Outi Uimari

Genome-wide genetic analyses highlight mitogen-activated protein kinase (MAPK) signaling in the pathogenesis of endometriosis.

Abstract STUDY QUESTION Do genome-wide association study (GWAS) data for endometriosis provide insight into novel biological pathways associated with its pathogenesis? SUMMARY ANSWER GWAS analysis uncovered multiple pathways that are statistically enriched for genetic association signals, analysis of Stage A disease highlighted a novel variant in MAP3K4, while top pathways significantly associated with all endometriosis and Stage A disease included several mitogen-activated protein kinase (MAPK)-related pathways. WHAT IS KNOWN ALREADY Endometriosis is a complex disease with an estimated heritability of 50%. To date, GWAS revealed 10 genomic regions associated with endometriosis, explaining <4% of heritability, while half of the heritability is estimated to be due to common risk variants. Pathway analyses combine the evidence of single variants into gene-based measures, leveraging the aggregate effect of variants in genes and uncovering biological pathways involved in disease pathogenesis. STUDY DESIGN, SIZE, DURATION Pathway analysis was conducted utilizing the International Endogene Consortium GWAS data, comprising 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry with genotype data imputed up to 1000 Genomes Phase three reference panel. GWAS was performed for all endometriosis cases and for Stage A (revised American Fertility Society (rAFS) I/II, n = 1686) and B (rAFS III/IV, n = 1364) cases separately. The identified significant pathways were compared with pathways previously investigated in the literature through candidate association studies. PARTICIPANTS/MATERIALS, SETTING, METHODS The most comprehensive biological pathway databases, MSigDB (including BioCarta, KEGG, PID, SA, SIG, ST and GO) and PANTHER were utilized to test for enrichment of genetic variants associated with endometriosis. Statistical enrichment analysis was performed using the MAGENTA (Meta-Analysis Gene-set Enrichment of variaNT Associations) software. MAIN RESULTS AND THE ROLE OF CHANCE The first genome-wide association analysis for Stage A endometriosis revealed a novel locus, rs144240142 (P = 6.45 × 10−8, OR = 1.71, 95% CI = 1.23–2.37), an intronic single-nucleotide polymorphism (SNP) within MAP3K4. This SNP was not associated with Stage B disease (P = 0.086). MAP3K4 was also shown to be differentially expressed in eutopic endometrium between Stage A endometriosis cases and controls (P = 3.8 × 10−4), but not with Stage B disease (P = 0.26). A total of 14 pathways enriched with genetic endometriosis associations were identified (false discovery rate (FDR)-P < 0.05). The pathways associated with any endometriosis were Grb2-Sos provides linkage to MAPK signaling for integrins pathway (P = 2.8 × 10−5, FDR-P = 3.0 × 10−3), Wnt signaling (P = 0.026, FDR-P = 0.026) and p130Cas linkage to MAPK signaling for integrins pathway (P = 6.0 × 10−4, FDR-P = 0.029); with Stage A endometriosis: extracellular signal-regulated kinase (ERK)1 ERK2 MAPK (P = 5.0 × 10−4, FDR-P = 5.0 × 10−4) and with Stage B endometriosis: two overlapping pathways that related to extracellular matrix biology—Core matrisome (P = 1.4 × 10−3, FDR-P = 0.013) and ECM glycoproteins (P = 1.8 × 10−3, FDR-P = 7.1 × 10−3). Genes arising from endometriosis candidate gene studies performed to date were enriched for Interleukin signaling pathway (P = 2.3 × 10−12), Apoptosis signaling pathway (P = 9.7 × 10−9) and Gonadotropin releasing hormone receptor pathway (P = 1.2 × 10−6); however, these pathways did not feature in the results based on GWAS data. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The analysis is restricted to (i) variants in/near genes that can be assigned to pathways, excluding intergenic variants; (ii) the gene-based pathway definition as registered in the databases; (iii) women of European ancestry. WIDER IMPLICATIONS OF THE FINDINGS The top ranked pathways associated with overall and Stage A endometriosis in particular involve integrin-mediated MAPK activation and intracellular ERK/MAPK acting downstream in the MAPK cascade, both acting in the control of cell division, gene expression, cell movement and survival. Other top enriched pathways in Stage B disease include ECM glycoprotein pathways important for extracellular structure and biochemical support. The results highlight the need for increased efforts to understand the functional role of these pathways in endometriosis pathogenesis, including the investigation of the biological effects of the genetic variants on downstream molecular processes in tissue relevant to endometriosis. Additionally, our results offer further support for the hypothesis of at least partially distinct causal pathophysiology for minimal/mild (rAFS I/II) vs. moderate/severe (rAFS III/IV) endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The genome-wide association data and Wellcome Trust Case Control Consortium (WTCCC) were generated through funding from the Wellcome Trust (WT084766/Z/08/Z, 076113 and 085475) and the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498). N.R. was funded by a grant from the Medical Research Council UK (MR/K011480/1). A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant WT098017). All authors declare there are no conflicts of interest.

