set of 25 candidate genes were studied in carefully selected case-control, family and twin settings. The objective of Study I was to pinpoint an OA-predisposing gene at the 2q11.2 region, which has previously been linked with hand OA using partially the same study sample (Leppävuori et al. 1999). In total 32 SNPs were genotyped in this region comprising 6 genes belonging to the interleukin 1 super-family. IL1R1 was associated with hand OA (p = 0.00091, with a significance threshold of p = 0.0021). The aim of Study II was to identify genetic variants in MMP8 and MMP9 genes that predispose to OA. Five different study cohorts including 1369 OA cases and 4445 controls were studied. Evidence for the role of MMP8 in knee OA was observed in one study sample (p = 0.0049, with a significance threshold of 0.0057) without statistically significant replication. The main association finding was found in the Finnish study sample for knee OA, while a similar tendency was observed in both the knee OA study sample of Spanish origin, and the hand OA sample of Finnish origin. The aim of Study III was to identify variants predisposing to hip OA from a preselected set of 25 biologically interesting candidate genes. Hips of the study subjects had been analyzed using magnetic resonance imaging (MRI). Variants in the COL9A2 and COL10A1 genes showed suggestive association (p = 0.0021 and p = 0.0015, with significance threshold of 0.00073). The change in the same codon of the COL9A2 has previously been shown to associate with a disc degeneration phenotype (Videman et al. 2009). In conclusion, the results of this thesis support the theory that inflammatory factors, cartilage breaking factors and originally poor quality of collagen associate with OA. Validation of all these results in larger study populations with more accurate genetic analysis are still needed for any further conclusions. Nivelrikko on monitekijäinen nivelruston rappeumasairaus. Sen pääasialliset oireet ovat kipu ja toiminnalliset nivelvaivat. Perintötekijät, naissukupuoli, ikä, lihavuus, aiemmat nivelvammat ja erilaiset fyysiset kuormitukset ovat nivelrikon riskitekijöitä. Taudin yleisyys vaihtelee 8 ja 60 %:n välillä riippuen tutkittavasta nivelestä, ikäryhmästä, väestöstä ja nivelrikon määritelmästä. Tauti vaikuttaa rustoon, luuhun ja nivelkalvoon, mutta taudin etiologia on vielä tuntematon. Jopa taudin alulle panevia tapahtumia tai varhaisia kudosmuutoksia ei vielä tunneta, mutta mekaanista kulutusta pidetään yhtenä tärkeimpänä tekijänä taudin synnyssä. Geneettisten tekijöiden merkitys tuki- ja liikuntaelinsairauksissa on yleisesti tunnustettu, mutta huonosti ymmärretty. Geneettiset tekijät selittävät käsi- ja polvinivelrikosta 39 - 65 %. Aiemmat assosiaatiotutkimukset ovat osoittaneet luun tiheyteen, tulehdukseen, ruston koostumukseen ja hajotukseen liittyvien geenien vaikuttavan nivelrikkoon, mutta useat tulokset ovat edelleen ristiriitaisia. Tämän väitöskirjatyön tavoitteena oli tunnistaa nivelrikolle altistavia geenejä. Tutkimus koostui käden, polven ja lonkan nivelrikkoa sairastavista henkilöistä (n = 1466) sekä näiden verrokkihenkilöistä (n = 4475). Yhteensä 25 ennalta valittua ehdokasgeeniä tutkittiin tarkoin valituissa tapaus-verrokki-, perhe- sekä kaksosasetelmissa. Ensimmäisen tutkimuksen tavoitteena oli osoittaa kromosomialueelta 2q11.2 nivelrikolle altistava geeni. Alueen oli aiemmassa suomalaistutkimuksessa havaittu kytkeytyvän käden nivelrikkoon aineistossa, joka oli osittain päällekkäinen tämän tutkimuksen aineiston kanssa (Leppävuori et al. 1999). Yhteensä 32 SNPiä (yhden emäksen