Polymorphisms in immune mediators associate with risk of cervical cancer

Objective

The immune system is critical for controlling the progression of HPV cervical disease and the development of cancer. This study aimed to identify cervical cancer susceptibility alleles in candidate immune-modulating genes.

Methods

Our family-based study involved a cohort of 641 probands (women with ICC/CIN III) and their biologic parents or siblings (641 trios). In the discovery phase (stage 1), involving 288 of the trios, 80 tag single nucleotide polymorphisms (SNPs) in 11 immune-modulating genes (IFNG, IFNGR1, IFNGR2, JAK1, JAK2, STAT1, STAT6, IL12A, TNF, LTA and LTB) were evaluated on the GoldenGate platform. We used the combined dataset for a total of 641 trios (stage 2) and the Taqman platform to validate the SNPs that had proved significant in the discovery dataset. The transmission disequilibrium test was used to detect significant shifts in allelic transmissions in the datasets.

Results

Two SNPs in JAK2 and one SNP in STAT6 showed significant allelic association with cervical cancer in the stage 1 discovery dataset and were replicated in the larger joint analysis stage 2 dataset (JAK2 rs10815144, P = 0.0029 and rs12349785, P = 0.0058; and STAT6 rs3024971, P = 0.0127). An additional SNP in exon 19 of JAK2 (rs2230724) was also examined in the combined dataset due to its strong linkage disequilibrium (LD) with rs10815144. It was also significant (P = 0.0335).

Conclusions

Our results suggest an association of SNPs in JAK2 and STAT6 with cervical cancer. This association should be investigated in additional cervical cancer populations.     

Polymorphic variants of DNMT3A and the risk of endometriosis

Objective

Overexpression of DNA methyltransferase 3A (DNMT3A) and aberrant methylation of various genes in eutopic endometrium have been demonstrated in women with endometriosis. We aimed to study whether DNMT3A polymorphisms could be a genetic risk factor for endometriosis and endometriosis-related infertility.

Study design

We studied 5 SNPs (rs2289195, rs7590760, rs13401241, rs749131 and rs1550117) located in the DNMT3A gene in 357 women with endometriosis and 640 controls.

Results

We did not observe significant differences between genotype and allele frequencies of rs2289195, rs7590760, rs13401241, rs749131 and rs1550117 SNPs in women with endometriosis, endometriosis-related infertility, and controls. The lowest p values of the trend test were observed for DNMT3A rs1550117 in endometriosis and endometriosis-related infertility (p_trend = 0.049 and p_trend = 0.055, respectively).

Conclusions

Our results did not supply evidence for the contribution of SNPs located in DNMT3A to either endometriosis or endometriosis-related infertility.     

The association of PTPN22 polymorphism with endometriosis: effect of genetic and clinical factors

Objective

To investigate the possible effect of clinical and genetic variables on the association between PTPN22 and endometriosis.

Methods

PTPN22, ACP₁ and p53 codon 72 genetic polymorphisms and duration of previous pharmacological treatment were studied. The study sample consisted of 132 women hospitalized for endometriosis diagnosed by laparoscopic intervention and histologically confirmed: 359 healthy blood donors were studied as controls. PTPN22, ACP₁ and p53 codon 72 genotypes were determined by DNA analysis. Discriminant statistical analysis, logistic regression analysis, chi square of independence, power test and linear correlation were performed using SPSS programs.

Results

A significant increase of PTPN22 *T allele in endometriosis is observed in women carrying ACP₁*C allele, in women carrying p53 codon 72 *Pro allele and in women with prolonged pharmacological treatment.

Conclusions

PTPN22 may not be a primary factor in the etiology of endometriosis but may cooperate with clinical and genetic factors influencing susceptibility and clinical course of disease. These new observations point to a multifactorial origin of endometriosis and help to explain the reported differences between human populations concerning the association between PTPN22 and endometriosis.     

Hypermethylation of miR-203 in endometrial carcinomas

Objectives

Aberrant expression of SOX4 in endometrial cancer has been identified and partially was contributed to hypermethylation of miR-129-2. Other miRNAs are suspected to influence SOX 4 as well. The current study seeks to identify other hypermethylated miRNAs that regulate SOX4 in endometrial carcinomas.

