MED12 : a novel player in uterine leiomyomas

Uterine leiomyomas, or fibroids, are benign tumors arising from the smooth muscle lining of the uterus, the myometrium. Although they represent one of the most common neoplasms in women with an estimated prevalence of 20-40% during the reproductive years, the molecular mechanisms underlying their tumorigenesis have remained relatively unknown. The aim of this thesis was to elucidate the molecular genetic characteristics of uterine leiomyomas using next-generation sequencing technology. Exome sequencing of 18 uterine leiomyomas and the respective normal myometrium from 17 Finnish (Caucasian) patients led to the identification of recurrent somatic mutations in mediator complex subunit 12 (MED12) gene. This, and further Sanger sequencing of 207 additional leiomyomas revealed that a remarkable 70% (159/225) of the tumors harbor MED12 mutations. MED12 is a component of the Mediator complex, which participates in the regulation of global as well as gene-specific transcription. All the observed mutations resided in exon 2 or the intron 1-exon 2 junction, an evolutionarily conserved region of the gene, suggesting that malfunction of the region contributes to tumorigenesis. This was the first time MED12 mutations have been implicated in human tumorigenesis. To validate the finding and determine the frequency of MED12 exon 2 mutations in another ethnic group, a series of 28 uterine leiomyomas from 18 South African patients underwent Sanger sequencing for the mutations. Altogether 50% (14/28) of the tumors displayed a mutation, indicating that MED12 mutations occur frequently also in uterine leiomyomas of South African women. Overall, the result confirms the role of these mutations in the growth and development of leiomyomas regardless of ethnicity. Original identification of MED12 exon 2 mutations took place in a series of histopathologically conventional uterine leiomyomas which account for approximately 90% of the tumors. To assess the frequency of MED12 mutations in rarer clinical leiomyoma subtypes, 103 histopathological uterine leiomyoma variants, as well as 34 uterine leiomyomas from 14 patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome, a disease caused by heterozygous germline mutations in fumarate hydratase (FH) gene, entered the study. Both the histopathological leiomyoma variants (17%; 18/103) and leiomyomas from HLRCC-patients (9%; 3/34) harbored MED12 mutations significantly less frequently than conventional leiomyomas (P less than 0.001), proposing that MED12 mutation positivity is a key characteristic of conventional leiomyomas. Of note, none of the MED12 mutation-positive tumors from HLRCC-patients displayed biallelic FH inactivation, a well-known attribute of tumors in HLRCC, suggesting that MED12 mutations and biallelic FH inactivation may be mutually exclusive. Exome sequencing of 27 uterine leiomyomas (12 MED12 mutation-negative and 15 MED12 mutation-positive) and their corresponding normal myometrium revealed no additional recurrent somatic mutations in either MED12 mutation-negative or -positive tumors. The result emphasizes the significance of MED12 mutations for the tumorigenesis of uterine leiomyomas. Alterations undetectable by exome sequencing, such as structural rearrangements, intronic