Geneetiline varieeruvus kui naisepoolse viljatuse eelsoodumuse mõjutaja ja võimalike uute biomarkerite allikas

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Üks tark mees on öelnud, et lapse saamine on üks ebatõenäoline protsess, ja arvestades seda, kui paljud paarid on viljatusega hädas, oli tal ilmselt õigus. Eestis on enamik naisepoolse viljatuse juhtudest tingitud peamiselt infektsioonide põhjustatud munajuhaviljatusest ja polütsüstiliste munasarjade sündroomist (PCOS), millele on iseloomulik meessuguhormoonide kõrgenenud tase ja insuliinresistentsus. Kunstlik viljastamine võib aidata sellistel juhtudel järglasi saada, kuid kahjuks on ravi tulemuslikkus ~30% ja sõltub suuresti sellest, kuidas munasarjad reageerivad ravi käigus kasutatavatele hormoonidele. Viljatuse molekulaarseid tagamaid on aastate jooksul palju uuritud ja on jõutud järeldusele, et individuaalne geneetiline varieeruvus mängib selle tekkes kindlasti olulist rolli. Geneetilise varieeruvuse uuringud võimaldavad leida ka biomarkereid, mis sobiksid reproduktiivpotentsiaali ja ehk isegi viljatusravi tulemuslikkuse ennustamiseks. Käesoleva töö üldine eesmärk oli hinnata seoseid erinevate geneetiliste variatsioonide ja PCOS-i ning munajuhaviljatuse vahel. Lisaks uurisime mitmeid menopausi algusajaga või munasarja funktsiooniga seotud geneetilisi variante, mis võiksid olla seotud reproduktiivpotentsiaali ja viljatusravi tulemuslikkuse parameetritega. Selle jaoks koguti tervete ja viljakusprobleemidega naiste DNA proovid ning analüüsiti 47 geneetilise variandi seost huvipakkuvate diagnooside ja kliiniliste tunnustega. Et teha kindlaks, kui suur osa munajuhaviljatuse juhtudest on tingitud urogenitaalsest klamüüdiainfektsioonist, määrati munajuhaviljatusega naiste vereseerumist klamüüdia-vastaste antikehade esinemine. Leidsime, et ~50% kõigist munajuhaviljatuse juhtudest võivad olla tingitud eelnevast klamüüdiainfektsioonist ja näitasime, et immuunvastust mõjutav mannoosi siduva lektiini geeni varieeruvus on ilmselt seotud munajuhaviljatuse geneetilise eelsoodumusega. Insuliini ja androgeeni retseptori geenide varieeruvus ei olnud seotud PCOS-iga. Lisaks näitasime, et menopausi algusaega mõjutavad geenivariandid on seotud munasarja funktsiooni ja viljatusravi tulemuslikkuse parameetritega, ja tulevikus võiks neid kombineerituna kliiniliste andmetega kaaluda võimalike biomarkeritena. Antud töö andis uut teavet naise viljakuse ja viljatuse geneetika kohta, kuid ühtlasi rõhutas edasiste uuringute vajalikkust.A wise man once said that the reproduction of mankind is a great marvel and mystery, and he was absolutely right, considering how many couples actually tackle with infertility. In Estonia, the majority of female infertility cases are due to tubal infertility (TFI) that is mostly caused by sexually transmitted infections, and polycystic ovary syndrome (PCOS), which is an endocrine disorder characterised by androgen excess and insulin resistance. In vitro fertilisation can help achieve pregnancy in these cases, but unfortunately the pregnancy rate per treatment cycle is only 30% and depends greatly on how the ovaries respond to the treatment. Over the years, great effort has been directed towards elucidating the molecular mechanisms behind the conditions causing infertility and it has been concluded that individual genetic variation is definitely one of the factors to blame. The studies of individual genetic variation form a good basis for finding biomarkers to predict natural reproductive function and perhaps even infertility treatment success. The general objective of the current thesis was to assess the associations between PCOS, TFI and selected candidate genes. In addition, several genetic variants that could be related to ovarian function and infertility treatment parameters were evaluated. Thus, DNA samples were collected from healthy women and infertile women and 47 genetic variants were studied in association with diagnoses of interest and different clinical parameters. In addition, to determine how many TFI cases are caused by a chlamydia infection, the prevalence of chlamydia-specific antibodies was measured in the blood sera of women with TFI. As a result, we found that ~50% of all TFI cases in Estonia might be caused by a previous chlamydia infection and identified genetic variants in the mannose binding lectin gene that modulate immune response and may be associated with susceptibility to TFI. No associations were found between selected variants in genes for insulin and androgen receptor and PCOS. However, we showed that genetic variants related to menopause timing are associated with ovarian function and infertility treatment parameters and could be considered as biomarker candidates if combined with clinical data. In conclusion, this study gave new knowledge about the genetics of female reproduction, but also highlighted the need for further research