Methods

Methylation levels of miRNA promoter regions were measured by combined bisulfite restriction analysis (COBRA) and pyrosequencing assays. Gene expression was determined by RT-qPCR. Methylation level of a miRNA locus was corrected with clinicopathologic factors for 252 gynecological specimens.

Results

In silico analysis identified 13 miRNA loci bound on the 3′-UTR of SOX4. Using COBRA assays, increased methylation of miR-203, miR-219-2, miR-596, and miR-618 was detected in endometrial cancer cells relative to those seen in a normal cell line and in normal endometrium. Transfection of a miR-203 mimic decreased SOX4 gene expression. Hypermethylation of miR-203 was detected in 52% of type I endometrioid endometrial carcinomas (n = 131) but was not seen in any of 10 uninvolved normal endometria (P < 0.001). Methylation status of miR-203 was significantly associated with microsatellite instability and MLH1 methylation in endometrial tumors (P < 0.001). Furthermore, hypermethylation of miR-203 was found in endometrioid and clear endometrial subtype tumors, but not in cervical squamous cell and ovarian carcinomas.

Conclusions

Hypermethylation of miR-203 is a frequent event in endometrial carcinomas and is strongly associated with microsatellite instability and MLH1 methylation status. Thus, miR-203 methylation level might represent a marker for patients with endometrioid and clear endometrial sub-cancers.     

Mycoplasma genitalium and preterm delivery at an urban community health center

Objective

To determine the prepartum prevalence of cervical Mycoplasma genitalium colonization and evaluate prospectively whether colonization is associated with preterm delivery among women from a racial/ethnic minority background with a high risk of delivering a low birth weight newborn and a high prevalence of sexually transmitted infections.

Methods

In a prospective cohort study at an urban community health center in Roxbury, MA, USA, 100 women receiving routine prenatal care for singleton pregnancies were enrolled between August 2010 and December 2011. Endocervical samples were tested for M. genitalium, and delivery data were collected.

Results

The prevalence of M. genitalium colonization at the first prenatal visit was 8.4%. The incidence of low birth weight was 16.7%. The incidence of preterm delivery among women who were known to have a live birth was 16.7%. The incidence of preterm delivery did not differ with respect to M. genitalium colonization. The crude odds ratio for preterm delivery among women with M. genitalium colonization versus those without was 1.27 (95% confidence interval, 0.02–14.78).

Conclusion

M. genitalium colonization was not associated with preterm delivery among women with a high incidence of low birth weight newborns and preterm delivery, and a high prevalence of sexually transmitted infections.     

BRCA1 and BRCA2 germline mutations in Moroccan breast/ovarian cancer families: novel mutations and unclassified variants

Objective

Breast cancer is the most common female cancer in Morocco. About 5 to 10% are due to hereditary predisposition and mutations in BRCA1 and BRCA2 genes are responsible for an important proportion of high-risk breast/ovarian cancer families. The relevance of BRCA1/2 mutations in the Moroccan population was not studied. The main objective of this study is to investigate the spectrum of BRCA1 and BRCA2 germline mutations in early onset and familial breast/ovarian cancer among Moroccan women.

Methods

We screened the entire coding sequences and intron/exon boundaries of BRCA1 and BRCA2 genes in 40 patients by direct sequencing.

Results

Nine pathogenic mutations were detected in ten unrelated families, five deleterious mutations in BRCA1 gene and four mutations in BRCA2 gene. Four novel mutations were found: one in BRCA1 (c.2805delA/2924delA) and three in BRCA2 (c.3381delT/3609delT; c.7110delA/7338delA and c.7235insG/7463insG). We also identified 51 distinct polymorphisms and unclassified variants (three described for the first time).

Conclusions

Our data suggest that BRCA1 and BRCA2 mutations are responsible for a significant proportion of familial breast cancer in Moroccan patients. Therefore full BRCA1/2 screening should be offered to patients with a family history of breast/ovarian cancer.     

