Arvutuslikud ja statistilised meetodid DNA sekveneerimisandmete analüüsimiseks ja rakendused TÜ Eesti Geenivaramu andmetel

Väitekirja elektrooniline versioon ei sisalda publikatsiooneTänapäeval võimaldavad teise põlvkonna sekveneerimisel (next-generation sequencing, NGS) põhinevad meetodid määrata inimese genoomi järjestusi suurtes kohortides. Seejuures toodetakse väga suuri andmemahtusid, mis tekitavad mitmeid väljakutseid nii informaatika kui statistika valdkonnas. TÜ Eesti Geenivaramu (TÜ EGV) on aastatel 2002-2011 kogunud enam kui 50 000 inimese geeniproovi ja käesoleval aastal lisandub veel 100 000. Praeguseks hetkeks on üle 5 500 geenidoonori DNA-d analüüsitud erinevate NGS meetoditega. Käesolevas doktoritöös on pakutud üldine raamistik TÜ EGV-s toodetud NGS-andmete töötluseks ning lisaks on uuritud, kuidas võimalikult hästi arvestada Eesti päritolu isikute geneetilist eripära. Üheks levinud NGS meetodiks on eksoomi ehk kõigi valku kodeerivate geenipiirkondade sekveneerimine, mis võimaldab efektiivselt leida harvu ja de novo geenivariante ja leiab seetõttu rakendust meditsiinigeneetikas mendeliaarsete haiguste geenimutatsioonide tuvastamisel. Doktoritöö esimeses osas on analüüsitud kolme Eesti perekonna andmeid ja kõigil kolmel juhul kindlaks tehtud potentsiaalne patogeenne mutatsioon, mis lubab tulevikus välja töötada paremaid ravimeetodeid. Samuti on läbi viidud genoomi sekveneerimisandmete analüüs kliinilise vere näitajatega. See analüüs tõi välja populatsioonipõhise biopanga eelised, mis lisaks rikkalikele genoomiandmetele sisaldab ka väärtuslikku informatsiooni erinevate haiguste ja tunnuste kohta. Uuringus tuvastati olulisi seoseid CEBPA geenivariantide ja basofiilide arvu vahel, kusjuures viimasel on roll mitmete autoimmuunhaiguste sümptomaatikas. Ülegenoomsete assotsiatsiooniuuringute võimsuse suurendamiseks kasutatakse puuduvate geenivariantide ennustamist ehk imputeerimist. Muutmaks just Eesti päritolu isikute andmeanalüüsi tõhusamaks, on kasutatud genoomi sekveneerimisandmeid eestlaste-spetsiifilise imputatsioonipaneeli loomiseks. Seejärel on imputeeritud puuduvaid geenivariante kolmel moel – kasutades nii eestlaste-spetsiifilist kui ka kahte multi-etnilist paneeli. Võrdlustulemused näitasid, et eestlaste-spetsiifilise paneeli kasutamisel õnnestub määrata rohkem parema kvaliteediga geenivariante ning loodud paneeli eelis tuleb eriti esile harvaesinevate variantide puhul.Next-generation sequencing (NGS) technology enables large-scale, routine sequencing in large cohorts. This thesis demonstrated that the analysis of NGS data has a huge potential in several fields, but also requires a massive computational power. Also, with the increase of data volumes, there is an incessant need for the development of computational and statistical methods. Covering the whole spectrum of protein-coding regions in a cost-effective way, exome sequencing opens new opportunities for quick and exact large-scale screenings. In the first part of the thesis we analysed three Estonian families with Mendelian diseases and detected potentially causative gene variants for each case. These projects highlighted that a tight collaboration between data scientists and medical geneticists can lead to findings with considerable impact in the research of rare genetic disorders and have the potential to lead to successful therapies in the future. Population-based biobanks provide numerous opportunities for expanding phenotypic datasets. We used additional blood cell measurements from the electronic medical records and our genome-wide scan detected previously undiscovered association with basophil counts near CEBPA gene, and highlighted their role in the autoimmune regulation. This example opens new dimensions for scanning underlying genetic basis for a variety of traits and diseases. To increase the resolution of genome-wide scans, imputation is routinely implemented to incorporate variants that are not directly genotyped. We had an opportunity to construct an imputation reference panel to Estonians based on genome sequencing data. We showed that the utilization of a population-specific reference panel provided significantly higher imputation confidence for rare variants compared to larger, multi-ethnic panels. In the downstream analysis, we observed a huge gain in gene-based rare variant testing. As one of the main results of this thesis, the Estonian-specific imputation reference panel is created, tested and ready to serve for a long time. This includes data processing in the framework of the ongoing initiative to invite 100,000 Estonians to join the Biobank cohort, with the purpose to develop efficient disease prevention and treatment guides for the implementation of personalized medicine

