MED12-geenin rooli kasvainten muodostumisessa

Mediator complex subunit 12 (MED12) is a component of the conserved Mediator complex and an important player in the regulation of general and gene specific transcription. Somatic MED12 mutations were associated with human tumorigenesis for the first time when exome sequencing of uterine leiomyomas identified highly specific mutations affecting exon 2 in as many as 70% of the tumors. Uterine leiomyomas are benign smooth muscle tumors estimated to affect up to 77% of reproductive-age women. Most of the tumors are sporadic, but uterine leiomyomas are also a feature of hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC), where biallelic inactivation of fumarate hydratase (FH) is driving the tumorigenesis. The overall aim of this thesis project was to take forward the finding of MED12 as a novel driver gene in myomagenesis and to analyze its role in other tumor types. Comprehensive MED12 exon 2 mutation screening in various tumor types identified mutations recurrently in uterine leiomyosarcoma and rarely in colorectal cancer. Findings demonstrate that these specific mutations are not restricted to benign tumors and suggest a possibility of some leiomyosarcomas to develop via benign leiomyoma precursor. Mutation analysis of MED12 exon 1 from tumor types where mutations in exon 2 have previously been reported revealed small in-frame deletions only in conventional uterine leiomyomas. Mutations in exon 1 led to similar global expression profile and mechanistic effects as previously observed with exon 2 mutations further emphasizing the role of MED12 in leiomyomagenesis. Assessment of the FH status and MED12 exon 1/2 mutation screening confirmed that biallelic FH inactivation and MED12 mutations are mutually exclusive both within HLRCC syndrome-associated and sporadic uterine leiomyomas. These results and the gene expression profiling of the tumors show that there are at least two distinct molecular mechanisms behind the development of uterine leiomyomas in HLRCC patients: biallelic FH inactivation and somatic MED12 mutations. A systematic database search identified few MED12 mutations affecting the leiomyoma-linked mutation hotspots in chronic lymphocytic leukemia (CLL), the most common form of leukemia in adults. Mutation screening of more than 700 samples revealed CLL as the first extrauterine cancer where specific MED12 exon 1 and 2 mutations have been observed at significant frequency. Furthermore, MED12 mutations associated with unmutated status of immunoglobulin heavy chain variable genes and elevated expression of 70 kD zeta-associated protein; well-characterized markers of poor prognosis in CLL. Functional analysis of the unusual MED12 exon 1 nonsense mutation identified in a patient with T cell acute lymphoblastic leukemia showed that mutant messenger ribonucleic acid escapes nonsense mediated decay and produces an N-terminally truncated protein. Mutation prevented protein s nuclear localization and prohibited its interactions with other Mediator complex components, which led to an identification of a nuclear localization signal occurring at the highly conserved N-terminal region of MED12. The results of this study further strengthen the role of MED12 in the pathogenesis of uterine leiomyomas. These findings demonstrate that MED12 mutations are not restricted to benign hormone-dependent solid tumors, but can be found also in malignant tumors and in hematological diseases. Our results also provide new knowledge about the structure and normal functions of the MED12 protein.MED12 (mediator complex subunit 12) -geenin koodaama proteiini on osa laajaa Mediaattori-kompleksia ja toimii solujen proteiinituotannon säätelijänä sekä yleisellä että geenikohtaisella tasolla. MED12-geenin mutaatiot yhdistettiin kasvainten muodostumiseen ensikertaa, kun somaattisia, geenin toiseen eksoniin sijoittuvia mutaatioita havaittiin jopa 70 %:ssa kohdun hyvänlaatuisista sileälihaskasvaimista, leiomyoomista. Kohdun leiomyoomat ovat hyvin yleisiä kasvaimia; niitä on arvioitu esiintyvän jopa 77 %:lla naisista 50 ikävuoteen mennessä. Tämän väitöskirjatutkimuksen tarkoituksena oli tarkastella MED12-geenin roolia kohdun leiomyoomien ja useiden muiden kasvaintyyppien kehityksen taustalla. MED12-geenin mutaatioiden ja niiden aiheuttamien molekyylitason muutosten tarkempi tuntemus eri kasvaintyypeissä voi parantaa diagnosointia ja molekyyligeneettistä luokittelua sekä edesauttaa yksilöityjen hoitomuotojen kehittämistä. Tutkimuksessa saavutettujen tulosten perusteella MED12-geenin toisen eksonin mutaatiot eivät rajoitu vain hyvänlaatuisiin kasvaimiin, vaan niitä esiintyy myös kohdun sileälihaskudoksen syövissä, leiomyosarkoomissa. Havainto tukee aiemmin esitettyä hypoteesia joidenkin leiomyoomien mahdollisesta pahanlaatuistumisesta. Lisäksi osoitimme leiomyoomissa esiintyvän mutaatioita myös geenin ensimmäisessä eksonissa ja niiden aikaansaamien molekulaaristen mekanismien olevan yhteneväisiä eksonin 2 mutaatioiden kanssa. Havainto viittaa rajatun toiminnallisen alueen sijoittumiseen proteiinin alkuosaan ja korostaa entisestään MED12-geenin merkitystä leiomyoomien muodostumisessa. Yleisimmin leiomyoomat ovat sporadisia, mutta pieni osa kasvaimista kehittyy fumaraattihydrataasi (FH) -geenin molempien alleelien inaktivoitumisen seurauksena ja liittyy usein perinnölliseen leiomyomatoosi ja munuaissyöpä -oireyhtymään (hereditary leiomyomatosis and renal cell cancer, HLRCC). Tutkimuksessa osoitimme somaattisten MED12-mutaatioiden sekä FH-geenin inaktivoitumisen olevan toisensa poissulkevia mekanismeja leiomyoomien kehittymisessä. HLRCC-potilaiden leiomyoomien taustalla on yleisimmin FH-geenin inaktivaatio, mutta satunnaisesti heillä esiintyy myös sporadisia, MED12-mutaatiopositiivisia leiomyyomia. Tutkimustulostemme perusteella krooninen lymfaattinen leukemia on ensimmäinen kohdun ulkopuolinen syöpätyyppi, jossa samoja eksonien 1 ja 2 mutaatioita esiintyy merkittävissä määrin. Lisäksi positiivisen MED12-mutaatiostatuksen osoitettiin assosioituvan merkitsevästi huonon tautiennusteen markkereihin. Tutkimalla akuutissa lymfaattisessa leukemiassa havaittua MED12-geenille epätyypillistä nonsense-mutaatiota tunnistimme tumalokalisaatiosignaalin proteiinin N-terminaalisessa päässä, ja osoitimme, että geenin alussa esiintyvästä nonsense-mutaatiosta huolimatta geenistä tuotetaan vaihtoehtoisen translaation aloituskohdan avulla lyhyempää proteiinituotetta. Tämän tutkimuksen tulokset vahvistavat entisestään MED12-geenin roolia kohdun leiomyoomien kehittymisessä ja osoittavat, että samoja mutaatioita esiintyy myös pahanlaatuisissa kasvaimissa ja veritaudeissa. Lisäksi tutkimus tuotti uutta tietoa MED12-proteiinin rakenteesta ja normaalista toiminnasta