Association of WNT4 polymorphisms with endometriosis in infertile patients

Purpose

Recently, several genome-wide association studies have demonstrated an association between endometriosis and markers located in or near to WNT4 gene. In order to assess the validity of the findings, we conducted a replication case–control study in a Brazilian population.

Methods

Genetic association study comprising 400 infertile women with endometriosis and 400 fertile women as controls. TaqMan allelic discrimination technique was used to investigate the relationship between endometriosis and four single-nucleotide polymorphisms (rs16826658, rs3820282, rs2235529, and rs7521902) in WNT4 gene. Genotype distribution, allele frequency, and haplotype analysis of the WNT4 polymorphisms were performed. A p value <0.05 was considered significant.

Results

The results revealed a significant association of rs16826658 (p = 7e-04) and rs3820282 (p = 0.048) single-nucleotide polymorphisms (SNPs) on WNT4 gene with endometriosis-related infertility, while rs2235529 and rs7521902 SNPs showed no difference between cases and controls.

Conclusions

Our results suggested that rs16826658 and rs3820282 polymorphisms on WNT4 gene might be involved in the pathogenesis of endometriosis in the infertile women studied. Analysis of WNT4 genetic variants might help to identify patients at high risk for disease development.     

Chemoprevention and prophylactic surgery in ovarian carcinoma

Introduction

Ovarian cancer is the leading cause of death from gynaecological malignancy, especially because of late diagnosis. The objective of the study was to provide the clinician with current concepts regarding prevention of ovarian cancer.

Material and methods

A computerized search of articles published was performed using the Medline database We performed a review of the literature (PubMed, Embase) using the following search terms (MeSH and non-MeSH): prevention, chemoprevention, chimioprevention, ovarian cancer, ovarian, ovary, carcinoma, tumor, tumour.

Results

Oral contraceptive and acetaminophen use may provide substantial protection against ovarian cancer, whereas aspirin, carotenoids and non-steroidal anti-inflammatory agents do not decrease the risk. However, to date, there is no recommendation concerning low risk population. At the opposite, young women (< 35–40 years old) presenting with BRCA1 or 2 mutation or Lynch syndrome may be counseled for chemoprevention using oral contraceptive. For high risk women over 35–40 years old, prophylactic bilateral salpingo-oophorectomy should be performed. Indeed, it has been showed that prophylactic surgery significantly decrease mortality rates in high risk women.

Conclusion

Large randomized studies are required to assess the efficacy of ovarian cancer chemoprevention in low risk women. High-risk women over 35–40 years old should be counseled for prophylactic salpingo-oophorectomy or for chemoprevention using oral contraceptive.     

Growth factor progranulin contributes to cervical cancer cell proliferation and transformation in vivo and in vitro

Objective

The growth factor progranulin (PGRN) is overexpressed in a number of tumors. We aimed to investigate the expression and role of PGRN in cervical cancer tumorigenesis.

Methods

PGRN expression and secretion was assessed in cells and normal and cancerous cervical tissues by Western blot analysis, ELISA or immunohistochemistry. The role of PGRN in cervical carcinogenesis was explored by cell-proliferation, colony-formation and tumor-growth assays. We assessed the role of PGRN-mediated signaling in the cervical cell with specific inhibitors.

Results

PGRN expression was upregulated in cervical cancer cell lines and tissue. PGRN promoted the transformation of human cervical mucosa epithelial H8 cells in vitro and tumor formation in vivo. Knockdown of PGRN expression in cervical cancer cells in vivo decreased cell proliferation and slowed tumor growth. PGRN stimulated cervical cell proliferation, and transformation was mediated, at least in part, by Akt and Erk signaling.

Conclusions

PGRN is overexpressed in cervical cancer and promotes the malignant growth and transformation of cervical cells. Therefore, PGRN plays a critical role in carcinogenesis of cervical cancer and shows promise for therapeutic strategies for cervical cancer.     

Expression of the imprinted IGF2 and H19 genes in the endometrium of cases with unexplained infertility

Objective

As genomic imprinting plays a critical role in the development of the placenta, the aim of this study was to detect whether the expression levels of the imprinted genes IGF2 and H19 in the endometrium differ between infertile and fertile women.