polymorfioita) analysoitiin tältä kuusi interleukiiniperheen geeniä sisältävältä alueelta. IL1R1-geenin todettiin assosioituvan käden nivelrikkoon (p = 0.00091, kun merkitsevyysraja oli p = 0.0021). Toisessa tutkimuksessa syvennyttiin matriksi metalloproteinaasi 8 (MMP8) ja matriksi metalloproteinaasi 9 (MMP9) -geeneihin polven nivelrikossa. Tutkimuksessa käytettiin viittä eri tutkimusaineistoa, jotka sisälsivät 1369 nivelrikkoa sairastavaa and 4445 tervettä verrokkia. Näiden avulla löydettiin assosiaatio MMP8-geeniin (p = 0.0049, kun merkitsevyysraja oli 0.0057), mutta löydös ei toistunut muissa aineistoissa tilastollisesti merkittävällä tasolla. Päälöydös tehtiin suomalaisessa polven nivelrikkoaineistossa, mutta myös suomalainen käden nivelrikkoaineisto ja espanjalainen polven nivelrikkoaineisto näyttivät samansuuntaista tulosta. Kolmannen osatyön tavoitteena oli tutkia nivelrikolle altistavia variantteja 25:stä biologisen merkityksensä vuoksi ennalta valitusta ehdokasgeenistä. Tutkimushenkilöiden lonkkanivelet oli analysoitu magneettisella resonanssikuvantamisella (MRI). Kaksi varianttia, geeneissä kollageeni 9 alfa 2 (COL9A2) sekä kollageeni 10 alfa 1 (COL10A1), näyttivät altistavan lonkan nivelrikolle (p = 0.0021 and p = 0.0015, kun merkitsevyysraja oli 0.00073). Tulos ei ollut tilastollisesti merkitsevä. Saman kodonin muutoksen COL9A2-geenissä on aiemmin todettu altistavan selän välilevyrappeumalle (Videman et al. 2009). Yhteenvetona voidaan todeta, että tämän väitöskirjatyön tulokset tukevat aiempaa teoriaa siitä, että tulehdukselliset ja rustoa hajottavat tekijät sekä jo alun alkaen huonolaatuinen kollageeni altistavat nivelrikolle. Nämä tulokset on kuitenkin validoitava laajemmissa aineistoissa ja geneettisiä analyysejä on tarkennettava varmempien johtopäätösten tekemiseksi.

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection. Mikrobit ovat tärkeä osa erilaisten biologisten kokonaisuuksien kuten ekosysteemien ja ihmiselimistön toimintaa. Näin ollen esimerkiksi sairastumisriski tai mikrobiyhteisön kyky muokata erilaisia aineita oikealla tavalla riippuu tiettyjen mikrobiryhmien läsnäolosta. Mikrobit ovat kuitenkin hyvin monimuotoisia, eikä valtaosaa pystytä viljelemään tehokkaasti. Siksi DNA ja muut biomolekyylit mahdollistavat luotettavampia tapoja havaita mikrobeja näytteestä. Tämän väitöskirjatyön tarkoituksena on ollut molekyylibiologisten menetelmien tutkiminen ja kehittäminen mikrobien tunnistamiseksi DNA-sekvenssin avulla ympäristö- ja potilasnäytteistä. Työssä on hyödynnetty DNA-koettimien entsymaattista ligaatioreaktiota yhdistettynä DNA-mikrosirutekniikkaan kohdesekvenssien havaitsemiseksi. Erityinen painopiste tutkimuksessa on ollut kohteen tunnistuksen tarkkuudella, mikä on ollut perinteisesti vaikeaa analysoitaessa monimutkaisia mikrobiyhteisöjä. Tuloksten perusteella sekä ympäristön homesienten että ihmisen papillomavirusten lajintunnistus on mahdollista noin 0,04% herkkyydellä polymeraasiketjureaktiolla (PCR) monistetusta näyte-DNA:sta. Analysoitaessa näyte-DNA:ta suoraan ilman monistusta herkkyys on arviolta 1% mitattuna luettujen sekvenssien kokonaismäärästä näytteessä. Lisäksi mikrosirujen signaalia on työssä pystytty parantamaan poistamalla laskennallisesti kontrollikoettimen mittaamaa virhettä sirulla. Tulosten perusteella voidaan olettaa, että DNA-mikrosirumenetelmä on käyttökelpoinen alusta mikrobien lajitason tannistukselle, ja että määräsuhteiden havaitseminen rajoitetulla vaihtelualueella on menetelmällä periaatteessa mahdollista. Menetelmä voisi korvata nykyisin vielä laajassa käytössä olevaa viljelypohjaista määritystä.