Uterine leiomyomas in hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome can be identified through distinct clinical characteristics and typical morphology

Introduction Hereditary leiomyomatosis and renal cell cancer (HLRCC) constitute a tumor susceptibility syndrome caused by germline mutations in the fumarate hydratase (FH) gene. The most common features are leiomyomas of the uterus and the skin. The syndrome includes a predisposition to early-onset, aggressive renal cell cancer. It is important to identify women with HLRCC among other uterine leiomyoma patients in order to direct them to genetic counseling and to identify other affected family members. Material and methods We conducted a nationwide historical study to identify typical clinical characteristics, uterine leiomyoma morphology, and immunohistochemistry for diagnosing HLRCC. The study included 20 women with a known FH germline mutation and 77 women with sporadic uterine leiomyomas. The patient records of all women were reviewed to obtain clinical details regarding their leiomyomas. Uterine leiomyoma tissue specimens from 43 HLRCC-related leiomyomas and 42 sporadic leiomyomas were collected and prepared for histology analysis. A morphologic description was performed on hematoxylin & eosin-stained tissue slides, and immunohistochemical analysis was carried out for CD34, Bcl-2, and p53 stainings. Results The women with HLRCC were diagnosed with uterine leiomyomas at a young age compared with the sporadic leiomyoma group (mean 33.8 years vs. 45.4 years, P < 0.0001), and their leiomyomas occurred as multiples compared with the sporadic leiomyoma group (more than four tumors 88.9% vs. 30.8%, P < 0.0001). Congruently, these women underwent surgical treatment at younger age compared with the sporadic leiomyoma group (mean 37.3 years vs. 48.3 years, P < 0.0001). HLRCC leiomyomas had denser microvasculature highlighted by CD34 immunostaining when compared with the sporadic leiomyoma group (112.6 mean count/high-power field, SD 20.8 vs. 37.4 mean count/high-power field, SD 21.0 P < 0.0001) and stronger anti-apoptotic protein Bcl-2 immunostaining when compared with the sporadic leiomyoma group (weak 4.7%, moderate 44.2%, strong 51.2% vs. 26.2%, 52.4%, 21.4%, respectively, P = 0.003). No differences were observed in p53 staining. Conclusions Women with HLRCC may be identified through the distinct clinical characteristics: symptomatic and numerous leioymyomas at young age, and morphologic features of FH-mutant leiomyomas, aided by Bcl-2 and CD34 immunohistochemistry. Further, distinguishing individuals with a germline FH mutation enables proper genetic counseling and regular renal monitoring.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