Study design

: Total RNA was extracted from 30 (15 unexplained infertile and 15 fertile) women's endometrial tissue. cDNA was synthesized from total RNAs of each sample. IGF2 and H19 mRNA expression levels were measured quantitatively using the Real Time PCR method. In order to determine the allelic expression of IGF2 and H19, genomic DNA was extracted from endometrial tissues.

Results

When compared with the control group, increased mRNA expression of IGF2 was detected (1.5-fold change, P = 0.015) in the unexplained infertility group. In contrast, H19 expression was lower in the infertility group as compared to the control group (4-fold change, P < 0.0001). Restriction analysis of cDNA-derived PCR product showed that all patients and controls indicated monoallelic expression of IGF2 and H19.

Conclusion

Our results showed that altered expression of these imprinted genes might affect implantation and that their timely and appropriate activation is important for proper functioning. To understand the molecular epigenetic basis of implantation and placental development, genomic imprinted genes should be further investigated.     

Risk management options elected by women after testing positive for a BRCA mutation

Objective

To assess the uptake of risk-reducing options for the management of ovarian and breast cancer risk in BRCA mutation carriers in a large community based integrated health system in Northern California.

Methods

A retrospective cohort of deleterious BRCA mutation carriers (1995–2012) was evaluated for consistency with NCCN guidelines for risk reducing salpingo-oophorectomy (RRSO) by age of 35–40, risk reducing mastectomy (RRM), as well as surveillance practices, including pelvic ultrasound, CA 125, mammogram, and breast MRI. Secondary outcomes included the use of chemoprevention and hormone replacement.

Results

Of the 305 eligible women, 170 were BRCA1 positive, and 135 were BRCA2 positive. Seventy four percent underwent RRSO with only 17% under age 40, while 44% underwent RRM. The median time from the test to both RRSO and RRM was 6 months. In the first year after BRCA diagnosis, 45% underwent a pelvic ultrasound, dropping to 2.3% by year 5. In year 1, 47% had a CA 125, dropping to 2% by year 5. The number of women undergoing annual MRI and mammogram fell similarly over time. Sixteen percent of BRCA carriers used oral contraceptives (OCPs) and only one patient used tamoxifen for chemoprevention.

Conclusion

Uptake of RRSO in BRCA carriers in a population based health system is high, however the majority of women do not have RRSO by the NCCN recommended age. Compliance with surveillance is low and rapidly declines even 1 year out from testing. Attention needs to be focused on the earlier identification of BRCA mutation carriers with consolidated and standardized care to improve risk reduction.     

Posicionamiento de la Asociación Española para el Estudio de la Menopausia sobre el uso clínico de la Cimicífuga racemosa en el climaterio

Objective

Due to an increasing interest in the symptoms related to menopause in western countries, the Spanish Menopause Society (SMS) has assessed the role of cimicifuga racemosa in relieving those symptoms based on the most reliable evidence available.

Material and methods

A meeting was held with a panel of experts, health scientists and researchers specialised in the field of phytotherapy. The studies selected were obtained through electronic search which included INTERNET, MEDLINE (1985-May 2008), and the Cochrane Controlled Clinical Trials Register.

Results

Most studies published in recent years have been carried out using an isopropanol extract of cimicifuga racemosa. The dose most commonly studied was 40mg/day and was shown to result in a moderate decrease in hot flushes, particularly in those women with the most intense hot flushes, and an improvement in their mood. When recommended doses are used there is no significant risk to the hepatic system. Available data on the effect of cimicifuga racemosa on cardiovascular disease, bones, cognitive function or skin are scarce or non-existent.

Conclusions

Cimicifuga racemosa is an effective treatment for relief of vasomotor symptoms, at least within a suitable population of peri- and postmenopausal women. Nevertheless, more accurate clinical trials which include sufficient numbers of patients and a longer follow-up are required.     

Association of polycystic ovary syndrome susceptibility single nucleotide polymorphism rs2479106 and PCOS in Caucasian patients with PCOS or hirsutism as referral diagnosis

Context

Polycystic ovary syndrome (PCOS) is the most common endocrine disease among premenopausal women. A recent study found association between three single nucleotide polymorphisms (SNPs) and PCOS in a cohort of Han Chinese women.

