Next-Generation Sequencing has opened the doors to nearly limitless amounts of genomic data, but the clinical utility of this data is not yet clear. From examining at sequencing data of known familial cancer genes in hereditary cancer patients, the NCGENES study found a clear molecular diagnosis in about 5% of patients and an uncertain molecular result in about 15% of patients. The remaining 80% of hereditary cancer patients received a negative result for the screening of known cancer genes. These latter patients were followed up by whole exome sequencing analysis, and the data was used to perform a research sweep to potentially identify mutation(s) in gene(s) that have yet to be clearly associated with their phenotype. Hereditary breast cancer has a relatively well-established set of susceptibility genes, yet a large percentage of the molecular etiology is still unknown. There are many genes that are good candidates for breast cancer genes based on their protein's function, but they may not actually contribute to breast cancer susceptibility. The ClinGen consortium is aiming to establish the clinical validity of gene-disease associations so that clinicians and patients can better interpret and utilize sequencing results. Six breast cancer susceptibility genes were evaluated using the ClinGen clinical validity framework with the goal of both evaluating the genes already on hereditary breast cancer panels and evaluating genes not yet widely tested to determine if there is enough evidence to support their role in disease to warrant widespread testing. These genes have varying levels of evidence supporting their role in breast cancer susceptibility. The variants in each of the six genes were compared between a cancer patient cohort and a non-cancer patient cohort enrolled in the NCGENES whole exome sequencing study. One likely pathogenic variant and several variants of unknown significance were identified in various genes, and the burden of variants in cancer cases versus controls was evaluated, although the controls were not matched to the cancer cohort in any way. Research sweeps were performed for patients with VUSs to ensure that there were no other mutations in genes that would better fit the phenotype. This thesis presents a method for evaluating gene-disease associations and for utilizing whole exome sequencing data to pinpoint a molecular diagnosis in hereditary breast cancer patients. Overall, it was found that the ClinGen method of evaluating clinical validity of gene-disease associations could be helpful when determining if variants are pathogenic or benign. A new gene, RINT1, was found to have enough evidence to be moderately associated with hereditary breast cancer and it was subsequently added to the diagnostic list so that all cancer patients will now be screened for RINT1 variants. In addition, it was found that two of the genes currently on the diagnostic list, RAD51C and RAD51D, have "disputed" evidence with respect to breast cancer susceptibility. Interestingly, they have much more evidence for an association with ovarian cancer, so if variants are found in these genes, the patient's phenotype should be considered when evaluating them. It was also shown that PALB2, an established breast cancer susceptibility gene, indeed is definitively associated with breast cancer, and the NCGENES cancer patients have more truncating variants than the controls, further validating the clinical validity assertion. Finally, an ovarian cancer patient with two interesting variants, one in SLX4 and one in GEN1, were evaluated. Studies showed that knocking out both of these genes' pathways was highly destructive to the cell. A VUS was found in each of these genes, and it was hypothesized that perhaps these two variants together may be sufficient to contribute to this patient's cancer susceptibility.