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.Peer reviewe

Breast tumors from CHEK2 1100delC-mutation carriers: genomic landscape and clinical implications

Introduction: Checkpoint kinase 2 (CHEK2) is a moderate penetrance breast cancer risk gene, whose truncating mutation 1100delC increases the risk about twofold. We investigated gene copy-number aberrations and gene-expression profiles that are typical for breast tumors of CHEK2 1100delC-mutation carriers. Methods: In total, 126 breast tumor tissue specimens including 32 samples from patients carrying CHEK2 1100delC were studied in array-comparative genomic hybridization (aCGH) and gene-expression (GEX) experiments. After dimensionality reduction with CGHregions R package, CHEK2 1100delC-associated regions in the aCGH data were detected by the Wilcoxon rank-sum test. The linear model was fitted to GEX data with R package limma. Genes whose expression levels were associated with CHEK2 1100delC mutation were detected by the bayesian method. Results: We discovered four lost and three gained CHEK2 1100delC-related loci. These include losses of 1p13.3-31.3, 8p21.1-2, 8p23.1-2, and 17p12-13.1 as well as gains of 12q13.11-3, 16p13.3, and 19p13.3. Twenty-eight genes located on these regions showed differential expression between CHEK2 1100delC and other tumors, nominating them as candidates for CHEK2 1100delC-associated tumor-progression drivers. These included CLCA1 on 1p22 as well as CALCOCO1, SBEM, and LRP1 on 12q13. Altogether, 188 genes were differentially expressed between CHEK2 1100delC and other tumors. Of these, 144 had elevated and 44, reduced expression levels. Our results suggest the WNT pathway as a driver of tumorigenesis in breast tumors of CHEK2 1100delC-mutation carriers and a role for the olfactory receptor protein family in cancer progression. Differences in the expression of the 188 CHEK2 1100delC-associated genes divided breast tumor samples from three independent datasets into two groups that differed in their relapse-free survival time. Conclusions: We have shown that copy-number aberrations of certain genomic regions are associated with CHEK2 mutation 1100delC. On these regions, we identified potential drivers of CHEK2 1100delC-associated tumorigenesis, whose role in cancer progression is worth investigating. Furthermore, poorer survival related to the CHEK2 1100delC gene-expression signature highlights pathways that are likely to have a role in the development of metastatic disease in carriers of the CHEK2 1100delC mutation

Somatic MED12 mutations are associated with poor prognosis markers in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. We performed systematic database search and identified highly specific MED12 mutations in CLL patients. To study this further, we collected three independent sample series comprising over 700 CLL samples and screened MED12 exons 1 and 2 by direct sequencing. Mutations were identified at significant frequency in all three series with a combined mutation frequency of 5.2% (37/709). Positive mutation status was found to be associated with unmutated IGHV and ZAP70 expression, which are well-known poor prognosis markers in CLL. Our results recognize CLL as the first extrauterine cancer type where 5'terminus of MED12 is mutated at significant frequency. Functional analyses have shown that these mutations lead to dissociation of Cyclin C-CDK8/19 from the core Mediator and to the loss of Mediator-associated CDK kinase activity. Additional studies on the role of MED12 mutation status as a putative prognostic factor as well as mutations' exact tumorigenic mechanism in CLL are warranted