Frequent DYSF rare variants/mutations in 152 Han Chinese samples with ovarian endometriosis.

PURPOSE: Endometriosis is a common chronic gynecological disease greatly affecting women health. Prior studies have implicated that dysferlin (DYSF) aberration might be involved in the pathogenesis of ovarian endometriosis. In the present study, we explore the potential presence of DYSF mutations in a total of 152 Han Chinese samples with ovarian endometriosis. METHODS: We analyze the potential presence of DYSF mutations by direct DNA sequencing. RESULTS: A total of seven rare variants/mutations in the DYSF gene in 10 out of 152 samples (6.6%) were identified, including 5 rare variants and 2 novel mutations. For the 5 rare variants, p.R334W and p.G941S existed in 2 samples, p.R865W, p.R1173H and p.G1531S existed in single sample, respectively; for the two novel mutations, p.W352* and p.I1642F, they were identified in three patients. These rare variants/mutations were absent or existed at extremely low frequency either in our 1006 local control women without endometriosis, or in the China Metabolic Analytics Project (ChinaMAP) and Genome Aggregation Database (gnomAD) databases. Evolutionary conservation analysis results suggested that all of these rare variants/mutations were evolutionarily conserved among 11 vertebrate species from Human to Fox. Furthermore, in silico analysis results suggested these rare variants/mutations were disease-causing. Nevertheless, we find no significant association between DYSF rare variants/mutations and the clinical features in our patients. To our knowledge, this is the first report revealing frequent DYSF mutations in ovarian endometriosis. CONCLUSION: We identified a high frequency of DYSF rare variants/mutations in ovarian endometriosis for the first time. This study suggests a new correlation between DYSF rare variants/mutations and ovarian endometriosis, implicating DYSF rare variants/mutations might be positively involved in the pathogenesis of ovarian endometriosis.

Mutation analysis of ZP1, ZP2, ZP3 and ZP4 genes in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35-50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.

The presence of KRAS, PPP2R1A and ARID1A mutations in 101 Chinese samples with ovarian endometriosis.

Endometriosis is a potential premalignant disorder. The underlying molecular aberrations, however, are not fully understood. A recent exome sequencing study found that 25% (10/39) of deep infiltrating endometriosis harbored cancer driver gene mutations. However, it is unclear whether these mutations also exist in ovarian endometriosis. Here, a total of 101 ovarian endometriosis samples were analyzed for the presence of these gene mutations, including KRAS, PPP2R1A, PIK3CA and ARID1A. In addition, 6 other cancer-associated genes (BRAF, NRAS, HRAS, ERK1, ERK2 and PTEN) were also analyzed. In total, four somatic mutations were identified in three out of 101 ovarian endometriotic lesions (4%, 4/101), including a KRAS p.G12V, a PPP2R1A p.S256F and two ARID1A nonsense mutations (p.Q403* and p.G1926*); while no mutations were identified in the remaining 7 genes (BRAF, NRAS, HRAS, ERK1, ERK2, PTEN and PIK3CA). Note that the KRAS G12V and ARID1A Q403* mutations co-occurred in a 36-year-old sample who had a high serum CA125 (308.4 U/mL) and a late menarche age (18-year-old). Additionally, no mutations in any of the 10 genes were identified in either the healthy eutopic endometrial tissues from 85 control individuals without endometriosis, or in 62 healthy ovarian tissues from ovarian cysts samples (without endometriosis). Our study revealed, for the first time, the presence of classical cancer driver gene mutations in ovarian endometriosis. Furthermore, the co-occurrence of KRAS and ARID1A mutations was identified in a single individual for the first time. The observations of cancer driver gene mutations in our ovarian endometriosis samples, together with several prior observations, further support the notion that endometriosis is a premalignant disorder.

Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

Downregulation of DNA methyltransferase 3 alpha promotes cell proliferation and invasion of ectopic endometrial stromal cells in adenomyosis.

Adenomyosis is a common benign gynecological condition in female reproductive tract and the detailed molecular etiology remains largely elusive. Previous studies implicated that deregulated expression of DNA (cytosine-5)-methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, might be involved in the pathogenesis of adenomyosis. Meanwhile, ectopic endometrial stromal cells (EESCs) were suggested to play crucial roles in adenomyosis. Herein, we evaluated the expression of DNMT3A protein in 36 ectopic endometriums with adenomyosis and 37 eutopic endometriums in controls with Western blotting (WB) or immunohistochemistry (IHC), we found that the expression of DNMT3A was significantly decreased in the ectopic endometriums and EESCs in adenomyosis relative to that of eutopic endometriums and EESCs in control samples, respectively. In addition, our functional assays revealed that overexpression of DNMT3A suppressed cell proliferation and invasion, while knockdown of DNMT3A enhanced cell proliferation and invasion in EESCs. Taken together, our results suggested that DNMT3A expression was decreased in ectopic endometriums and EESCs in adenomyosis, and we provided the first evidence that decreased DNMT3A expression in EESCs facilitated the development of adenomyosis via enhanced cell growth and invasion.

A novel somatic MAPK1 mutation in primary ovarian mixed germ cell tumors.

A recent exome-sequencing study revealed prevalent mitogen-activated protein kinase 1 (MAPK1) p.E322K mutation in cervical carcinoma. It remains largely unknown whether ovarian carcinomas also harbor MAPK1 mutations. As paralogous gene mutations co­occur frequently in human malignancies, we analyzed here a total of 263 ovarian carcinomas for the presence of MAPK1 and paralogous MAPK3 mutations by DNA sequencing. A previously unreported MAPK1 p.D321N somatic mutation was identified in 2 out of 18 (11.1%) ovarian mixed germ cell tumors, while no other MAPK1 or MAPK3 mutation was detected in our samples. Of note, OCC­115, the MAPK1­mutated sample with bilateral cancerous ovaries affected, harbored MAPK1 mutation in the right ovary while retained the left ovary intact, implicating that the genetic alterations underlying ovarian mixed germ cell tumor may be different, even in patients with similar genetic backgrounds and tumor microenvironments. The results of evolutionary conservation and protein structure modeling analysis implicated that MAPK1 p.D321N mutation may be pathogenic. Additionally, mutations in protein phosphatase 2 regulatory subunit α (PPP2R1A), ring finger protein 43 (RNF43), DNA directed polymerase ε (POLE1), ribonuclease type III (DICER1), CCCTC­binding factor (CTCF), ribosomal protein L22 (RPL22), DNA methyltransferase 3α (DNMT3A), transformation/transcription domain­associated protein (TRRAP), isocitrate dehydrogenase (IDH)1 and IDH2 were not detected in ovarian mixed germ cell tumors, implicating these genetic alterations may be not associated with MAPK1 mutation in the development of this malignancy. The present study identified a previously unreported MAPK1 mutation in ovarian mixed germ cell tumors for the first time, and this mutation may be actively involved in the tumorigenesis of this disease.