Abstract Musculoskeletal diseases, such as osteoarthritis (OA), lumbar disc degeneration (LDD) and osteoporosis (OP), are common complex disorders affected by both environmental and genetic factors. OA and LDD are degenerative diseases affecting joints and spine and Modic changes (MC) are a specific phenotype of LDD. OP is a disorder causing bone fragility. There are families with a history of early onset cartilage degradation, disc disorders and bone fragility as well as rare, more severe disorders with these traits as part of the phenotype. The aim of this study was to identify predisposing genetic factors in Finnish families with three different musculoskeletal phenotypes and to investigate the use of whole exome sequencing (WES) as a tool. Six families were studied here, three diagnosed with hip and knee OA, two with MC and one with primary OP. Using WES together with in silico and in vitro analyses we identified new candidate genes. In the two OA families we identified family specific variants, c.-127G>T in the 5’UTR of FIP1L1 and p.Arg210Gly in OLIG3. We observed expression of these genes in human bone and cartilage. Both FIP1L1 and OLIG3 participate in the regulation of transcription. Family specific variants were also found in both families with MC: p.Gln1611fs in HSPG2 and p.Glu553Lys in MAML1. HSPG2 encodes for an important structural protein in the disc and MAML1 is a transcription factor. The family with primary OP had previously been reported to carry a heterozygous COL1A2 deletion leading to nonsense-mediated mRNA decay. In the WES we identified an additional change that may contribute to the phenotype: p.Arg428* in ZNF528. We showed experimentally that the variant leads to expression of a truncated form of ZNF528 in the nucleus. ZNF528 binding sites are located near genes associated with bone phenotypes. We identified twelve potential target genes for ZNF528 that were differentially expressed in patients’ cells compared to controls. Altogether, we identified five new candidate genes for the studied phenotypes demonstrating that WES can be used as a tool in studying complex musculoskeletal phenotypes in families. One of the identified candidate genes, HSPG2, encodes a structural protein, whereas, OLIG3, FIP1L1, MAML1 and ZNF528, participate in the regulation of transcription supporting the importance of regulatory mechanisms in the pathogenesis of musculoskeletal diseases. / Tiivistelmä Tuki- ja liikuntaelinsairaudet, kuten nivelrikko, välilevyrappeuma ja osteoporoosi, ovat yleisiä, monitekijäisiä sairauksia. Nivelrikko ja välilevynrappeuma ovat eteneviä nivelten ja selkärangan sairauksia. Modic muutokset ovat välilevyn ja nikaman välisten päätelevyjen muutoksia. Osteoporoosi on luuta haurastuttava sairaus. Varhaisessa iässä ilmenevää ruston haurastumista, välilevyn sairauksia tai luun haurautta tavataan myös suvuittain esiintyvinä sairauksina tai vakavien harvinaisten sairauksien oireina. Tutkimuksen tarkoitus oli tunnistaa altistavia geneettisiä tekijöitä kolmelle tuki- ja liikuntaelimistön sairaudelle suomalaisissa perheissä käyttäen eksomisekvensointi-menetelmää. Aineisto koostui kuudesta perheestä: kolmessa oli diagnosoitu lonkan ja polven nivelrikko, kahdessa selän välilevyjen Modic muutoksia ja yhdessä primaarinen vaikea selän osteoporoosi. Tunnistimme uusia ehdokasgeenejä käyttäen eksomisekvensointi-menetelmää sekä in silico ja in vitro analyysejä. Kahdessa nivelrikkoperheessä tunnistimme perhekohtaiset variantit kahdessa geenissä: c.-127G>T variantin FIP1L1 geenin säätelyalueella ja p.Arg210Gly variantin OLIG3 geenissä. Osoitimme, että nämä traskription säätelyyn osallistuvat geenit ilmenevät ihmisen luu- ja rustokudoksessa. Perhekohtaiset variantit havaittiin myös perheissä, joilla oli todettu Modic muutoksia: p.Gln1611fs HSPG2 -geenissä ja p.Glu553Lys MAML1 -geenissä. HSPG2 koodaa välilevylle tärkeää rakenneproteiinia ja MAML1 on transkriptiota säätelevä tekijä. Primaarista osteoporoosia sairastavalla perheellä oli aiemmin havaittu heterotsygootti, geenituotteen hajottamiseen johtava deleetio, COL1A2 -geenissä. Eksomisekvensoinnlla havaitsimme mahdollisesti taudin ilmiasuun lisäksi vaikuttavan muutoksen ZNF528 -geenissä. Osoitimme kokeellisesti, että havaittu variantti johtaa lyhentyneen proteiinin tuottoon solussa. ZNF528 on transkriptiotekijä, jolle tunnistimme kaksitoista mahdollista kohdegeeniä ja havaitsimme että niiden tuotto oli muuttunut potilaiden soluissa kontrollisoluihin verrattuna. Tunnistimme viisi uutta ehdokasgeeniä kolmessa eri sairaudessa eksomisekvensointi-menetelmän avulla. Yksi tunnistetuista geeneistä, HSPG2, koodaa rakenneproteiinia, ja muut osallistuvat transkription säätelyyn. Tämä tukee käsitystä säätelytekijöiden tärkeydestä TULE sairauksien synnyssä.

