Christian Schneeberger

PROGINS receptor gene polymorphism is associated with endometriosis

OBJECTIVE To investigate the association between the 306-base pair insertion polymorphism in intron G of the progesterone receptor (PROGINS) and endometriosis. DESIGN Case-control study. SETTING Tertiary care center. PATIENT(S) Ninety-five white women with surgically diagnosed and histologically confirmed endometriosis and 107 white women without endometriosis (controls). INTERVENTION(S) Determination of PROGINS was performed by polymerase chain reaction and gel electrophoresis. MAIN OUTCOIME MEASURE(S): Frequency and distribution of the PROGINS allele. RESULT(S) Frequencies of the mutant allele T2 was 0.17 among women with endometriosis and 0.08 among controls (odds ratio, 2.41 [CI, 1.31-4.53]). Homozygosity for allele T2 was present in 3.2% of women with endometriosis and 0.9% of controls. CONCLUSION(S) PROGINS appears to be associated with endometriosis in white persons.

Expression of inducible nitric oxide synthase in human breast cancer depends on tumor grade.

Expression of inducible nitric oxide synthase (iNOS) by tumor cells has been suggested to abrogate metastasis in several tumor models, whereas constitutive NOS expression correlated positively with tumor grade in human breast carcinoma. Whether or not expression of one of the various NOS isoforms could predict the prognosis of breast cancer, however, has not been established. In the present report we investigated the cellular distribution of NOS isoforms in a series of benign and malignant breast tumors and in normal breast tissue. Immunohistochemistry revealed that in samples of benign disease the number of iNOS + epithelial cells or total epithelial cells was 69 ± 16% (n=50). In samples of grade II invasive ductal breast carcinomas the number of iNOS+ tumor cells or total tumor cells was 62 ± 20 (n=40), compared to 12 ± 9 (n=40) in samples of grade III carcinomas (P < 0.0001). iNOS protein was also identifiable in most of the epithelial cells of normal breast tissue (n=4). In contrast, eNOS protein was restricted to vascular endothelial cells in all of the specimens studied. Since the presence of tumor cell iNOS protein is inversely related to the tumor’s metastatic potential, we conclude that endogenous tumor cell mediated iNOS expression might have an inhibitory effect on the metastatic process in breast cancer.

Genetic modelling of the estrogen metabolism as a risk factor of hormone-dependent disorders

Estradiol is a pleiotropic hormone, involved in the etiology of a wide variety of diseases. Over the last decade individual genetic variability of the estradiol metabolism has been described as a significant contributor to disease susceptibility with variations depending on ethnic background. Among others, genetic variations of genes encoding cytochrome P450 (CYP) enzymes play an important role in this regard. Mutant alleles of the CYP 1A1 gene are major modulators of lung cancer risk among smokers, mediate gender differences in lung cancer susceptibility, and have been associated with an elevated risk for developing breast, prostate, colorectal, and oral squamous cell cancer. Variants of the CYP 1B1 gene modulate the risk for developing prostate, ovarian, lung, and breast cancer. Also, mutations in the CYP 1B1 gene are the major genetic determinant of congenital glaucoma. Mutant CYP 17 alleles are associated with serum and plasma levels of steroid hormones, use of hormone replacement therapy, and the development of endometrial, prostate, and breast cancer. Available data indicate that the protective effect against breast cancer of a later age at menarche is limited to wild-type CYP 17 allele carriers. Among women with the polycystic ovary syndrome, carriage of mutant CYP 17 alleles is sufficient to aggravate the clinical presentation of the disease. Molecular variants of the CYP 19 gene are associated with an increased risk for developing breast cancer, advanced breast cancer stages, and tumor aromatase production. Carriage of a mutant catechol-O-methyltransferase allele is associated with breast cancer, neurologic disorders such as Parkinsons disease, and modulates behavior among patients with schizophrenia, alcoholics and the general population. In summary, the available evidence points to genes that encode estrogen-metabolizing enzymes as strong hereditary determinants of the susceptibility to benign as well as malignant conditions.

