Pathological repeat variation at the SCA17/TBP gene in south Indian patients.

Despite the intense debate around the repeat instability reported on the large group of neurological disorders caused by trinucleotide repeat expansions, little is known about the mutation process underlying alleles in the normal range, diseases range, large normal alleles (LNAs). In this study, we assessed the CAG repeats at SCA17 in 188 clinical SCA patients and 100 individuals without any neurological signs. This highly polymorphic population displayed high variability in the CAG repeats and ranged from 19-38 CAG repeats in patients with mode of 20 and 19-32 CAG repeats in controls with mode of 24. The triplet repeat expansion was not detected in any of the 188 patients, as per the reference pathogenic range (>43 repeats); however, 2.7% of the patients had >33 CAG repeats with a clinical phenotype close to what is expected of SCA 17 patients. The findings of this study implicate a more sophisticated interpretation of SCA17 gene and raise the question about the diagnostic thresh hold between normal and expanded repeats in our population.

Role of dynamic and mitochondrial mutations in neurodegenerative diseases with ataxia: lower repeats and LNAs at multiple loci as alternative pathogenesis.

Spinocerebellar ataxia is a growing group of hereditary neurodegenerative diseases for which ≥30 different genetic loci have been identified. In this study, we assessed the repeats at eight spinocerebellar ataxia (SCA) loci in 188 clinical SCA patients and 100 individuals without any neurological signs. Results from the present study were able to identify 16/188 (8.5%) clinical ataxia patients with repeat expansions in the pathological range of SCA genes, with the majority having expansion at the SCA1, 2, and 3 loci. The present study further evaluated two mitochondrial mutations associated with ataxia, i.e., T8993G and A8344G. Six patients were identified with A8344G mutation and none had the mutation in ATPase 6 gene; however, G8994A variation was found in three cases. Overall, three cases had triplet repeat expansions as well as mitochondrial (mt) mutations, which indicates potential association of triplet repeat expansions and mitochondrial mutations. Both the molecular analysis of several SCA loci and two relevant mt mutations indicated that the majority of ataxia cases were still undiagnosed; hence, the following hypotheses were proposed and tested based on available data: (i) lower repeats than normal range and (ii) large normal alleles (LNAs) at multiple loci may be an alternative basis for disease pathogenesis.

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.

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.

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.

Estrogen receptor-alpha gene (T/C) Pvu II polymorphism in endometriosis and uterine fibroids.

Endometriosis and fibroids are estrogen-dependent benign pathologies of the uterus, which account for infertility and pelvic pain along with dysmenorrhea in women. Suppression of the disease and recurrence after discontinuing hormone therapy strongly suggests that these are responsive to hormones, especially estrogen, which acts via its receptor. A T/C SNP in intron 1 and exon 2 boundary of estrogen receptor (ER) alpha gene recognized by PvuII enzyme has been associated with several female pathologies like breast cancer, osteoporosis, endometriosis and fibroids in various ethnic groups. The aim of the present study was to assess this ER alpha T/C polymorphism in endometriosis and fibroid patients from Asian Indian population. Genomic DNA was isolated from 367 women, who included 110 cases of endometriosis, 142 cases of uterine fibroids and 115 healthy age matched women volunteers. PCR was carried out to amplify ER alpha gene followed by restriction digestion with Pvu II. Results indicate a significant association of C allele with both endometriosis [OR=2.6667, 95% CI=1.4166 to 5.0199; p < 0.05] and fibroids [2.0833, 95% CI=1.1327 to 3.8319; p < 0.05]. Further studies are needed in larger population to establish ERalpha C allele as a risk marker for endometriosis and fibroids in Asian Indian women. Ethnicity, race, diet etc may play a role in susceptibility to endometriosis and fibroids and further studies are warranted in this area.

Polymorphic (CAG)n repeats in the androgen receptor gene: a risk marker for endometriosis and uterine leiomyomas.

BACKGROUND: Endometriosis and uterine leiomyomas are leading hormone responsive, benign uterine disorders responsible for high morbidity in women of reproductive age group. A polymorphic (CAG)n repeat length located in exon 1 of the androgen receptor (AR) gene has been proposed as a risk marker for both endometriosis and leiomyomas in some ethnic groups. The present study was carried out to assess the frequency of AR (CAG)n repeat polymorphism as a risk marker for endometriosis and uterine leiomyomas in Asian Indian women. METHODS: DNA was isolated from peripheral blood samples of 331 subjects, which include 90 endometriosis cases, 140 cases of leiomyomas and 101 healthy age- and sex-matched controls. PCR was carried out to amplify exon 1 of the AR gene. All the PCR amplicons were analysed initially on 2% agarose gel electrophoresis, followed by bidirectional sequencing to calculate the number CAG repeats in individuals. RESULTS: The CAG repeat ranges detected in endometriosis cases were 4-33 (Mode-19) and in leiomyomas cases 5-34 (Mode-20), whereas in controls it was 5-34 (Mode-22). A distinct variation was observed in the three groups at 14, 18, 19, 20 and 22 (CAG)n repeats, which were statistically analyzed using chi-square and odds ratio tests. 19 CAG repeats were found to be higher in endometriosis cases (19.09%) when compared with controls (9.04%), while 20 CAG repeats were higher in leiomyomas cases (14.02%) compared to controls (6.14%). A statistically significant (P < 0.05) association was observed in 19 and 20 CAG repeats in endometriosis and leiomyomas, respectively. CONCLUSION: This is the first report from an Asian Indian population proposing that 19 and 20 CAG repeats of the AR gene are associated with endometriosis and leiomyoma and can be regarded as high-risk markers.