Viie aasta elulemus pärast plaanilist aordi aneurüsmi ravi avatud ja endovaskulaarsel meetodil

Eesti Arst 2023; 102(6–7):35

Five-year survival after elective open and endovascular aortic aneurysm repair

Background and objective: Current evidence suggests short-term survival benefit from endovascular aneurysm repair (EVAR) versus open surgical repair (OSR) in elective abdominal aortic aneurysm (AAA) procedures, but this benefit is lost during long-term follow-up. The aim of this study was to compare short- and mid-term all-cause mortality in patients with non-ruptured aneurysm treated by OSR and EVAR; and to assess the rate of complications and reinterventions, as well as to evaluate their impact on survival. Methods: The medical records of the non-ruptured AAA patients undergoing OSR or EVAR between 1 January 2011 and 31 December 2019 at Tartu University Hospital, Estonia, were retrospectively reviewed. We gathered survival data from the national registry (mean follow-up period was 3.7 +/- 2.3 years). Results: A total of 225 non-ruptured AAA patients were treated operatively out of whom 95 (42.2%) were EVAR and 130 (57.8%) were OSR procedures. The difference in estimated all-cause mortality between the OSR and EVAR groups at day 30 was statistically irrelevant (2.3% vs 0%; p = 0.140), but OSR patients showed statistically significantly higher 5 year survival compared with EVAR patients (75.3% vs 50.0%, p = 0.002). Complication and reintervention rates for the EVAR and OSR groups did not differ statistically (26.3% vs 16.9%, p = 0.122; 10.5% vs 11.5%, p = 0.981, respectively). Multivariate analysis revealed that greater aneurysm diameter (p = 0.012), EVAR procedure (p = 0.016), male gender (p = 0.023), and cerebrovascular diseases (p = 0.028) were independently positively associated with 5-year mortality. Conclusions: Thirty-day mortality, and complication and reintervention rates for EVAR and OSR after elective AAA repair were similar. Although the EVAR procedure is an independent risk factor for 5-year mortality, higher age and greater proportion of comorbidities among EVAR patients may influence not only the choice of treatment modality, but also prognosis.Peer reviewe

Migreeniga inimeste elukvaliteet ning selle hindamine

Migreen on inimese elu ning selle kvaliteeti vägagi mõjutav haigus. Migreeniga inimeste elukvaliteeti hindavate uuringute võrdluse muudab keeruliseks aga see, et uuringutes on kasutatud väga erinevaid küsimustikke ja skaalasid ning puuduvad piisavad andmed nende tulemuste omavaheliste seoste kohta.Uuringu eesmärk oli võrdlevalt hinnata migreeniga inimeste elukvaliteedi hindamiseks kasutatavate küsimustike (üldine tervise mõju hindav, peavaluspetsiifiline ja migreenispetsiifiline küsimustik) tulemusi ning migreeniga inimeste elukvaliteeti.Mittejuhuslikustatud läbilõikeuuringus võrreldi RAND-36 (ingl 36-item Health Survey) küsimustikuga saadud, uuringurühma kuulunud migreeniga patsientide (n = 105) tulemusi üldrahvastiku migreenita kontrollrühma (n = 176) sama küsimustiku tulemustega. Küsimustikuga RAND-36 saadud tulemisi võrreldi nii HIT-6 (ingl Headache Impact Test 6) kui ka MIDAS küsimustikega saadud tulemustega.Kõigi kolme küsimustiku omavaheliste korrelatsioonide võrdlemisel selgus, et nii peavaluspetsiifilise HIT-6 kui ka migreenispetsiifilise MIDAS (ingl Migraine Disability Assesment Survey) küsimustiku tulemused korreleerusid RAND-36 üldise elukvaliteedi küsimustiku tulemustega. Arvestades seda, et üldise elukvaliteedi küsimustike täitmine on aeganõudvam, soovitame kasutada igapäevases kliinilises praktikas migreenispetsiifilisi küsimustikke.Eesti Arst 2016; 95(3):155–16