Association of in vitro invasiveness and gene expression of estrogen receptor, progesterone receptor, pS2 and plasminogen activator inhibitor-1 in human breast cancer cell lines

The invasive potential of tumor cells is usually tested either by in vitro invasion assays which evaluate cell spreading ability in basement membrane‐like matrices or by in vivo invasion assays in nude mice. Both methods are laborious and time‐consuming. Tumor invasiveness is accompanied by the changes in expression of various genes. The invasive behavior of cells is therefore represented by certain gene expression patterns. The purpose of this study was to investigate whether expression patterns of several genes are characteristic for the invasiveness of cultured cells. We examined the mRNA levels of estrogen receptor (ER), progesterone receptor (PR), estrogen inducible pS2 and plasminogen activator inhibitor‐1 (PAI‐1) in 23 cell lines derived from benign and malignant breast tissues using a competitive reverse transcription‐polymerase chain reaction (cRT‐PCR) system. We also evaluated the invasiveness of these cell lines by their ability to penetrate into a collagen‐fibroblast matrix. We demonstrate that the gene expression pattern of breast cell lines is clearly associated with its in vitro invasiveness. In general, cells with ER, PR, pS2 but no PAI‐1 expression showed a non‐invasive phenotype, while cells expressing PAI‐1 mRNA but not ER mRNA are invasive. Our study indicates that the invasiveness of breast cancer cell lines is characterized by PAI‐1 gene expression and the lack of ER mRNA. This suggests that PAI‐1 may participate in the invasive process.

Interleukin 1 receptor antagonist polymorphism in women with idiopathic recurrent miscarriage

Abstract Objective: Proinflammatory cytokines have been described as etiologic factors in idiopathic recurrent miscarriage. We investigated the relation between idiopathic recurrent miscarriage and polymorphisms in the gene encoding for the interleukin 1 receptor antagonist, an indigenous modulator of proinflammatory immune response. Design: Prospective case control study. Setting: Academic research institution. Patient(s): One hundred five women with a history of three or more consecutive pregnancy losses before 20 weeks of gestation and 91 healthy, postmenopausal controls with at least two live births and no history of pregnancy loss. Intervention(s): Peripheral venous puncture. Main Outcome Measure(s): Polymerase chain reaction was performed to identify the different alleles of the gene encoding for interleukin 1 receptor antagonist. Result(s): Allele frequencies among women with idiopathic recurrent miscarriage and controls were 0.34 and 0.11, respectively, for the polymorphic allele 2 ( P =.002; odds ratio: 7.4, confidence interval: 2.9–10.8) and .05 and .05, respectively, for the polymorphic allele 3 ( P =.6; odds ratio: 1.3, confidence interval: 0.8–2.3). Allele 2 was present in homozygous form in 9% of women with idiopathic recurrent miscarriage. In contrast, 1% of the control women were homozygous for this allele ( P Conclusion(s): These data support a role for allele 2 of the gene encoding for interleukin 1 receptor antagonist as genetic determinant of idiopathic recurrent miscarriage.

Simultaneous expression of nitric oxide synthase and estrogen receptor in human breast cancer cell lines

SummaryFor various human tumor cell lines (neuroblastoma, cervix carcinoma) the presence of constitutive nitric oxide synthase (cNOS) has been documented. Here, for the first time, we report about cNOS expression in 10 of 16 human breast cancer cell lines. cNOS expression correlates strongly with expression of estrogen receptor (ER). Competitive reverse transcription — polymerase chain reaction (cRT-PCR) was used to detect cNOS and ER mRNA expression. Our findings suggest that estradiol could stimulate constitutive NO release in breast tissue, where it acts as a free radical.

Analysis of an interleukin-6 gene promoter polymorphism in women with endometriosis by pyrosequencing.

Interleukin (IL)-6 has been implicated in the etiology of endometriosis. A single nucleotide polymorphism (SNP) at position - 174 in the IL-6 gene promoter appears to influence IL-6 transcription rates in vitro and basal IL-6 levels in vivo. We determined the genotype and the allele frequencies of the -174 IL-6 promoter polymorphism and the corresponding IL-6 serum levels in women with endometriosis. The pyrosequencing technique was used to assess the IL-6 genotypes in 94 women with histologically confirmed endometriosis (study group). A series of 70 healthy women without history of uterine disease served as clinical controls (control group). Allele frequencies for the G allele among women with and without endometriosis were 59.6% and 55.0%, respectively (P =.430; odds ratio [OR] 0.83, 95% confidence interval [CI] 0.53, 1.29). Homozygotes for the protective allele C were present in 17.0% of women with endometriosis and in 18.6% of controls were homozygous for the protective allele C (P =.797; OR 0.90, 95% CI 0.40, 2.02). When patients with various disease manifestations were compared, we found an association between the -174 G allele and chocolate cysts (P =.037). Serum levels of IL-6 were significantly higher in women with endometriosis than in controls (P <.001), with highest levels in women with chocolate cysts. There was no association between serum IL-6 levels and IL-6 genotype. The IL-6 promoter polymorphism -174 G/C does not contribute significantly to overall disease susceptibility but does predispose the carrier to distinct endometriosis with chocolate cysts. A genetically determined high IL-6 response might play a pathogenic role in this disease condition.

