Mutations in the PTEN tumor gene and risk of endometriosis: a case-control study.

STUDY QUESTION: Are mutations in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene associated with endometriosis? SUMMARY ANSWER: Loss of heterozygosity (LOH) at the 10q23.3 locus, PTEN somatic mutations and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway are associated with endometriosis. WHAT IS KNOWN ALREADY: Endometriosis has a strong genetic basis. Recent genome-wide association and linkage studies have reported a significant association of endometriosis with 7p15.2, 9p21 and 10q23-26 loci. PTEN, which maps to 10q23.3, acts as a tumor suppressor gene through the action of its phosphatase protein product, phosphatase and tensin homolog (PTEN). This phosphatase is involved in the regulation of the cell cycle, and mutations of PTEN are a step in the development of many cancers. STUDY DESIGN, SIZE, DURATION: A total of 1252 subjects of Indian origin (endometriosis patients = 752; controls = 500) were recruited to participate in this case-control study. Recruitment took place from 2001 to 2009 at Institute of Reproductive Medicine (IRM), Kolkata, India; Infertility Institute and Research Centre (IIRC), Secundrabad, India and Vasavi Medical and Research Centre, Hyderabad, India. PARTICIPANTS/MATERIALS, SETTING, METHODS: LOH on 10q, 9p and 7p was analyzed in analogous ectopic-eutopic endometria along with blood samples from 32 advanced stage endometriosis patients by PCR-GeneScan analysis. Genotyping of PTEN was carried out on genomic DNA of analogous ectopic-eutopic endometria (n = 32) as well as blood samples from 720 patients and 500 controls by PCR-sequencing analysis to explore somatic and germ-line mutations, respectively. The levels and distribution of PTEN, p-Akt, p-Bad and p27 were analyzed in the eutopic endometria of patients (n = 5) and controls (n = 5) using western-blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: PCR-GeneScan analysis revealed a higher LOH frequency at 10q23.3 (84.4%) compared with other loci analyzed, hence we focused our attention on PTEN. PCR-sequencing analysis revealed seven novel somatic mutations and 23 germ-line polymorphisms in patients. Among somatic mutations, a frame-shift insertion at 10:89692992-89692993 (in the functionally important N-terminal phosphatase domain of PTEN) occurred in 11 of the 32 ectopic endometria. Western-blot and immunohistochemical analysis revealed decreased PTEN and increased p-Akt and p-Bad levels in eutopic endometria of patients compared with controls (all comparisons, P < 0.0001). Furthermore, PTEN loss was more frequent in the nucleus than in the cytoplasm. Expression of p27 did not differ between patients and controls. LIMITATIONS, REASONS FOR CAUTION: Protein analysis was performed in eutopic endometrial samples from only a small number of patients and controls. In future investigations, a larger sample size should be used and the role of the other genes involved in the PTEN-PI3K/Akt signal transduction pathway should be analyzed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings revealed a possible involvement of the PTEN-PI3K/Akt-Bad axis in the pathogenesis of endometriosis, which may facilitate the discovery of suitable pathway inhibitors for disease treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Science & Engineering Research Board (SERB), India (Lr No: SR/FT/LS-188/2009) to BM. The authors have no competing interests to declare.

No evidence for the role of somatic mutations and promoter hypermethylation of FH gene in the tumorigenesis of nonsyndromic uterine leiomyomas.

Fumarate hydratase (FH) gene is reported to have specific involvement in syndromic uterine tumors, but its role in nonsyndromic forms is still unclear. Hence, the present study has aimed to screen the role of promoter methylation status and mutations in exon 2 and 7 regions of FH gene in the genesis of nonsyndromic uterine leiomyomas. Leiomyoma and myometrium tissues were collected from 85 hysterectomized uterine specimens. DNA from each of the biopsy was subjected to PCR, methylation-specific restriction assay, and DNA sequencing. In silico analysis was carried out to identify the impact of sequence variants on the protein structure. Chi-square (χ (2)) test was used to compare the promoter methylation proportions of leiomyoma and myometrium tissues. No sequence variants were observed in exon 2 region, but three novel heterozygous germ line sequence variants, i.e., c.1010A > C, c.1021 G > A, and c.1066 T > C in exon 7 region of the FH gene were detected in 14/85 (16.5 %) of the cases examined. In silico analysis results showed that c.1010A > C and c.1021 G > A mutations damage the structure and function of FH, whereas c.1066 T > C mutation is mostly tolerant or neutral. No significant difference of FH promoter methylation status between the leiomyoma (11.76 %) and myometrium (5.88 %) tissues was observed (P = 0.176). Therefore, it is concluded that somatic mutations in FH do not show pronounced effect in nonsyndromic uterine leiomyomas compared to that of their syndromic counterparts. However, higher frequency of FH mutations in leiomyoma cases raises the need to conduct larger number of prospective case-control and family-based studies to assess them as risk markers to nonsyndromic leiomyomas.

Enhanced transcription of estrogen receptor α and mitochondrial cytochrome b genes in uterine leiomyomas.