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults, affecting more than two million people worldwide. It manifests as a chronic inflammation in the central nervous system (CNS) and causes demyelination and neurodegeneration. Depending on the location of the demyelinated plaques and axonal loss, a variety of symptoms can be observed including deficits in vision, coordination, balance and movement. With a typical age of onset at 20-40 years, the social and economic impacts of MS on lives of the patients and their families are considerable. Unfortunately the current treatments are relatively inefficient and the development of more effective treatments has been impeded by our limited understanding of the causes and pathogenesis of MS. Risk of MS is higher in biological relatives of MS patients than in the general population. Twin and adoption studies have shown that familial clustering of MS is explained by shared genetic factors rather than by shared familial environment. While the involvement of the human leukocyte antigen (HLA) genes was first discovered four decades ago, additional genetic risk factors have only recently been identified through genome-wide association studies (GWAS). Current evidence suggests that MS is a highly polygenic disease with perhaps hundreds of common variants with relatively modest effects contributing to susceptibility. Despite extensive research, the majority of these risk factors still remain to be identified. In this thesis the aim was to identify novel genes and pathways involved in MS. Using genome-wide microarray technology, gene expression levels in peripheral blood mononuclear cells (PBMC) from 12 MS patients and 15 controls were profiled and more than 600 genes with altered expression in MS were identified. Three of five selected findings, DEFA1A3, LILRA4 and TNFRSF25, were successfully replicated in an independent sample. Increased expression of DEFA1A3 in MS is a particularly interesting observation, because its elevated levels have previously been reported also in several other autoimmune diseases. A systematic review of seven microarray studies was then performed leading to identification of 229 genes, in which either decreased or increased expression in MS had been reported in at least two studies. In general there was relatively little overlap across the experiments: 11 of the 229 genes had been reported in three studies and only HSPA1A in four studies. Nevertheless, these 229 genes were associated with several immunological pathways including interleukin pathways related to type 2 and type 17 helper T cells and regulatory T cells. However, whether these pathways are involved in causing MS or related to secondary processes activated after disease onset remains to be investigated. The 229 genes were also compared with loci identified in published MS GWASs. Single nucleotide polymorphisms (SNP) in 17 of the 229 loci had been reported to be associated with MS with P-value less than 0.0001 including variants in CXCR4 and SAPS2, which were the only loci where evidence for correlation between the associated variant and gene expression was found. The CXCR4 variant was further tested for association with MS in a large case-control sample and the previously reported suggestive association was replicated (P-value is 0.0004). Finally, common genetic variants in candidate genes, which had been selected on the basis of showing association with other autoimmune diseases (MYO9B) or showing differential expression in MS in our study (DEFA1A3, LILRA4 and TNFRSF25), were tested for association with MS, but no