Comparison of p53 Mutational Status with mRNA and Protein Expression in a Panel of 24 Human Breast Carcinoma Cell Lines

We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines. Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH). A functional yeast assay (FASAY) was used to screen for inactivating mutations. Positive results were subsequently verified by DNA sequencing. Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry. The DNA content of the cell lines ranged from 0.85 to 2.58. Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies. The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles. The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations. Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results. However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident. The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression. Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.

Ten estrogen- related polymorphisms and endometriosis : A study of multiple gene-gene interactions

OBJECTIVE: Genetic as well as hormonal factors are known to influence the development and clinical course of endometriosis. We aimed to investigate the association among 10 single nucleotide polymorphisms (SNPs) involved in the estrogen metabolism and endometriosis and to develop a multiple genetic model. METHODS: In a case–control study, we investigated the genotype frequencies of 10 estrogen metabolizing SNPs in 32 patients with endometriosis and 790 healthy controls using sequencing-on-chip-technology with solid-phase polymerase chain reaction on oligonucleotide microarrays: catechol-O-methyltransferase, Val158Met G->A, 17-β-hydroxysteroid dehydrogenase type 1 (HSD17), vlV A->C, cytochrome P450 (CYP), 17 A2 allele T->C, CYP1A1 MspI RFLP T->C, CYP1A1 Ile462Val A->G, CYP19 Arg264Cys C->T, CYP19 C1558T C->T, CYP 1B1 Leu432Val, CYP1B1 Asn453Ser, and estrogen receptor alpha IVS1 –401>C. Associations and 2-way interaction models between SNPs were calculated by stepwise logistic regression models. RESULTS: In a univariate model, HSD17 vlV A->C was associated with a significantly increased risk of endometriosis (P = .004; odds ratio 3.9, 95% confidence interval 1.6–9.8). When all 2-way interactions of investigated SNPs were ascertained, no significant interactions among SNPs were observed. In a multivariate model, HSD17 vlV A->C was also significantly associated with endometriosis (P = .002). CONCLUSION: We present data on multiple SNPs in patients with endometriosis indicating an association between HSD17 gene variation and the disease. Although not able to demonstrate interaction models of SNPs, we provide evidence of HSD17 vlV A->C as a low penetrance genetic marker of endometriosis. LEVEL OF EVIDENCE: II-2

Tumor Necrosis Factor-α Promotor Polymorphisms and Endometriosis

Objective: To explore whether having the mutant tumor necrosis factor (TNF)2 (G-308*A) and TNFA-A (G-238*A) alleles in the TNF-α gene promotor region is higher in women with endometriosis, we determined the respective genotype and allele frequencies in a retrospective case-control study. Methods: Polymerase chain reaction was performed to identify the G-308A and G-238A promotor polymorphisms in 92 women with surgically and histologically confirmed endometriosis. A series of 69 healthy women without a history of endometriosis served as clinical controls. Results: The allele frequnecies of the TNF2 polymorphism were 0.13 and 0.16 in women with endometriosis and in the control group, respectively, and the frequencies of the TNFA-A polymorphisms in women with endometrisosis and in the control group were 0.04 and 0.05, respectively, with no significant difference between the study and control groups. The TNF2 polymorphism was present in the homozygous form (TNF2/2) in 4.3% of women with endometriosis and in 2.9% of controls (P = .7). No TNFA-A homozygotes (TNFAA/A) were detected. Conclusion: We studied TNF-α promotor gene variants among women with endometriosis and found that having the G-308A TNF-α and the G-238A TNF-α polymorphism was not associated with endometriosis in a white population.