Markers of Inflammation, Oxidative Stress, and Fibrosis in Patients with Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in clinical practice. The pathogenesis of AF is linked to inflammatory reaction and oxidative stress, which leads to fibrosis of the atria and progression of the disease. The purpose of this study was to define the role of several biomarkers of inflammation, fibrosis, and oxidative stress (OxS). We included 75 patients with paroxysmal/persistent AF, who were admitted for electrical cardioversion or pulmonary vein isolation (PVI). High-sensitivity C-reactive protein (hsCRP), galectin-3 (Gal-3), myeloperoxidase (MPO), oxidized low-density lipoprotein (oxLDL), and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured before the procedures. We compared the results with those of 75 healthy age-, sex-, and blood pressure-matched individuals. The patients were followed up for 1 year after the intervention to establish the recurrence of AF and its association with the measured markers. Patients with AF had higher MPO (52.6 vs. 36.2 ng/ml, p < 0.001) and NT-proBNP (209.0 vs. 28.0 pg/ml, p < 0.001) compared to healthy subjects. Also, they showed significantly higher levels of hsCRP (1.5 vs. 1.1 mg/l, p=0.001) and Gal-3 (11.4 vs. 9.7 mg/l, p=0.003), while there was no difference found in oxLDL (71.5 vs. 71.7 U/l, p=0.449). MPO (OR=1.012, p=0.014), hsCRP (OR=1.265, p=0.026), and weight (OR=1.029, p=0.013) were independently associated with AF in a multivariable logistic regression analysis. Patients with successful maintenance of sinus rhythm (SR) for one year had lower baseline MPO (40.5 vs. 84.3 ng/ml, p=0.005) and NT-proBNP (127.5 vs. 694.0 pg/ml, p < 0.001) compared to patients with recurrent AF episodes, but there was no difference in hsCRP, Gal-3, or oxLDL between them. MPO (OR=0.985, p=0.010) was independently associated with AF recurrence during the follow-up period when adjusted for cofounders. Patients with AF had increased markers of inflammation and fibrosis, while there was no increase detected in the OxS marker oxLDL. MPO was independently associated with AF in a multivariate model. Inflammatory and fibrotic mechanisms are important factors in electrical and structural remodelling progress in the atria of patients with AF.Peer reviewe

Omics-informed CNV calls reduce false-positive rates and improve power for CNV-trait associations

Copy-number variations (CNV) are believed to play an important role in a wide range of complex traits, but discovering such associations remains challenging. While whole-genome sequencing (WGS) is the gold-standard approach for CNV detection, there are several orders of magnitude more samples with available genotyping microarray data. Such array data can be exploited for CNV detection using dedicated software (e.g., PennCNV); however, these calls suffer from elevated false-positive and -negative rates. In this study, we developed a CNV quality score that weights PennCNV calls (pCNVs) based on their likelihood of being true positive. First, we established a measure of pCNV reliability by leveraging evidence from multiple omics data (WGS, transcriptomics, and methylomics) obtained from the same samples. Next, we built a predictor of omics-confirmed pCNVs, termed omics-informed quality score (OQS), using only PennCNV software output parameters. Promisingly, OQS assigned to pCNVs detected in close family members was up to 35% higher than the OQS of pCNVs not carried by other relatives (p < 3.0 x 10(-90)), outperforming other scores. Finally, in an association study of four anthropometric traits in 89,516 Estonian Biobank samples, the use of OQS led to a relative increase in the trait variance explained by CNVs of up to 56% compared with published quality filtering methods or scores. Overall, we put forward a flexible framework to improve any CNV detection method leveraging multi-omics evidence, applied it to improve PennCNV calls, and demonstrated its utility by improving the statistical power for downstream association analyses.Peer reviewe

Atrial fibrillation is associated with increased central blood pressure and arterial stiffness