Family and twin studies have shown a heritable component to seasonal affective disorder (SAD). While a few studies have examined individual genetic variants in SAD, many methodological issues exist in the current literature. First, most studies combined major depression (MDD) and bipolar (BD) cases in the genetic analysis of SAD. This makes it difficult to differentiate the effect from MDD and BD. Second, most studies adopted a candidate gene approach and used fairly small sample sizes. This does not allow for testing across a wide variety of genes, and it yields less robust P-values. Third, healthy controls have been used, but not case comparisons, which makes it difficult to differentiate the effects of seasonality from that of the primary illness (MDD and BD). To overcome these issues, seasonal MDD and BD cases were separated into two different studies in this thesis; sample sizes for both studies are the largest in the current SAD molecular genetics literature; GWAS was used to test for potential risk loci in a hypothesis-free fashion; case comparisons were incorporated to exclude potential genetic contributions related generally to the primary diseases themselves (MDD and BD). For MDD, we performed a GWAS with 562 seasonal MDD cases and 1,225 comparison cases with non-seasonal MDD. Subjects were drawn from two iterations of the Genetics of Recurrent Early Onset Depression (GenRED) study. Seasonal cases were those whose depressive episodes typically started in fall or winter. A mega-analysis of the two GWAS datasets was done using SNPTEST. We found that two single nucleotide polymorphisms (SNPs), rs149882931 and rs77073398, on chromosome 16p12.1 were associated with seasonal depression, at a genome-wide significant level (OR= 1.66, P= 3.59 x 10-8 and OR=1.62, 4.76 x 10-8, respectively). Since SAD is more prevalent in females, a female-specific analysis was carried out. The two variants were more significant in this analysis: P=2.18x10-9 (OR=1.89) and P=2.79x10-9 (OR=1.82), respectively, and a significant sex-by-SNP interaction was observed. These SNPs are located in a conserved intergenic region between the genes HS3ST4 and C16orf82. The protein product of HS3ST4 modifies the side chains of heparan sulfate proteoglycans. We therefore tested the hypothesis that the heparan sulfate biosynthesis pathway would be enriched in nominally significant SNPs using the SNP ratio test, and found evidence for such enrichment (P=0.008, SNP ratio test, P=0.027, SKAT). For BD, the GWAS analysis of 818 seasonal BD cases and 1,515 healthy controls showed that BD-S is most strongly associated with two SNPs within the ZBTB20 genes. BD subjects were drawn from NIMH Bipolar Genetics Study (BIGS), and seasonal cases were defined as those with depressive episodes starting in fall or winter. An association study was carried out with SNPTEST, and we found two single nucleotide polymorphisms (SNPs) in the intronic region of ZBTB20 gene to be associated with BD-S (rs7646282, OR=2.34, P= 7.23 x 10-8 and rs139459337, OR=2.37, 8.05 x 10-8). A similar case-only study was carried out with 818 BD-S cases and 1239 cases without seasonal depressive symptoms (non-BDS), though no SNP was found to be significantly associated in this analysis. rs7646282 is the strongest SNP in cis-association with ZBTB20 gene expression, and ZBTB20 has been shown to affect the neural development of the hippocampus, a brain region implicated in the pathophysiology of BD. Finally, we sought to determine whether there is a role for circadian rhythm genes in BD susceptibility. In this study, we used a discovery set of 189 exome-sequenced BD patients and 105 healthy controls to look for circadian genes associated with BD. We found the DRD2 gene to be the circadian gene most strongly associated with BD. Among the rare damaging variants in the DRD2 gene, the S311C variant was the predominant SNP. To test whether this variant segregates in family members with BD, we genotyped the family members of probands from the discovery sample. This data was used for a linkage and family-based association study. Even though the linkage analysis was only very weakly positive, the family-based association study showed significant segregation of the variant in family members with BD (P< 0.05). To follow up on this finding, we further genotyped 2,185 unrelated BD cases and 1,982 healthy controls. We found no support for the S311C variant in this replication dataset. Sub-phenotype study of psychotic features and mood-incongruence also did not show significant association. Meta-analysis with 2,994 BD cases and 3,661 controls, however, revealed no association between the S311C variant and BD.

Abstract Undefined severe neurological and multi-organ diseases are rare as single diseases, but as a group of diseases, they are responsible for significant morbidity, impaired quality of life and mortality, emphasizing the importance of neuroscience research and its translation into novel diagnostic and treatment strategies. Molecular karyotyping and whole-exome sequencing were used to identify three novel disease-causing genes, GLE1, NHLRC2 and MYH7B, in Northem Finnish families having children with undefined progressive neuromuscular diseases. Functional studies on GLE1, NHLRC2, and MYH7B were conducted in order to understand better the impact of these mutations. The studies revealed that the cellular localization of GLE1 was impaired due to a mutation in the coding gene. The NHLRC2 is involved in many biological processes and its dysfunction has a role in the development of a novel FINCA disease