variants, and epigenetic events, probably have an impact to the development of MED12 mutation-negative lesions. The discovery of MED12 mutations is a giant step forward in understanding the pathogenesis of uterine leiomyomas, hopefully leading to improved diagnosis, personalized medical treatments, and prognosis in the future.Kohdun hyvänlaatuiset sileälihaskasvaimet, eli myoomat, ovat naisten yleisimpiä kasvaimia, joiden esiintyvyys hedelmällisessä iässä on arviolta 20-40 %. Kasvainten yleisyydestä huolimatta niiden syntyyn ja kehitykseen vaikuttavista tekijöistä on tiedetty varsin vähän. Tämän väitöskirjatyön tavoitteena oli kartoittaa kohdun myoomien molekyyligeneettistä taustaa hyödyntämällä uuden sukupolven sekvensointiteknologiaa. Sekvensoimalla kaikki ihmisen genomin proteiinia koodaavat alueet ( eksomisekvensointi ) 18:sta suomalaisten potilaiden kohdun myoomasta ja niitä vastaavasta normaalikudosnäytteestä tunnistimme toistuvia somaattisia geenivirheitä eli mutaatioita geenissä nimeltä MED12 (mediator complex subunit 12). Tulokset vahvistettiin suuremmassa, yhteensä 207 myooman näytejoukossa, mikä paljasti mutaatioita kyseisessä geenissä jopa 70 %:lla tutkituista kasvaimista. MED12-geenin tuottama proteiini on osa suurempaa proteiinikompleksia, joka osallistuu transkription säätelyyn soluissa. Kaikki havaitut mutaatiot sijaitsivat hyvin kapealla, evolutiivisesti konservoituneella alueella geenissä viitaten siihen, että tämän alueen häiriintynyt toiminta on mahdollisesti keskeisessä asemassa kasvainten kehittymisen kannalta. Tämä oli ensimmäinen kerta, kun MED12-mutaatiot on yhdistetty ihmisen kasvainten kehittymiseen. Osoitimme lisäksi MED12-mutaatioiden olevan yleisiä myös eteläafrikkalaisten naisten kohdun myoomissa (50 %), mikä vahvistaa mutaatioiden merkitystä kasvainten synnyssä ja kehityksessä etnisyydestä riippumatta. MED12-mutaatioiden frekvenssiä tarkasteltiin myös harvinaisemmissa myooma-alaluokissa, jotka vastaavat noin 10 % kaikista kohdun myoomista. Tutkimus käsitti yhteensä 103 histopatologialtaan poikkeavaa myoomaa sekä 34 myoomaa perinnöllistä leiomyomatoosi ja munuaissyöpä-oireyhtymää (hereditary leiomyomatosis and renal cell cancer, HLRCC) sairastavilta potilailta. MED12-mutaatioita havaittiin sekä histopatologisissa myooma-alatyypeissä että HLRCC-potilaiden myoomissa merkittävästi vähemmän kuin yleisissä myoomissa. Tulosten perusteella mutaatiot näyttäisivät olevan ominaisia nimenomaan yleisille myoomille. Eksomisekvensoimalla 27 kohdun yleistä myoomaa (12 MED12-mutaationegatiivista ja 15 MED12-mutaatiopositiivista) ja niitä vastaavaa normaalikudosnäytettä emme tunnistaneet muita toistuvasti mutatoituneita geenejä kasvaimissa. Tulos korostaa entisestään MED12-mutaatioiden roolia kohdun myoomien kehityksessä. MED12-mutaationegatiivisten myoomien taustalla saattaa esiintyä tekijöitä, joita ei voida eksomisekvensoinnilla havaita, kuten kromosomien rakenteelliset tai geenien introniset muutokset. MED12-mutaatioiden löytyminen on tärkeä edistysaskel kohti kohdun myoomien synnyn ja kehityksen ymmärtämistä sekä niiden taustalla piileviä mekanismeja. Uusi tieto myoomien kehitykseen vaikuttavista molekyyligeneettisistä tekijöistä saattaa tulevaisuudessa johtaa kasvainten parantuneeseen diagnostiikkaan, prognoosiin sekä yksilöityihin lääkehoitoihin