evidence of association was found. In conclusion, through a systematic review of genome-wide expression studies in MS we have identified several promising candidate genes and pathways for future studies. In addition, we have replicated a previously suggested association of a SNP variant upstream of CXCR4 with MS. Keywords: autoimmune disease, common variant, CXCR4, DEFA1A3, HSPA1A,gene expression, genetic association, GWAS, MS, multiple sclerosis, systematic review Pesäkekovettumatauti eli multippeli skleroosi (MS) on nuorten aikuisten yleisin neurologisiin häiriöihin johtava syy. Maailmanlaajuisesti sitä sairastaa yli kaksi miljoonaa ihmistä. Tauti ilmenee keskushermoston kroonisena tulehduksena ja johtaa myeliinikatoon ja keskushermoston rappeutumiseen. Oireet riippuvat leesioiden sijainnista keskushermostossa ja saattavat kohdistua esimerkiksi näköaistiin, tasapainoon ja koordinaatio- ja liikuntakykyyn. MS-tauti puhkeaa yleensa 20-40 vuoden iässä, joten sen sosiaaliset ja ekonomiset vaikutukset potilaisiin ja näiden lähimmäisiin ovat merkittävät. Tehokkaiden hoitojen kehittämistä on vaikeuttanut se, että taudin syyt ja mekanismit tunnetaan toistaiseksi huonosti. Todennäköisyys sairastua MS-tautiin on suurempi MS-potilaiden biologisilla sukulaisilla kuin populaatiossa keskimäärin. Kaksos- ja adoptiotutkimuksissa on osoitettu, että taudin keskittyminen perheisiin selittyy jaetuilla perinnöllisillä alttiustekijöillä. Vaikka immunologisiin soluihin liittyvien HLA-varianttien yhteys MS-tautiin osoitettiin jo lähes neljä vuosikymmentä sitten, uusia geneettisiä alttiustekijöitä on onnistuttu paikantamaan vasta viime vuosina koko genomin laajuisten assosiaatiotutkimusten avulla. Nykyisten tutkimusten valossa MS-alttiuteen vaikuttavia geenivariantteja näyttäisi kuitenkin olevan jopa satoja ja suurin osa näistä on edelleen tunnistamatta. Tämän väitöskirjan tavoitteena oli tunnistaa uusia MS-tautiin liittyviä geenejä ja reaktioreittejä. Geenien ilmentymistasoja verrattiin ensin periferaalisissa mononukleaarisissa immuunisoluissa MS-potilaiden ja verrokkien välillä käyttäen mikrosirutekniikkaa, joka mahdollistaa kaikkien tunnettujen geenien samanaikaisen tarkastelun. Yli 600 geenin ilmentymisen havaittiin poikkeavan merkitsevästi MS-potilaiden ja verrokkien välillä. Tunnistetuista geeneistä viisi valittiin toistotutkimukseen, jossa kolmen geenin (DEFA1A3, LILRA4 ja TNFRSF25) tapauksessa ilmentymisero potilaiden ja verrokkien välillä toistui. Näistä geeneistä DEFA1A3 on erityisen kiinnostava, sillä sen ilmentymistason on havaittu olevan verrokkeja korkeampi myös muissa autoimmuunisairauksissa. Koska vastaavia mikosirututkimuksia on tehty MS-taudissa myös aiemmin, yhtenä väitöskirjan tavoitteena oli verrata tuloksia näistä tutkimuksista. Vertailuun otettiin kaikkiaan seitsemän tutkimusta, mukaan lukien tämän väitöskirjan osana tehty tutkimus. Tavoitteena oli tunnistaa geenit, joissa ilmentymisen oli raportoitu poikkeavan MS-potilaiden ja verrokkien välillä ainakin kahdessa tutkimuksessa. Vaikka tällaisia geenejä tunnistettiin 229, yleisesti ottaen tutkimustulosten välillä oli kuitenkin vähän päällekkäisyyttä: vain 11 geeniä oli raportoitu kolmessa tutkimuksessa ja ainoastaan yksi, HSPA1A, neljässä tutkimuksessa. Reaktioreittianalyysin avulla osoitettiin, että tunnistetut 229 geeniä assosioituvat merkittävästi useisiin mielenkiintoisiin immunologisiin reaktioreitteihin. Monet näistä liittyvät tiettyihin T-solutyppeihin, joiden merkitystä sekä MS-taudin alttiuden että taudin etenemisen ja patogeneesin kannalta on tulostemme perusteella syytä tutkia lisää. 