Strong family history of uterine leiomyomatosis warrants fumarate hydratase mutation screening

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome characterized by cutaneous and uterine leiomyomas and renal cell cancer. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. A Finnish family with nine closely related women with uterine leiomyomas was detected by an alert gynecologist. No cutaneous or renal cell tumors were reported in the family when it was referred to genetic analyses. Samples were available from seven patients, and a novel germline FH mutation was detected in five of them. Mutation carriers were symptomatic, had multiple tumors and were diagnosed at an early age. This study emphasizes the importance of considering FH mutation screening when gynecologists encounter families with multiple severe uterine leiomyoma cases. Due to possibility of phenocopies more than one patient should be tested. Early mutation detection allows regular screening of the mutation carriers and enables early detection of possible highly aggressive renal tumors. It may also affect family planning as multiple myomas at early age may significantly reduce fertility.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas

Background:Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients’ myomagenesis.Methods:MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients’ (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA).Results:Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients’ MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours.Conclusions:Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients’ uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Do symptomatic endometriosis and uterine fibroids appear together

OBJECTIVES: Endometriosis and uterine fibroids are common gynecological disorders in fertile women. It has been suggested that these two disorders may be associated with each other. In this study, we tested whether this connection exists. In addition, we wanted to evaluate whether they both affect fertility independently of each other. MATERIALS AND METHODS: The prevalence of endometriosis and uterine fibroids was investigated in three groups of patients: Symptomatic patients requiring surgery either for endometriosis (n=182), or for uterine fibroids (n=240) and asymptomatic patients undergoing laparoscopic sterilization (n=183). The prevalences were examined in three age groups: 35–39 yrs, 40–44 yrs and ≥ 45 yrs. The significance of both diagnoses on fertility was assessed using logistic regression analysis. RESULTS: Uterine fibroids were detected in 25.8% (47/182) of patients with endometriosis. Endometriosis was detected in 19.6% (47/240) of patients with uterine fibroids. 5.5% (10/183) women undergoing sterilization had endometriosis and 19.3% (17/183) had uterine fibroids. Both uterine fibroids and endometriosis were, independently of each other, related to subfertility (OR, 95% CI: 3.8, 2.3–6.5; 6.8, 4.0–11.6, respectively). CONCLUSIONS: The results suggest that symptomatic endometriosis appears together with symptomatic uterine fibroids. Both diseases seem to decrease female fertility independently of each other.

Controlled ovarian hyperstimulation leads to high progesterone and estradiol levels during early pregnancy

STUDY QUESTIONnAre there differences in estrogen and progesterone secretion in singleton pregnancies, up to Week 11, between spontaneous pregnancies, after controlled ovarian hyperstimulation and fresh embryo transfer (COH + ET) and after frozen embryo transfer in a spontaneous cycle (FET)?nnnSUMMARY ANSWERnSerum progesterone and estradiol (E2) concentrations after COH + ET were higher in early pregnancy, lasting up to Week 7-8, than FET and spontaneous pregnancies, while hormone levels after FET did not differ from spontaneous pregnancies.nnnWHAT IS ALREADY KNOWNnThe risk of adverse perinatal outcomes after COH + ET seems to be increased when compared with spontaneous pregnancies. One of the reasons suggested for this is related to ovarian hyperstimulation.nnnSTUDY DESIGN, SIZE, DURATIONnThis was a prospective cohort study consisting of three different groups of pregnant women which were followed-up weekly until Week 11 of their pregnancies. The spontaneous pregnancy group consisted of 41 women, the COH + ET group consisted of 39 and the FET group consisted of 30 women.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWomen in the control group with spontaneous conception were recruited from local prenatal clinics. Women in the COH + ET and FET groups were recruited from the Reproductive Unit of Oulu University Hospital. At each visit, a three-dimensional ultrasonography was performed to examine the ovarian volumes and vascularization. A blood sample was drawn to analyse progesterone and E2 levels. The pregnancy outcome was included in the analysis.nnnMAIN RESULTS AND THE ROLE OF CHANCEnAt pregnancy Week 5, the serum progesterone levels were higher after the COH + ET (median 312, inter-quartile range 183-480 nmol/l), when compared with the spontaneous (63, 52-80 nmol/l; P < 0.001) and FET (74, 48-96 nmol/l; P < 0.001) pregnancies. At Week 11, the P (189, 124-260 nmol/l) was still higher in the COH + ET group (FET 101, 78-120 nmol/l, P < 0.001; spontaneous 115, 80-139 nmol/l, P < 0.01) than the other two groups. The E2 levels at Week 5 were also significantly higher after COH + ET (4.1, 2.2-6.6 nmol/l) than in the spontaneous pregnancies (1.1, 0.7-1.6 nmol/l, P < 0.001) or after FET (0.7, 0.6-0.9 nmol/l, P < 0.001). The volume of the ovaries and the intraovarian vasculature in the COH + ET group were significantly higher when compared with the other two groups (P < 0.001). The birthweight was negatively correlated with the serum P (R -0.340, P < 0.01) and E2 (R= -0.275, P < 0.05) in pregnancy Weeks 5-8. In the multivariate analysis evaluating the factors affecting birthweight of the newborn, the significant factors were the length of gestation, maternal height and progesterone or E2 secretion during Weeks 5-8.nnnLIMITATIONS, REASONS FOR CAUTIONnBecause of the low number of patients in this study, larger cohort studies are required to confirm the findings.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe findings here indicate that COH-induced increased luteal activity should be evaluated by measuring steroid levels or the ovarian size or vascularity, rather than number of oocytes retrieved. If unphysiologically high steroid activity during pregnancy after COH contributes to the risk of adverse perinatal outcomes after fresh embryo transfer, milder stimulation protocols or even freezing of all of the embryos should be considered.nnnSTUDY FUNDING/COMPETING INTERESTSnThis study was supported by a research grant from the Academy of Finland. The authors declare no conflicts of interest.