Transcription of lignocellulose-decomposition associated genes, enzyme activities and production of ethanol upon bioconversion of waste substrate by Phlebia radiata

Previously identified twelve plant cell wall degradation-associated genes of the white rot fungus Phlebia radiata were studied by RT-qPCR in semi-aerobic solid-state cultures on lignocellulose waste material, and on glucose-containing reference medium. Wood-decay-involved enzyme activities and ethanol production were followed to elucidate both the degradative and fermentative processes. On the waste lignocellulose substrate, P. radiata carbohydrate-active enzyme (CAZy) genes encoding cellulolytic and hemicellulolytic activities were significantly upregulated whereas genes involved in lignin modification displayed a more complex response. Two lignin peroxidase genes were differentially expressed on waste lignocellulose compared to glucose medium, whereas three manganese peroxidase-encoding genes were less affected. On the contrary, highly significant difference was noticed for three cellulolytic genes (cbhI_1, eg1, bgl1) with higher expression levels on the lignocellulose substrate than on glucose. This indicates expression of the wood-attacking degradative enzyme system by the fungus also on the recycled, waste core board material. During the second week of cultivation, ethanol production increased on the core board to 0.24 g/L, and extracellular activities against cellulose, xylan, and lignin were detected. Sugar release from the solid lignocellulose resulted with concomitant accumulation of ethanol as fermentation product. Our findings confirm that the fungus activates its white rot decay system also on industrially processed lignocellulose adopted as growth substrate, and under semi-aerobic cultivation conditions. Thus, P. radiata is a good candidate for lignocellulose-based renewable biotechnology to make biofuels and biocompounds from materials with less value for recycling or manufacturing.Peer reviewe

Loss of ATRX/DAXX expression and alternative lengthening of telomeres in uterine leiomyomas

Background Uterine leiomyomas (ULs) are the most common gynecologic tumors and affect 3 of every 4 women by the age of 50 years. The majority of ULs are classified as conventional tumors, whereas 10% represent various histopathological subtypes with features that mimic malignancy. These subtypes include cellular and mitotically active ULs and ULs with bizarre nuclei. Uterine leiomyosarcoma (ULMS), the malignant counterpart of UL, is an aggressive cancer with poor overall survival. The early diagnosis and preoperative differentiation of ULMS from UL are often challenging because their symptoms and morphology resemble one another. Recent studies have shown frequent loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX) expression in ULMS, and this is often associated with an alternative lengthening of telomeres (ALT) phenotype. Methods To investigate ATRX and DAXX expression and the presence of ALT in UL subtypes, immunohistochemical and telomere-specific fluorescence in situ hybridization analyses were performed. The study material consisted of 142 formalin-fixed, paraffin-embedded tissue samples representing various UL subtypes and 64 conventional ULs. Results A loss of ATRX or DAXX and/or ALT was detected in 6.3% of the histopathological UL subtype samples (9 of 142). Two patients whose ULs showed either ATRX loss or ALT were later diagnosed with a pulmonary smooth muscle tumor. Pulmonary tumors displayed molecular alterations found in the corresponding uterine tumors, which indicated metastasis to the lungs. All conventional ULs displayed normal ATRX, DAXX, and telomeres. Conclusions These results highlight the differences between conventional and histopathologically atypical ULs and indicate that some UL subtype tumors may harbor long-term malignant potential. Cancer 2018;124:4650-4656. (C) 2018 American Cancer Society.Peer reviewe

Comparison of 2SC, AKR1B10, and FH Antibodies as Potential Biomarkers for FH-deficient Uterine Leiomyomas

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome caused by germline fumarate hydratase (FH) mutations and characterized by uterine and cutaneous leiomyomas and renal cell cancer. Currently, there is no generally approved method to differentiate FH-deficient uterine leiomyomas from other leiomyomas. Here, we analyzed 3 antibodies (S-(2-succino)-cysteine [2SC], aldo-keto reductase family 1, member B10 [AKR1B10], and FH) as potential biomarkers. The study consisted of 2 sample series. The first series included 155 formalin-fixed paraffin-embedded uterine leiomyomas, of which 90 were from HLRCC patients and 65 were sporadic. The second series included 1590 unselected fresh frozen leiomyomas. Twenty-seven tumors were from known HLRCC patients, while the FH status for the remaining 1563 tumors has been determined by copy number analysis and Sanger sequencing revealing 45 tumors with monoallelic (n=33) or biallelic (n=12) FH loss. Altogether 197 samples were included in immunohistochemical analyses: all 155 samples from series 1 and 42 available corresponding formalin-fixed paraffin-embedded samples from series 2 (15 tumors with monoallelic and 7 with biallelic FH loss, 20 with no FH deletion). Results show that 2SC performed best with 100% sensitivity and specificity. Scoring was straightforward with unambiguously positive or negative results. AKR1B10 identified most tumors accurately with 100% sensitivity and 99% specificity. FH was 100% specific but showed slightly reduced 91% sensitivity. Both FH and AKR1B10 displayed also intermediate staining intensities. We suggest that when patient's medical history and/or histopathologic tumor characteristics indicate potential FH-deficiency, the tumor's FH status is determined by 2SC staining. When aberrant staining is observed, the patient can be directed to genetic counseling and mutation screening.Peer reviewe