229 geenistä ainoastaan 17 on aiemmin raportoitu assosioituvan MS-tautiin, mikä viittaisi siihen että nämä 229 geeniä eivät välttämättä ole merkittäviä taudin alttiuden lisäämisen kannalta, mutta voivat liittyä taudin mekanismeihin taudin puhkeamisen jälkeen. 17 geenistä ainoastaan SAPS2- ja CXCR4-geenien tapauksessa havaitsimme yhteyden geenin ilmentymisen ja geneettisen riskivariantin välillä siten, että molemmissa tapauksissa geenin ilmentyminen oli koholla riskialleelin kantajilla. Lopuksi väitöskirjassa testattiin tämän CXCR4-lokuksessa sijaitsevan variantin assosiaatiota MS-tautiin tuhansien henkilöiden potilas-kontrolli aineistossa ja aiemmissa tutkimuksissa havaittu assosiaatio toistettiin (P-arvo on 0.0004). Lisäksi tutkittiin valituissa ehdokasgeeneissä sijaitsevien yleisten geneettisten varianttien yhteyttä MS-tautiin. Edellä mikrosirututkimuksessa tunnistettujen DEFA1A3-, LILRA4- ja TNFRSF25-geenien lisäksi tarkasteltiin MYO9B-geeniä, jonka oli aiemmin havaittu assosioituvan muihin autoimmuunisairauksiin. Merkitsevää yhteyttä MS-taudin ja tarkasteltujen geneettisten varianttien välillä ei kuitenkaan havaittu. Yhteenvetona, tässä väitöskirjassa on geenien ilmentymistutkimusten ja niiden systemaattisen katsauksen avulla tunnistettu useita lupaavia ehdokasgeenejä (mm. DEFA1A3, LILRA4, TNFRSF25 ja HSPA1A) ja erityisesti T-soluihin liittyviä reaktioreittejä. Lisäksi väitöskirjassa on toistettu aiemmin raportoitu assosiaatio MS-taudin ja CXCR4-lokuksessa sijaitsevan geneettisen variantin välillä. Tunnistettujen geenien rooli MS-taudin patogeneesissä on kuitenkin toistaiseksi tuntematon, joten tarkempia jatkotutkimuksia tarvitaan. Avainsanat: CXCR4, DEFA1A3, HSPA1A, geenien ilmentyminen, geneettinen assosiaatio, GWAS, MS-tauti, multippeli skleroosi, pesäkekovettumatauti, yleinen variantti

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Both environmental factors and several predisposing genes are required to generate MS. Despite intensive research these risk factors are still largely unknown, the pathogenesis of MS demyelination is poorly understood, and no curative treatment exists. Both prevalence and familial occurrence of MS are exceptionally high in a Finnish population subisolate, Southern Ostrobothnia, presumably due to enrichment of predisposing genetic variants within this region. Previous linkage scan on MS pedigrees from Southern Ostrobothnia detected three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in other populations have also provided independent evidence for the location of MS susceptibility genes in these regions. Further, these loci are syntenic to the experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents. In this thesis work an effort was made to localize MS predisposing alleles of the linked loci outside the HLA region by studying familial MS cases from the Southern Ostrobothnia isolate. Analysis of the 5p locus revealed one region, flanking the complement component 7 (C7) gene. The identified relatively rare haplotype seems to have a fairly large effect on genetic susceptibility of MS (frequency MS 12%, controls 4%; p=0.000003, OR=2.73). Evidence for association with alleles of the region and MS was seen also in more heterogeneous populations. Convincingly, plasma C7 protein levels and complement activity correlated with the risk haplotype identified. The finding stimulated us to study other complement cascade genes in MS. No evidence for association could be observed with the complement component coding genes outside 5p. A scan of the 17q locus provided evidence for association with variants of