and in fibrosis. Furthermore, mutations in MYH7B in the myosin family have now been connected to encephalomyopathies. Mutations in GLE1, NHLRC2 and MYH7B are involved in encephalomyopathies and neurodegeneration, stressing the important role of these genes in normal psychomotor development Analyses of these previously uncharacterized disease-causing gene mutations provided new insights into the etiologies behind these diseases, representing a relevant starting point for resolving the pathomechanisms underpinning these disorders. The newly-discovered human disease-causing genes and the novel phenotypes of childhood onset neuromuscular diseases provide the possibility for offering the relevant families preclinical diagnostics and may be beneficial in the identification of similar clinical phenotypes all around the world. / Tiivistelmä Yksittäiset, määrittelemättömät, vaikeat neurologiset monielinsairaudet ovat harvinaisia. Sen sijaan neurologisten ja monielinsairauksien alle ryhmittyvät taudit ovat merkittävä syy useisiin sairauksiin, jotka heikentävät elämänlaatua ja aiheuttavat kuolleisuutta. Tästä johtuen neurotieteiden tutkimus ja saatujen tulosten soveltaminen diagnostiikassa ja hoitomuotojen kehittämisessä on hyvin tärkeää. Molekyylikaryotyypitys- ja eksomisekvensointi-menetelmiä hyödynnettiin etsittäessä taudin syytä eteneville neuromuskulaarisairauksille pohjoissuomalaisissa perheissä. Tutkimuksessa tehtiin lisäksi funktionaalisia kokeita GLE1-, NHLRC2- ja MYH7B-proteiineilla, jotta ymmärrettäisiin paremmin löydettyjen mutaatioiden vaikutus potilaiden sairauksiin. Havaittiin, että GLE1-mutaatio vaikutti proteiinin solunsisäiseen paikantumiseen. NHLRC2-proteiini puolestaan on mukana useissa solun biologisissa prosesseissa ja sen toiminnanhäiriö vaikuttaa FINCA-taudin ja fibroosin kehittymiseen. MYH7B-myosiinigeenimutaatio puolestaan yhdistettiin ensimmäistä kertaa enkefalomyopatiaan. Havaittujen tautigeenien; GLE1, NHLRC2 ja MYH7B, vaikutus enkefalomyopatioissa ja neurodegeneraatiossa kertoo, että kyseisillä geeneillä on hyvin todennäköisesti tärkeä rooli ihmisen kehityksessä. Kyseisten, aiemmin tuntemattomien sairautta-aiheuttavien geenimutaatioiden analysointi lisäsi tietoa sairauksien etiologiasta ja loi pohjan tautimekanismien ratkaisemiselle tulevaisuudessa. Työssä esitettyjä uusia sairautta-aiheuttavia geenejä ja uusia karakterisoituja lapsuusiän neuromuskulaarisairauksien ilmiasuja voidaan hyödyntää perheille tarjotun sikiödiagnostiikan lisäksi myös muiden potilaiden samankaltaisen taudinkuvan diagnosoinnissa maailmanlaajuisesti.

Lower motor neuron diseases and peripheral neuropathies are two groups of diseases that include multiple rare disorders where many causes are unknown and definitive treatments are unavailable. Understanding the molecular etiology of these genetic diseases provides an opportunity for rapid diagnosis, preconception genetic counseling and, in a subset, direction for the development of future treatment options. The recent introduction of whole exome sequencing (WES) marks a new era in Mendelian genetic disease research as the majority of the coding region of the genome can be sequenced in a timely and cost-effective manner. In this study, WES was used to investigate the molecular etiology of a cohort of 37 patients presenting with lower motor neuron disease or peripheral neuropathy. A molecular diagnosis was determined for seven patients informing the diagnostic utility of WES. Novel phenotypes were found for three genes originally associated with a different disorder. Finally, the foundation has been laid, through the use of functional studies and large scale data-sharing, to identify novel disease-causing genes for lower motor neuron disease and peripheral neuropathy.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13442号 / 論医博第2241号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 藤田 恭之, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chenopodium quinoa, a South American pseudocereal, has valuable agricultural traits such as salt tolerance and drought tolerance, and it has beneficial nutritional properties such as high protein content and a complete amino acid profile. However, problems including disease susceptibility, low harvest index, lodging, seed shattering, low heat tolerance, and saponin content plague quinoa. Genetic resources for quinoa are needed to fix these problems and make quinoa more available throughout the world. We used ethyl methanesulfonate (EMS) to create a mutant population of QQ74 quinoa (USDA GRIN PI 614886) of 5,030 mutant families. We did whole exome sequencing (WES) on 44 mutant families. Using the recently published quinoa reference genome and MAPS, a mutation detection pipeline, we found a mutation rate of 11.35 mutations/Mb in these families. We also used whole genome sequencing (WGS) to calculate a mutation rate of 21.67 mutations/Mb in an additional nine mutant families. To demonstrate the utility of this population as a genetic resource, we found an EMS-induced nonsense mutation in the betalain synthesis pathway that prevents red betacyanins from accumulating in the hypocotyl of quinoa. With the mutation rates in our population, we calculate that analysis of 300 mutant families will yield 3-7 mutations in any gene of interest, which will facilitate forward and reverse genetic studies in quinoa.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12920号 / 論医博第2095号 / 新制||医||1010(附属図書館) / 32130 / (主査)教授 髙橋 良輔, 教授 松田 文彦, 教授 瀬原 淳子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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