2022. aasta inimgeneetikas

Eesti Arst 2023; 102(1):46–4

Whole exome sequencing of benign pulmonary metastasizing leiomyoma reveals mutation in the BMP8B gene

Background: Benign metastasizing leiomyoma (BML) is an orphan neoplasm commonly characterized by pulmonary metastases consisting of smooth muscle cells. Patients with BML have usually a current or previous uterine leiomyoma, which is therefore suggested to be the most probable source of this tumour. The purpose of this case report was to determine the possible genetic grounds for pulmonary BML. Case presentation: We present a case report in an asymptomatic 44-year-old female patient, who has developed uterine leiomyoma with subsequent pulmonary BML. Whole exome sequencing (WES) was used to detect somatic mutations in BML lesion. Somatic single nucleotide mutations were identified by comparing the WES data between the pulmonary metastasis and blood sample of the same BML patient. One heterozygous somatic mutation was selected for validation by Sanger sequencing. Clonality of the pulmonary metastasis and uterine leiomyoma was assessed by X-chromosome inactivation assay. Conclusions: We describe a potentially deleterious somatic heterozygous mutation in bone morphogenetic protein 8B (BMP8B) gene (c.1139A > G, Tyr380Cys) that was identified in the pulmonary metastasis and was absent from blood and uterine leiomyoma, and may play a facilitating role in the metastasizing of BML. The clonality assay confirmed a skewed pattern of X-chromosome inactivation, suggesting monoclonal origin of the pulmonary metastases.Peer reviewe

Endometrioos – anamneesist diagnoosini

Uuringu eesmärk. Töö eesmärgiks oli anda ülevaade aastatel 2005–2008 endometrioosile viitavate kaebustega naistearsti vastuvõtule pöördunud ning diagnostilise laparoskoopia/laparotoomia läbinud patsientide küsitluslehtede andmetest ning leida võimalikke endometrioosiga seotud kaebuseid ja riskitegureid. Metoodika. Retrospektiivne uuring, mille valimi moodustasid TÜ Kliinikumi naistekliinikus laparoskoopia/laparotoomia läbinud 170 naist, kellest 118-l (69,4%) diagnoositi endometrioos ja 52-l (30,6%) haigust ei tuvastatud. Patsiendid, kellel operatsiooni käigus endometrioosi ei tuvastatud, moodustasid edasistes analüüsides kontrollrühma. Tulemused ja järeldused. Uuringus osalenud endometrioosi ja kontrollrühma naiste üld- ja reproduktiivanamneesis olulisi erinevusi ei leitud, kuid endometrioosiga patsiendid kaebasid mõnevõrra sagedamini kogu menstruatsiooni kestel esinevaid alakõhuvalusid ning menstruatsiooniaegset valulikkust või veritsust defekatsioonil. Kontrollrühma naised olid sagedamini suitsetajad (32,7% vs 17,8%, p = 0,02) ning olid rohkem põdenud sugulisel teel levivaid haigusi (48,1% vs 25,4%, p = 0,01). Võrreldes endometrioosi mõõduka-raske vormiga esines minimaalse-kerge vormi korral sagedamini viljatust (vastavalt 34,7% ja 76,8%; p vs 27,5%; p = 0,02). Kokkuvõttes leidsime, et kogutud anamneesi ja kaebuste alusel on raske ennustada endometrioosi esinemist ja raskusastet. Diagnoos kinnitatakse vaid laparoskoopilise ja histoloogilise leiu põhjal. Endometrioosi kergemad vormid on aga seotud naise reproduktiivfunktsiooni häiretega – viljatuse ja spontaanabortidega. Eesti Arst 2011; 90(7):321–32