Genome-wide association analysis identifies 27 novel loci associated with uterine leiomyomata revealing common genetic origins with endometriosis

Uterine leiomyomata (UL), also known as uterine fibroids, are the most common neoplasms of the reproductive tract and the primary cause for hysterectomy, leading to considerable impact on women’s lives as well as high economic burden1,2. Genetic epidemiologic studies indicate that heritable risk factors contribute to UL pathogenesis3. Previous genome-wide association studies (GWAS) identified five loci associated with UL at genome-wide significance (P < 5 × 10−8)4–6. We conducted GWAS meta-analysis in 20,406 cases and 223,918 female controls of white European ancestry, identifying 24 genome-wide significant independent loci; 17 replicated in an unrelated cohort of 15,068 additional cases and 43,587 female controls. Aggregation of discovery and replication studies (35,474 cases and 267,505 female controls) revealed six additional significant loci. Interestingly, four of the 17 loci identified and replicated in these analyses have also been associated with risk for endometriosis – another common gynecologic disorder. These findings increase our understanding of the biological mechanisms underlying UL development, and suggest overlapping genetic origins with endometriosis.

Abstract 4800: Identification of candidate predisposition genes for familial uterine leiomyomas

Uterine leiomyomas are benign smooth muscle tumors that cause considerable morbidity. They are among the most common human tumors and are the leading indication for hysterectomy. Genetic factors play an important role in the development of these hormone-dependent tumors. First-degree relatives of uterine leiomyoma patients are at increased risk for developing leiomyomas. Twin studies have shown that an inherited predisposition is likely to contribute to the formation of these lesions. Furthermore, the clinical and molecular features of familial leiomyomas differ from those of sporadic cases. Thus far, the only known high risk predisposition defect is dominantly inherited loss of function mutations in fumarate hydratase (FH) that underlie a tumor predisposition syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The aim of this study is to identify novel candidate high risk susceptibility genes for uterine leiomyomas. The study material consists of five FH mutation-negative families, which have at least four individuals diagnosed with uterine leiomyomas, originating from Northern Finland. Genomic DNA extracted from peripheral blood lymphocytes from one affected individual from each family has been exome sequenced. Exome sequencing data have been analyzed for rare non-synonymous variants with allele frequencies less than 0.1% in 4 019 controls. All protein-truncating variants as well as missense and splice site variants affecting the same gene in at least two individuals that are predicted to be protein-damaging have been included for further analysis. Preliminarily 25 candidate susceptibility genes have been shortlisted. Relevant variant calls will be verified by direct sequencing, followed by segregation analysis and assessment of loss of heterozygosity in the corresponding tumor samples. Identification of novel genes predisposing to familial uterine leiomyomas may provide a better understanding into the tumorigenic mechanisms and could lead to new prevention approaches and personalized medical treatments in the future. Citation Format: Hanna-Riikka Heinonen, Outi Uimari, Jaana Tolvanen, Markku Ryynanen, Lauri A. Aaltonen, Pia Vahteristo. Identification of candidate predisposition genes for familial uterine leiomyomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2015-4800