Whole Exome Sequencing in a Random Sample of North American Women with Leiomyomas Identifies MED12 Mutations in Majority of Uterine Leiomyomas

et al. (2012) Whole Exome Sequencing in a Random Sample of North American Women with Leiomyomas Identifies MED12 Mutations in Majority of Uterine Leiomyomas. PLoS ONE 7(3): e33251. doi:10.1371/journal.pone.0033251 Whole Exome Sequencing in a Random Sample of North American Women with Leiomyomas Identifies MED12 Mutations in Majority of Uterine Leiomyomas Megan M. McGuire 0 1 Alexander Yatsenko 0 1 Lori Hoffner 0 1 Mirka Jones 0 1 Urvashi Surti 0 1 Aleksandar 0 1 Noam Shomron, Tel Aviv University, Israel 0 Patient information This study was approved by the Institutional Review Board of the University of Pittsburgh as an exempt study (Pittsburgh , PA 1 1 Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Gynecologic Pathology, Magee-Womens Hospital of UPMC , Pittsburgh, Pennsylvania , United States of America Uterine leiomyomas (uterine fibroids) arise from smooth muscle tissue in the majority of women by age 45. It is common for these clonal tumors to develop from multiple locations within the uterus, leading to a variety of symptoms such as pelvic pain, abnormal uterine bleeding, and infertility. We performed whole exome sequencing on genomic DNA from five pairs of leiomyomas and corresponding normal myometrium to determine genetic variations unique to leiomyomas. Whole exome sequencing revealed that the gene encoding transcription factor MED12 (Mediator complex subunit 12) harbored heterozygous missense mutations caused by single nucleotide variants in highly conserved codon 44 of exon 2 in two of five leiomyomas. Sanger re-sequencing of MED12 among these five leiomyomas confirmed the two single nucleotide variants and detected a 42 base-pair deletion within exon 2 of MED12 in a third leiomyoma. MED12 was sequenced in an additional 143 leiomyomas and 73 normal myometrial tissues. Overall, MED12 was mutated in 100/148 (67%) of the genotyped leiomyomas: 79/148 (53%) leiomyomas exhibited heterozygous missense single nucleotide variants, 17/148 (11%) leiomyomas exhibited heterozygous in-frame deletions/insertion-deletions, 2/148 (1%) leiomyomas exhibited intronic heterozygous single nucleotide variants affecting splicing, and 2/148 (1%) leiomyomas exhibited heterozygous deletions/ insertion-deletions spanning the intron 1-exon 2 boundary which affected the splice acceptor site. Mutations were not detected in MED12 in normal myometrial tissue. MED12 mutations were equally distributed among karyotypically normal and abnormal uterine leiomyomas and were identified in leiomyomas from both black and white American women. Our studies show an association between MED12 mutations and leiomyomas in ethnically and racially diverse American women. - Funding: Magee-Womens Research Institute and Foundation provided the funding that has supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Uterine leiomyomas, better known as fibroid tumors, are clinically apparent in nearly 25% of women by age 45, and they cause major morbidity in American women. More than 200,000 surgeries are performed each year to either remove the leiomyomatous tumors (myomectomy) or the entire uterus (hysterectomy) [1,2]. These tumors are responsive to steroid hormones such as estrogen and progesterone, and they often shrink following menopause [3]. Approximately half of all leiomyomas are asymptomatic, while the rest cause pelvic pressure and pain, menometrorrhagia, anemia, premature labor and infertility. These symptoms are intensified by the common occurrence of multiple tumors within a single uterus, often necessitating surgical intervention [4]. Previous genetic analyses of leiomyomas have established that approximately one half of these tumors have an abnormal karyotype, while the other half are karyotypically normal. We recently showed that a subset of karyotypically normal leiomyomas contained microdeletions and microduplications, but there is still a large proportion of karyotypically normal leiomyomas that have no known lesions [5]. In karyotypically abnormal leiomyomas, cytogenetic aberrations commonly include deletions in 7q, trisomy of chromosome 12, and various translocations between chromosomes 12 and 14 involving the high mobility group AT-hook 2 (HMGA2) gene at 12q15, which encodes a transcriptional regulator [6,7,8,9]. Cytogenetic abnormalities are likely a reflection of general genomic instability as is true for other tumors. Mutations in leiomyomas that cause such genomic instabilities are unknown. Furthermore, heterozygous germline mutations in fumarate hydratase (FH) can cause a rare disorder of hereditary leiomyomatosis and renal cell carcinoma (HLRCC [MIM 150800]) [10,11]. However, fumarate hydratase does not appear to play an important role in the non-syndromic forms of uterine leiomyomas [12]. IRB# PRO11120169), and informed consent was not obtained. All leiomyoma and myometrial samples were derived from specimens that were originally obtained for clinical treatment or pathology purposes only, and specimens did not contain any personal identifiers or linkage codes. Samples were collected from women who were diagnosed with uterine leiomyomas and underwent medically indicated abdominal hysterectomy. Chromosome analysis was performed using standard G-banding technique. Whole exome sequencing on the initial five pairs of samples from five different individuals was performed on randomly selected, karyotypically normal leiomyomas. Subsequent DNA sequencing was performed on 143 randomly selected leiomyomas from our biobank of more than 500 samples. The histology of all samples was reviewed by a board certified gynecologic pathologist. Nucleic acid extraction and preparation Genomic DNAs from all leiomyomas and corresponding myometrial samples were extracted from 100 mg of freshly frozen tissue using the Gentra Puregene Blood Kit (QIAGEN, Valencia, CA, USA), according to the manufacturers protocol. RNA was extracted from a randomly selected group of leiomyoma samples which harbored MED12 mutations. Tissue samples stored at 280uC were placed in chilled RNAlaterH-ICE (Invitrogen, Carlsbad, CA, USA) for at least 16 hrs at 220uC to allow for tissue thawing under preservative conditions prior to RNA isolation. TRIzolH Reagent (Invitrogen, Carlsbad, CA, USA) was used to carry out RNA isolations according to the manufacturers protocol. RNA samples were converted to cDNA using SuperScriptH III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), according to the manufacturers protocol. Whole exome capture and DNA sequencing Ten genomic DNA samples (five leiomyomas and five corresponding myometrial samples) were subjected to in-solution exome enrichment via the SureSelect (...truncated)