Leveraging Northern European population history : novel low-frequency variants for polycystic ovary syndrome

STUDY QUESTION Can we identify novel variants associated with polycystic ovary syndrome (PCOS) by leveraging the unique population history of Northern Europe? SUMMARY ANSWER We identified three novel genome-wide significant associations with PCOS, with two putative independent causal variants in the checkpoint kinase 2 (CHEK2) gene and a third in myosin X (MYO10). WHAT IS KNOWN ALREADY PCOS is a common, complex disorder with unknown aetiology. While previous genome-wide association studies (GWAS) have mapped several loci associated with PCOS, the analysis of populations with unique population history and genetic makeup has the potential to uncover new low-frequency variants with larger effects. STUDY DESIGN, SIZE, DURATION A population-based case-control GWAS was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS We identified PCOS cases from national registers by ICD codes (ICD-10 E28.2, ICD-9 256.4, or ICD-8 256.90), and all remaining women were considered controls. We then conducted a three-stage case-control GWAS: in the discovery phase, we had a total of 797 cases and 140 558 controls from the FinnGen study. For validation, we used an independent dataset from the Estonian Biobank, including 2812 cases and 89 230 controls. Finally, we performed a joint meta-analysis of 3609 cases and 229 788 controls from both cohorts. Additionally, we reran the association analyses including BMI as a covariate, with 2169 cases and 160 321 controls from both cohorts. MAIN RESULTS AND THE ROLE OF CHANCE Two out of the three novel genome-wide significant variants associating with PCOS, rs145598156 (P = 3.6x10(-8), odds ratio (OR) = 3.01 [2.02-4.50] minor allele frequency (MAF) = 0.005) and rs182075939 (P = 1.9x10(-16), OR = 1.69 [1.49-1.91], MAF = 0.04), were found to be enriched in the Finnish and Estonian populations and are tightly linked to a deletion c.1100delC (r(2) = 0.95) and a missense I157T (r(2) = 0.83) in CHEK2. The third novel association is a common variant near MYO10 (rs9312937, P = 1.7 x 10(-8), OR = 1.16 [1.10-1.23], MAF = 0.44). We also replicated four previous reported associations near the genes Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4), DENN Domain Containing 1A (DENND1A), FSH Subunit Beta (FSHB) and Zinc Finger And BTB Domain Containing 16 (ZBTB16). When adding BMI as a covariate only one of the novel variants remained genome-wide significant in the meta-analysis (the EstBB lead signal in CHEK2 rs182075939, P = 1.9x10(-16), OR = 1.74 [1.5-2.01]) possibly owing to reduced sample size. LARGE SCALE DATA The age- and BMI-adjusted GWAS meta-analysis summary statistics are available for download from the GWAS Catalog with accession numbers GCST90044902 and GCST90044903. LIMITATIONS, REASONS FOR CAUTION The main limitation was the low prevalence of PCOS in registers; however, the ones with the diagnosis most likely represent the most severe cases. Also, BMI data were not available for all (63% for FinnGen, 76% for EstBB), and the biobank setting limited the accessibility of PCOS phenotypes and laboratory values. WIDER IMPLICATIONS OF THE FINDINGS This study encourages the use of isolated populations to perform genetic association studies for the identification of rare variants contributing to the genetic landscape of complex diseases such as PCOS. STUDY FUNDING/COMPETING INTEREST(S) This work has received funding from the European Union's Horizon 2020 research and innovation programme under the MATER Marie Sklodowska-Curie grant agreement No. 813707 (N.P.-G., T.L., T.P.), the Estonian Research Council grant (PRG687, T.L.), the Academy of Finland grants 315921 (T.P.), 321763 (T.P.), 297338 (J.K.), 307247 (J.K.), 344695 (H.L.), Novo Nordisk Foundation grant NNF17OC0026062 (J.K.), the Sigrid Juselius Foundation project grants (T.L., J.K., T.P.), Finska Lakaresallskapet (H.L.) and Jane and Aatos Erkko Foundation (H.L.). The funders had no role in study design, data collection and analysis, publishing or preparation of the manuscript. The authors declare no conflicts of interest.Peer reviewe

DNA methylation changes in endometrium and correlation with gene expression during the transition from pre-receptive to receptive phase

