Type 1 diabetes mellitus (T1DM) is associated with reduced fetal growth in early pregnancy, but a contributing role of the placenta has remained elusive. Thus, we investigated whether T1DM alters placental development in the first trimester. Using a protein array, the level of 60 cell-cycle-related proteins was determined in human first trimester placental tissue (gestational week 5–11) from control (n = 11) and T1DM pregnancies (n = 12). Primary trophoblasts (gestational week 7–12, n = 32) were incubated in the absence (control) or presence of hyperglycemia (25 mM D-glucose) and hyperosmolarity (5.5 mM D-glucose + 19.5 mM D-mannitol). We quantified the number of viable and dead trophoblasts (CASY Counter) and assessed cell cycle distribution (FACS) and trophoblast invasion using a transwell assay. T1DM was associated with a significant (p < 0.05) downregulation of Ki67 (−26%), chk1 (−25%), and p73 (−26%). The number of viable trophoblasts was reduced under hyperglycemia (−23%) and hyperosmolarity (−18%), whereas trophoblast invasion was increased only under hyperglycemia (+6%). Trophoblast cell death and cell cycle distribution remained unaffected. Collectively, our data demonstrate that hyperglycemia decreases trophoblast proliferation as a potential contributing factor to the reduced placental growth in T1DM in vivo.

SNP309T>G (rs2279744) and SNP285G>C (rs117039649) in the MDM2 promoter are thought to have opposite effects on the binding of transcription factor SP1 (specificity protein 1), and consequently on MDM2 expression, p53 levels, cancer risk, age at onset, and prognosis. Here, we genotyped SNP309 and SNP285 in 406 Austrian breast cancer patients and 254 female controls. The SNP309GG genotype was associated with an increased breast cancer risk in p53 negative (OR, 1.82; 95% CI, 1.09–3.03; p = 0.02), but not p53 positive or unselected patients. In contrast, the SNP309TT genotype was associated with an earlier age at onset (TT, 57.0 ± 12.9; TG, 58.6 ± 13.9; GG, 59.7 ± 15.0 years; p = 0.048). 31% of SNP309TT, 26% of TG, and 13% of GG tumors were p53 positive (p = 0.034), indicating a lower selective pressure to mutate TP53 in the presence of the G-allele. Moreover, SNP309TT patients exhibited a shortened metastasis-free survival in multivariable analysis. Censoring carriers of the SNP285C-allele hardly altered the strength of these associations of SNP309, thus challenging the proposed antagonistic function of SNP285C towards SNP309G. The minor SNP285C-allele tended to be non-significantly associated with an increased breast cancer risk and a poor disease-free and metastasis-free survival, which may be bystander effects of its complete linkage disequilibrium with SNP309G. We conclude that the SNP309G-allele attenuates the p53-response and leads to a higher breast cancer risk, but also to a later onset of breast cancer and a trend towards a good prognosis.

Telomerase reverse transcriptase (TERT) plays a key role in the maintenance of telomere DNA length. The rs10069690 single nucleotide variant, located in intron 4 of TERT, was found to be associated with telomere length and the risk of estrogen receptor-negative but not–positive breast cancer. This study aimed at analysis of the association of rs10069690 genotype and TERT expression with the risk, age at onset, prognosis, and clinically and molecularly relevant subtypes of breast cancer. Accordingly, rs10069690 was genotyped in a hospital-based case-control study of 403 female breast cancer patients and 246 female controls of a Central European (Austrian) study population, and the mRNA levels of TERT were quantified in 106 primary breast tumors using qRT-PCR. We found that in triple-negative breast cancer patients, the minor rs10069690 TT genotype tended to be associated with an increased breast cancer risk (OR, 1.87; 95% CI, 0.75–4.71; p = 0.155) and was significantly associated with 11.7 years younger age at breast cancer onset (p = 0.0002), whereas the CC genotype was associated with a poor brain metastasis-free survival (p = 0.009). Overall, our data show that the rs10069690 CC genotype and a high TERT expression tended to be associated with each other and with a poor prognosis. Our findings indicate a key role of rs10069690 in triple-negative breast cancer.

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5–intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5–intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon–intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing.

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.

In this focused genetic case–control study, we analyzed two functional single-nucleotide variants (SNVs) associated with breast cancer risk (rs2046210, rs9383590) and one risk SNV for an implantation defect and infertility (rs9340799) for their association with endometriosis susceptibility, progression and ESR1 gene regulation in endometriosis patients. The rs2046210, rs9383590 and rs9340799 SNVs were genotyped in 153 endometriosis patients and 87 control subjects with Caucasian ancestry. We analyzed the association of all SNVs with endometriosis susceptibility in all patients and in subgroups and assessed the concordance between the SNVs. Quantitative reverse transcription PCR was used to determine ESR1 gene expression in the eutopic endometrial tissue of the controls and endometriosis patients. The heterozygous rs2046210 GA genotype was associated with significantly increased endometriosis risk, particularly in younger, leaner and infertile women and with an increased ESR1 gene expression in the eutopic endometrium of these patients, compared to controls. The minor AA genotype of rs2046210 was identified as a potential risk factor for endometriosis progression in women with mild endometriosis. The results from this analysis indicate that rs2046210 may be a functional genetic variant associated with endometriosis development and progression.