Objective

To investigate the association between rs13405728 (LHCGR gene), rs13429458 (THADA gene) and rs2479106 (DENND1A gene), PCOS, hirsutism and metabolic and hormonal parameters in a well characterized cohort of Caucasian patients of Danish descendant with PCOS or hirsutism.

Study design

Patients underwent clinical examination, hormone analyses, oral glucose tolerance test and transvaginal ultrasound. Genetic variation was tested using allelic discrimination by real-time PCR.

Patients

268 patients referred to The Department of Endocrinology, Odense University Hospital, Denmark with PCOS or hirsutism between 1997 and 2011. Two hundred and forty-eight healthy females were included as controls.

Results

Genotype distributions and allele frequencies of rs13405728, rs13429458, and rs2479106 were comparable in patients and controls. The rs2479106 G allele was associated with a decreased PCOS susceptibility. None of the SNPs were associated with hirsutism or increased metabolic parameters.

Conclusions

The rs2479106 G allele was associated with decreased PCOS susceptibility, thus confirming previously reported findings of association between rs2479106 and PCOS. Metabolic and hormonal parameters were comparable between genotypes of rs13405728 and rs2479106.     

Lack of association of KATNAL1 gene sequence variants and azoospermia in humans

Purpose

A recent experiment indicated that a loss of function mutation in the murine Katnal1 gene resulted in male factor infertility due to premature exfoliation of spermatids. This study investigated the relevance of this gene to infertility in humans.

Methods

Multiple methods of genetic analysis were employed to investigate whether mutations in human KATNAL1 have a causative role in male infertility. This was a genetic association study, which included DNA samples from 105 men with non-obstructive azoospermia (NOA) and 242 anonymous sperm donor controls. 28 commercially available TaqMan SNP assays were used to haplotype samples from both groups and genetically tag regions of interest across the entire gene. AmpliSeq primers were then designed for identified regions so that targeted next-generation sequencing (NGS) could be used to identify causative variants.

Results

Four SNPs in the 3’UTR demonstrated a putative association with NOA. The AmpliSeq primers designed for the 3’UTR provided 83 % coverage of the 7,202 basepairs within the regions of interest. Variant sites were analyzed against genetic models to identify sequence polymorphisms which associated with NOA. No variants met standard criteria for significance when tested between the groups.

Conclusions

This study suggests a lack of association of KATNAL1 gene sequence variants and azoospermia in humans.     

Interleukin-12 p40 gene (IL12B) polymorphisms and the risk of cervical caner in Korean women

Objective

The aim of this study was to investigate whether IL12B polymorphisms might be associated with increased risk and invasiveness of cervical cancer in Korean women.

Study design

Peripheral blood samples from patients with invasive cervical cancer (n = 154) and non-cancer controls (n = 191) were used to detect three biallelic IL12B polymorphisms at IVS2 −912, IVS4 +314, 3′UTR +1188 sites by performing SNaPshot assay. Allelic frequencies, genotype distributions, and haplotype patterns in the case group were compared with those in the control group. The relationships between these polymorphisms and cancer invasiveness were also evaluated by collating the clinicopathologic parameters including FIGO stage, lymph node status, histologic type, and parametrial invasion. The used analytic methods are chi-square test and logistic regression analysis.

Results

Allelic frequencies of cases (G, 0.853; A, 0.147) were not significantly different from controls (G, 0.796; A, 0.204) in IVS2 −912G/A SNP (P = 0.054). GG genotype of IVS2 −912G/A SNP showed increased risk for cervical cancer compared with AA genotype (P = 0.040). The IVS2 −912G:IVS4 + 314A haplotype, IVS2 −912G:IVS4 +314A:3′UTR +1188A haplotype, and IVS2 −912G:IVS4 +314A:3′UTR +1188C haplotype were also significantly associated with increased risk for cervical cancer. A subgroup analysis of the clinicopathologic parameters in cancer group also showed that there is no significant association between IL12B polymorphisms and cervical cancer invasiveness.