The relative expression levels of estrogen receptor α (ERα) and mitochondrial cytochrome b (MTCYB) transcripts and their association with ERα, -397T > C gene polymorphism was determined in premenopausal uterine leiomyomas and myometrium tissues to gain an insight into the role of ER-mediated action of estrogen on mitochondrial gene transcription. Both ERα and MTCYB transcripts were overexpressed in leiomyomas compared with myometrium tissues with 9.18 ± 0.79 folds and 5.24 ± 0.48 folds, respectively. ERα demonstrated ≥1.7 folds overexpression expressed over MTCYB (p < 0.001). Genotype correlation with transcript expression revealed that leiomyomas with CC genotype had significantly increased levels of ERα with 11.9 ± 1.02 folds as compared with 6.46 ± 0.56 folds seen in CT and TT genotypes together (p < 0.001). Interestingly, MTCYB transcript levels were also >1.9 folds overexpressed in leiomyomas with the CC genotype as compared with leiomyomas with other genotypes (p < 0.01).Significant elevation of ERα and MTCYB transcript levels in premenopausal leiomyomas and its association with ERα, -397 CC genotype suggests the mitochondrial-mediated role of estrogen as the promoter of leiomyoma tumorigenesis.

Mitochondrial genome variations in advanced stage endometriosis: a study in South Indian population.

BACKGROUND: Endometriosis is a chronic gynecological benign disease that shares several features similar to malignancy. Mitochondrial DNA (mtDNA) mutations have been reported in all most all types of tumors. However, it is not known as to whether mtDNA mutations are associated with endometriosis. METHODOLOGY: We sequenced the entire mitochondrial genome of analogous ectopic and eutopic endometrial tissues along with blood samples from 32 advanced stage endometriosis patients to analyze the role of somatic and germ-line mtDNA variations in pathogenesis of endometriosis. All ectopic tissues were screened for tumor-specific mtDNA deletions and microsatellite instability (MSI). We also performed mtDNA haplogrouping in 128 patients and 90 controls to identify its possible association with endometriosis risk. PRINCIPAL FINDINGS: We identified 51 somatic (novel: 31; reported: 20) and 583 germ-line mtDNA variations (novel: 53; reported: 530) in endometriosis patients. The A13603G, a novel missense mutation which leads to a substitution from serine to glycine at the codon 423 of ND5 gene showed 100% incidence in ectopic tissues. Interestingly, eutopic endometrium and peripheral leukocytes of all the patients showed heteroplasmy (A/G; 40-80%) at this locus, while their ectopic endometrium showed homoplasmic mutant allele (G/G). Superimposition of native and mutant structures of ND5 generated by homology modeling revealed no structural differences. Tumor-specific deletions and MSI were not observed in any of the ectopic tissues. Haplogrouping analysis showed a significant association between haplogroup M5 and endometriosis risk (P: 0.00069) after bonferroni correction. CONCLUSIONS: Our findings substantiate the rationale for exploring the mitochondrial genome as a biomarker for the diagnosis of endometriosis.

p53 and risk of endometriosis in Indian women.

AIM: To investigate the role of loss of heterozygosity (LOH), single nucleotide polymorphisms (SNPs), and the expression of gene p53 in the pathogenesis of endometriosis. METHODS: LOH at the p53 gene locus (17p13.1) was examined in matched ectopic and eutopic endometrial tissues from 31 endometriosis patients by polymerase chain reaction (PCR)-GeneScan analysis. The genotyping of selected p53 SNPs (n=10) was carried out on genomic DNA of blood from endometriosis patients (n=720) and controls (n=500) by PCR sequencing. The p53 expression levels were analyzed in the endometrial tissues from endometriosis patients (n=5) and controls (n=4) by Western blot and immunohistochemical analysis. RESULTS: LOH was observed at the 17p13.1 locus (38.7%) in the ectopic endometrium but not in the eutopic endometrium of patients. The genotype (p=0.909) and allele (p=0.729) distribution of the p53 codon Arg72Pro polymorphism was not significantly different between patients and controls. The polymorphism was not observed at codon 47 along the other SNPs studied. There was no preferential loss of either "Arg72" or "Pro72" alleles among the LOH-positive heterozygous cases. In addition, decreased p53 expression was observed more often in the endometrium of patients than in controls. CONCLUSIONS: p53 SNPs are not associated with endometriosis in Indian women. However, LOH and reduced expression of p53 are related with the risk of endometriosis in Indian women.

Detection of somatic mutations and germline polymorphisms in mitochondrial DNA of uterine fibroids patients.

To identify the role of mitochondrial DNA (mtDNA) mutations in uterine fibroids patients, genomic DNA isolated from paired myometrium and fibroid tissues was screened for mutations. The present study represents the first investigation to report that 10.4% of uterine fibroids cases had either mtDNA mutations or polymorphisms or both. Among the 14 mitochondrial sequence variants identified, seven are somatic mutations (A3327C, G3352A, G3376A, G3380A, G3421A, T15312G, and C15493G) and the remaining (G3316A, C3342A, C3442T, T10205A, A10188G, A10229C, and A10301T) are gene polymorphisms. Somatic mutations were both homo- and heteroplasmic in nature. Of the seven somatic mutations located in the MTND1 and MTCYB genes, five (71.42%) are nonsynonymous in nature, whereas four (57.14%) of the polymorphisms located in MTND1 and MTND3 genes are found to be nonsynonymous. Sequence variants such as G3380A, G3421A, T15312G, G3376A, and G3316A have been earlier described in different human pathologies, but the remaining are novel ones. Mitochondrial somatic mutations and polymorphisms may predispose women to an earlier onset of degenerative cellular processes, which impair oxidative phosphorylation capacity and thereby promote tumorigenesis in uterine smooth muscle cells. Detection of mtDNA sequence variations in fibroid patients raises the need for larger case-control studies to screen the whole mitochondrial genome and evaluate as a future diagnostic biomarker in fibroid patients.