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and beta blockers (BBs) are the drugs of choice for rate or rhythm control in these patients. The purpose of this study was to describe differences in arterial stiffness (AS), central blood pressure (cBP), and the role of BBs on cBP in patients with AF compared to healthy individuals. The authors included 76 patients with paroxysmal/persistent AF. Carotid-femoral pulse wave velocity (PWV) and cBP were measured and compared with data from 75 healthy individuals. Patients with AF had higher PWV (8.0 m/s vs. 7.2 m/s, p < .001), central systolic blood pressure (cSBP) (118 mm Hg vs. 114 mm Hg, p = .033), central pulse pressure (cPP) (39 mm Hg vs. 37 mm Hg, p = .035) and lower pulse pressure amplification (PPA) (1.24 vs. 1.30, p = .015), without differences in peripheral blood pressure (pBP) and heart rate (HR). AF patients had significantly increased PWV (beta= 0.500, p = .010, adjusted R-2 = 0.37) after adjustment for confounding factors. The use of BBs significantly reduced PPA (beta = -0.059, p = .017, adjusted R-2 = 0.30). AF patients have higher PWV, cSBP, cPP, and lower PPA, compared to healthy patients. These findings support the role of AS in the development of AF. Use of BBs is related to a potential adverse effect on cBP.Peer reviewe

A genome-wide association study of outcome from traumatic brain injury

Background Factors such as age, pre-injury health, and injury severity, account for less than 35% of outcome variability in traumatic brain injury (TBI). While some residual outcome variability may be attributable to genetic factors, published candidate gene association studies have often been underpowered and subject to publication bias.& nbsp;Methods We performed the first genome-and transcriptome-wide association studies (GWAS, TWAS) of genetic effects on outcome in TBI. The study population consisted of 5268 patients from prospective European and US studies, who attended hospital within 24 h of TBI, and satisfied local protocols for computed tomography.& nbsp;Findings The estimated heritability of TBI outcome was 0.26. GWAS revealed no genetic variants with genome-wide significance (p < 5 x 10(-8)), but identified 83 variants in 13 independent loci which met a lower pre-specified sub-genomic statistical threshold (p < 10(-5)). Similarly, none of the genes tested in TWAS met tissue-wide significance. An exploratory analysis of 75 published candidate variants associated with 28 genes revealed one replicable variant (rs1800450 in the MBL2 gene) which retained significance after correction for multiple comparison (p = 5.24 x 10(-4)).& nbsp;Interpretation While multiple novel loci reached less stringent thresholds, none achieved genome-wide significance. The overall heritability estimate, however, is consistent with the hypothesis that common genetic variation substantially contributes to inter-individual variability in TBI outcome. The meta-analytic approach to the GWAS and the availability of summary data allows for a continuous extension with additional cohorts as data becomes available.& nbsp;Copyright (C)& nbsp;2022 Published by Elsevier B.V.Peer reviewe

Comprehensive population-based genome sequencing provides insight into hematopoietic regulatory mechanisms

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high-coverage whole-genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional 14,904 samples. Using up to 7,134 samples with available phenotype data, our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic datasets provided evidence for causal mechanisms at several loci, including at a previously undiscovered basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.Peer reviewe

Precise, Genotype-First Breast Cancer Prevention : Experience With Transferring Monogenic Findings From a Population Biobank to the Clinical Setting

Although hereditary breast cancer screening and management are well accepted and established in clinical settings, these efforts result in the detection of only a fraction of genetic predisposition at the population level. Here, we describe our experience from a national pilot study (2018-2021) in which 180 female participants of Estonian biobank (of >150,000 participants in total) were re-contacted to discuss personalized clinical prevention measures based on their genetic predisposition defined by 11 breast cancer-related genes. Our results show that genetic risk variants are relatively common in the average-risk Estonian population. Seventy-five percent of breast cancer cases in at-risk subjects occurred before the age of 50 years. Only one-third of subjects would have been eligible for clinical screening according to the current criteria. The participants perceived the receipt of genetic risk information as valuable. Fluent cooperation of project teams supported by state-of-art data management, quality control, and secure transfer can enable the integration of research results to everyday medical practice in a highly efficient, timely, and well-accepted manner. The positive experience in this genotype-first breast cancer study confirms the value of using existing basic genomic data from population biobanks for precise prevention.Peer reviewe