The inner uterine lining (endometrium) is a unique tissue going through remarkable changes each menstrual cycle. Endometrium has its characteristic DNA methylation profile, although not much is known about the endometrial methylome changes throughout the menstrual cycle. The impact of methylome changes on gene expression and thereby on the function of the tissue, including establishing receptivity to implanting embryo, is also unclear. Therefore, this study used genome-wide technologies to characterize the methylome and the correlation between DNA methylation and gene expression in endometrial biopsies collected from 17 healthy fertile-aged women from pre-receptive and receptive phase within one menstrual cycle. Our study showed that the overall methylome remains relatively stable during this stage of the menstrual cycle, with small-scale changes affecting 5% of the studied CpG sites (22,272 out of studied 437,022 CpGs, FDR <0.05). Of differentially methylated CpG sites with the largest absolute changes in methylation level, approximately 30% correlated with gene expression measured by RNA sequencing, with negative correlations being more common in 5 ' UTR and positive correlations in the gene 'Body' region. According to our results, extracellular matrix organization and immune response are the pathways most affected by methylation changes during the transition from pre-receptive to receptive phase.Peer reviewe

Uniting biobank resources reveals novel genetic pathways modulating susceptibility for atopic dermatitis

Publisher Copyright: © 2021 The AuthorsBackground: Atopic dermatitis (AD) is a common chronic inflammatory skin disease with high heritability. Previous genome-wide association studies have identified several loci predisposing to AD. These findings explain approximately 30% of the variance in AD susceptibility, suggesting that further work is required to fully understand the genetic underpinnings. Objective: We sought to gain additional understanding of the genetic contribution to AD risk by using biobank resources. Methods: We completed a genome-wide meta-analysis of AD in 796,661 individuals (Ncases = 22,474) from the FinnGen study, the Estonian Biobank, and the UK Biobank. We further performed downstream in silico analyses to characterize the risk variants at the novel loci. Results: We report 30 loci associating with AD (P < 5 × 10−8), 5 of which are novel. In 2 of the novel loci, we identified missense mutations with deleterious predictions in desmocollin 1 and serpin family B member 7, genes encoding proteins crucial to epidermal strength and integrity. Conclusions: These findings elucidate novel genetic pathways involved in AD pathophysiology. The likely involvement of desmocollin 1 and serpin family B member 7 in AD pathogenesis may offer opportunities for the development of novel treatment strategies for AD in the future.Peer reviewe

Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity

STUDY QUESTION Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? SUMMARY ANSWER The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. WHAT IS ALREADY KNOWN Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. STUDY DESIGN, SIZE, DURATION Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. PARTICIPANTS/MATERIAL, SETTING, METHODS Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23–36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. MAIN RESULTS AND THE ROLE OF CHANCE The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10−6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. LARGE-SCALE DATA The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. LIMITATIONS REASONS FOR CAUTION Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. WIDER IMPLICATIONS OF THE FINDINGS The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial ‘omics’ studies, where tissue heterogeneity could potentially influence the results. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests

Uniting biobank resources reveals novel genetic pathways modulating susceptibility for atopic dermatitis

Publisher Copyright: © 2021 The AuthorsBackground: Atopic dermatitis (AD) is a common chronic inflammatory skin disease with high heritability. Previous genome-wide association studies have identified several loci predisposing to AD. These findings explain approximately 30% of the variance in AD susceptibility, suggesting that further work is required to fully understand the genetic underpinnings. Objective: We sought to gain additional understanding of the genetic contribution to AD risk by using biobank resources. Methods: We completed a genome-wide meta-analysis of AD in 796,661 individuals (Ncases = 22,474) from the FinnGen study, the Estonian Biobank, and the UK Biobank. We further performed downstream in silico analyses to characterize the risk variants at the novel loci. Results: We report 30 loci associating with AD (P < 5 × 10−8), 5 of which are novel. In 2 of the novel loci, we identified missense mutations with deleterious predictions in desmocollin 1 and serpin family B member 7, genes encoding proteins crucial to epidermal strength and integrity. Conclusions: These findings elucidate novel genetic pathways involved in AD pathophysiology. The likely involvement of desmocollin 1 and serpin family B member 7 in AD pathogenesis may offer opportunities for the development of novel treatment strategies for AD in the future.Peer reviewe