the protein kinase C alpha (PRKCA) gene (p=0.0001). Modest evidence for association with PRKCA was observed also in Canadian MS families. Finally we used a candidate gene based approach to identify potential MS loci. Mutations of DAP12 and TREM2 cause a recessively inherited CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located in MS regions on 6p and 19q, and we tested them as potential candidate genes in the Finnish MS sample. No evidence for association with MS was observed. This thesis provides an example of how extended families from special populations can be utilized in fine-mapping of the linked loci. A first relatively rare MS variant was identified utilizing the strength of a Finnish population subisolate. This variant seems to have an effect on activity of the complement system, which has previously been suggested to have an important role in the pathogenesis of MS. Multippeliskleroosi (MS-tauti) on krooninen tulehduksellinen sairaus, jonka puhkeamisen aiheuttavat ympäristötekijät ja useat alttiusgeenit yhdessä. Intensiivisestä tutkimuksesta huolimatta suurin osa näistä MS-taudin riskitekijöistä on kuitenkin vielä tunnistamatta, MS-taudissa tapahtuvan keskushermoston valkean aineen tuhon syntymekanismit ovat huonosti ymmärrettyjä, eikä MS-taudille ole olemassa parantavaa hoitoa. MS-taudin esiintyvyys ja suvuittainen esiintyminen ovat poikkeuksellisen korkeat erään väestöisolaatin alueella Etelä-Pohjanmaalla. Tämä johtuu luultavasti taudin perinnöllisten alttiustekijöiden rikastumisesta alueelle. Suomalaistutkimuksissa on aiemmin tunnistettu kolme pääasiallista MS- geenipaikkaa kromosomeissa 5p, 6p (HLA) ja 17q. Myös muissa väestöissä tehdyt tutkimukset tukevat löydöstä. Tässä väitöskirjatutkimuksessa pyrittiin paikantamaan MS-taudille altistavia geenivariantteja HLA-alueen ulkopuolisilta kytkentäalueilta tutkimalla MS-sukuja Etelä-Pohjanmaan isolaattialueelta. Kromosomin 5p alttiusalueen suhteellisen harvinaisen, komplementti komponentti 7 (C7) geeniä sivuavan haplotyypin havaittiin liittyvän MS-alttiuteen (p=0,000003; OR=2,73). Assosiaatio pystyttiin vahvistamaan myös muissa väestöissä, ja plasman C7-proteiinitasojen ja komplementtiaktiivisuuden havaittiin korreloivan tunnistetun riskihaplotyppin kantajuuteen. Tulos viittaa siihen, että tunnistetulla alueella sijaitsee MS-taudin riskiä selkeästi lisäävä tekijä. Löydös kannusti tutkimaan myös muiden komplementtigeenien osuutta MS-taudissa. Kromosomin 5p ulkopuolisten kompementtigeenien ei kuitenkaan todettu liittyvän MS-alttiuteen suomalaisessa väestössä. Kromosomin 17q alttiusalueen analyysissä havaittiin proteiinikinaasi C alfa -geenin (PRKCA) varianttien liittyvän MS-alttiuteen sekä suomalaisilla että kanadalaisilla (p=0,0001). Väitöskirjan viimeisessä osatyössä tutkittiin MS-taudin ehdokasgeenejä. DAP12 ja TREM2 geenien mutaatiot aiheuttavat keskushermoston valkean aineen taudin, PLOSL:n. Koska kyseiset geenit sijaitsevat kromosomien 6p ja 19q alueilla, joiden on havaittu kytkeytyvän myös MS-tautiin, selvitimme, altistavatko DAP12 ja TREM2 myös MS-taudille. Näiden geenien ei havaittu liittyvän MS-alttiuteen. Tässä väitöskirjassa kuvataan, miten erityisväestöjen sukuaineistoja voidaan hyödyntää alttiusvarianttien paikantamiseksi kytkentäalueilta. Tutkimuksessa tunnistettiin suomalaista isolaattia aineistona hyödyntäen ensimmäinen suhteellisen harvinainen MS-taudille altistava geenivariantti. Tämä variantti vaikuttaa säätelevän komplementtisysteemiä, jolla on jo aiemmin arveltu olevan tärkeä rooli MS-taudin synnyssä.

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.