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5 end nonsense mutation (c.97G > T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin- and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD. (C) 2017 Wiley Periodicals, Inc.Peer reviewe

Exome and immune cell score analyses reveal great variation within synchronous primary colorectal cancers

BACKGROUND: Approximately 4% of colorectal cancer (CRC) patients have at least two simultaneous cancers in the colon. Due to the shared environment, these synchronous CRCs (SCRCs) provide a unique setting to study colorectal carcinogenesis. Understanding whether these tumours are genetically similar or distinct is essential when designing therapeutic approaches. METHODS: We performed exome sequencing of 47 primary cancers and corresponding normal samples from 23 patients. Additionally, we carried out a comprehensive mutational signature analysis to assess whether tumours had undergone similar mutational processes and the first immune cell score analysis (IS) of SCRC to analyse the interplay between immune cell invasion and mutation profile in both lesions of an individual. RESULTS: The tumour pairs shared only few mutations, favouring different mutations in known CRC genes and signalling pathways and displayed variation in their signature content. Two tumour pairs had discordant mismatch repair statuses. In majority of the pairs, IS varied between primaries. Differences were not explained by any clinicopathological variable or mutation burden. CONCLUSIONS: The study shows major diversity within SCRCs. Rather than rely on data from one tumour, our study highlights the need to evaluate both tumours of a synchronous pair for optimised targeted therapy.Peer reviewe

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility(1), and are a common cause of hysterectomy(2). They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA2(3). Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex(4), and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice(5). Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.Peer reviewe

Exome Sequencing of Uterine Leiomyosarcomas Identifies Frequent Mutations in TP53, ATRX, and MED12

Uterine leiomyosarcomas (ULMSs) are aggressive smooth muscle tumors associated with poor clinical outcome. Despite previous cytogenetic and molecular studies, their molecular background has remained elusive. To examine somatic variation in ULMS, we performed exome sequencing on 19 tumors. Altogether, 43 genes were mutated in at least two ULMSs. Most frequently mutated genes included tumor protein P53 (TP53; 6/19; 33%), alpha thalassemia/mental retardation syndrome X-linked (ATRX; 5/19; 26%), and mediator complex subunit 12 (MED12; 4/19; 21%). Unlike ATRX mutations, both TP53 and MED12 alterations have repeatedly been associated with ULMSs. All the observed ATRX alterations were either nonsense or frameshift mutations. ATRX protein levels were reliably analyzed by immunohistochemistry in altogether 44 ULMSs, and the majority of tumors (23/44; 52%) showed clearly reduced expression. Loss of ATRX expression has been associated with alternative lengthening of telomeres (ALT), and thus the telomere length was analyzed with telomere-specific fluorescence in situ hybridization. The ALT phenotype was confirmed in all ULMSs showing diminished ATRX expression. Exome data also revealed one nonsense mutation in death-domain associated protein (DAXX), another gene previously associated with ALT, and the tumor showed ALT positivity. In conclusion, exome sequencing revealed that TP53, ATRX, and MED12 are frequently mutated in ULMSs. ALT phenotype was commonly seen in tumors, indicating that ATR inhibitors, which were recently suggested as possible new drugs for ATRX-deficient tumors, could provide a potential novel therapeutic option for ULMS.Peer reviewe

Uterine Leiomyoma-Linked MED12 Mutations Disrupt Mediator-Associated CDK Activity

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