Conclusions

This study suggests that IVS2 −912GG genotype and IVS2 −912G:IVS4 +314A haplotype of IL12B gene are associated with increased risk for cervical cancer in Korean women.     

Association of TP53 codon 72 polymorphism with susceptibility to ovarian carcinomas in Serbian women

Objectives

Finding a potential genetic factor associated with a deadly disease, such as ovarian carcinoma, is of particular importance. The aim of this study was to examine the role of the TP53 codon 72 polymorphism in ovarian carcinoma development in Serbian women.

Study design

47 wild-type TP53 gene ovarian carcinoma samples and 70 cervical smears from gynecologically healthy women were analyzed. DNA was extracted by a salting-out procedure. Codon 72 polymorphism was assessed by PCR-RFLP method. χ², Fisher exact test and odds ratio were used for statistical analysis.

Results

The distribution of Arg/Arg, Arg/Pro and Pro/Pro genotypes of codon 72 of the TP53 gene was: 46.8%, 46.8% and 6.4%, respectively in the ovarian carcinomas and 64.3%, 31.4% and 4.3%, respectively in the control group. We observed an increased risk for the development of ovarian carcinoma for Pro homozygotes in relation to heterozygotes plus Arg homozygotes (OR = 1.52; 95% CI 0.29–7.89) and a higher one for Pro/Pro plus Arg/Pro genotype in relation to Arg homozygotes (OR = 2.04; 95% CI 0.96–4.34).

Conclusion

The results showed no association between codon 72 TP53 gene polymorphism and risk for development of ovarian carcinoma in Serbian women. However, this observation requires further analysis of a larger case–control study group.     

Absence of activating somatic mutations of PI3KCA and AKT1 genes in South Indian women with endometriosis

Objective

To investigate whether the PI3KCA and AKT1 gene influences the risk of developing endometriosis in South Indian women.

Study design

Mutations in exon 9 and 20 of PI3KCA gene and E17K mutation in exon 4 of AKT1 gene were tested for association in a case-control study between eutopic and ectopic endometrium tissue from 30 endometriosis cases and eutopic endometrium tissue from 30 controls. The genotype frequencies of these mutations were compared using polymerase chain reaction and direct sequencing analysis of tissue DNA.

Results

The analysis did not reveal any activating somatic mutations in either PI3KCA or AKT1 gene in the cases.

Conclusion

In the present study we could not observe any mutation in PI3KCA and AKT1 gene, indicating that these mutations are rarely associated with endometriosis in South Indian women.     

Adult granulosa cell tumours (GCT): Clinicopathological outcomes including FOXL2 mutational status and expression

Objectives

The aim of this research was to use nucleic acids isolated from formalin-fixed paraffin-embedded (FFPE) tissue to investigate the diagnostic potential and prognostic significance of FOXL2 in adult-type GCTs, particularly as a marker of identifying early stage patients that are likely to relapse.

Methods

We performed a retrospective review of GCT patients referred to the Auckland Gynae-Oncology Multidisciplinary Team from 1955 to 2012. Baseline characteristics, clinical course, histopathology and survival data was recorded. Using nucleic acids extracted from FFPE tumour blocks, FOXL2 mutation status and expression was determined by DNA sequencing and RT-qPCR, respectively, and correlated with clinical data.

Results

57 adult GCT patients were identified, however FFPE tumour blocks were available for only 37 of these patients. Sequencing results confirmed the presence of the FOXL2 mutation in 70% of patients. FOXL2 mutation positive adult tumours showed a trend towards higher FOXL2 expression than wildtype adult tumours, particularly in stage I patients (p = 0.051). In addition, patients with homozygous FOXL2 mutations had a significantly higher relapse rate (p = 0.04). There was no significant correlation between FOXL2 mutation status or FOXL2 expression and any other clinical variables.

Conclusions

FFPE tumour blocks are a valuable resource of molecular information, especially when studying rare tumours such as GCTs. The FOXL2 mutation appears to have some diagnostic potential, however additional work in a larger cohort needs to be completed to confirm the prognostic significance of this gene mutation, and its expression.