Technical note: developmental validation of a novel 41-plex Y-STR system for the direct amplification of reference samples.

The SureID® PathFinder Plus is a new 6-dye, 41-plex Y-STR system that includes the 17 loci from the Yfiler® kit (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) plus 14 rapidly mutating Y-STR loci (DYS449, DYS481, DYS518, DYS527a/b, DYS533, DYS549, DYS570, DYS576, DYS627, DYF387S1a/b, and DYF404S1), and 10 low-medium mutation loci (DYS388, DYS444, DYS447, DYS460, DYS522, DYS557, DYS593, DYS596, DYS643, and DYS645). The inclusion of the 14 rapidly mutating Y-STR loci improves the discrimination of related individuals. Conversely, the 10 low-medium mutation loci are suitable not only for familial searching but also for providing a higher refinement in the construction of Y chromosome phylogenetic relationships among lineages. The 41-plex Y-STR system is designed for direct amplification of reference samples, such as blood samples on an FTA® Card, gauze, tissue, or cotton substrates as well as hair root or buccal samples on swabs. We performed developmental validation work including accuracy, stability, stutter precision, species specificity, sensitivity, PCR inhibitors, reproducibility, parallel testing of the system, and suitability for use on DNA mixtures. In addition, mutations of the loci were analyzed by 754 DNA-confirmed father-son pairs. The results demonstrate that this kit, developed in-house, is time-efficient, accurate, reliable, and highly informative for forensic database, familial searching, and distinguishing related males.

Forensic and phylogenetic analyses of populations in the Tibetan-Yi corridor using 41 Y-STRs.

Y-chromosome haplotypes of 527 non-related males (176 Han, 186 Tibetan, and 165 Yi) in the Tibetan-Yi corridor were analyzed using SureID® PathFinder Plus. In the populations of Han, Tibetans, and Yi, the haplotype diversity was 0.9989, 0.9981, and 0.9993, respectively, and the discrimination capacity was 0.9148, 0.8925, and 0.9576, respectively. Phylogenetic relationships among 12 studied ethnic groups and 7 other ethnic groups in the Tibetan-Yi corridor were investigated. Both multi-dimensional scaling analysis and phylogenetic reconstructions indicated that Tibetans appeared separated from the Han and Yi ethnic groups in the Tibetan-Yi corridor. Their genetic homogeneity or heterogeneity has not entirely been affected by their geographical distance and linguistic origin.

Histone 3 Trimethylation of IGFBP-7 Gene Promoter by Expression of D5 Stat5a in Breast Epithelial Cells.

As a very important transcription factor, signal transducer and activator of transcription 5a (Stat5a) has been reported to be involved in human reproductive cancers such as breast, prostate and ovarian cancer. However, up to date, the exact role of Stat5a in breast cancer is still not clear. The data reported are conflicting. D5 Stat5a is a variant of Stat5a we cloned recently. This newly cloned variant behave like its full length counterpart in terms of dimerization, being activated by prolactin and nuclear translocation, however it also behave differently in terms of effect on cell proliferation and interaction with other transcription factors. In the present study, we examined its effect on cell proliferation of cultured breast cancer cells (MCF-10A and MCF-7) by using adenovirus-mediated gene transfer and MTS technology. Also, quantitative real time polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation assay (ChIP) and Western blot were used to probe the possible changes of insulin-like growth factor binding protein-7 (IGFBP-7) expression including mRNA and protein, and the epigenetic changes with overexpression of this newly cloned variant. The results clarified that D5 Stat5a (1) behaves as a promoting factor to the cell proliferation of MCF-10A and MCF-7, (2) induces enhancer of zeste homology 2 (EZH2) expression in breast epithelial cells, as well as histone 3 trimethylation of IGFBP-7 promoter region, and (3) lower IGFBP-7 expression was detected in breast cancer tissue. Taking together, we concluded that the mitogenic effect of D5 Stat5a on breast cells is, at least partly, through up-regulation of histone methyltransferase, EZH2, and therefore inhibiting IGFBP-7 expression by increasing H3K27Me3 of IGFBP-7 promoter region.

Histone trimethylation of the p53 gene by expression of a constitutively active prolactin receptor in prostate cancer cells.

Prolactin (PRL) is a pituitary polypeptide hormone characterized by multiple biological actions including stimulation of growth in the prostate, breast and ovarian epithelial cells. A sizable body of reports has presented evidences to indicate the involvement of PRL in the pathogenic process of cancers of the reproductive system, such as prostate and breast cancers. PRL exerts its effects by dimerizing its receptor (PRLR) on the plasma membrane, and initiating cellular Jak-Stat signal pathway. We have previously cloned from prostate cancer cells a natural variant of PRLR in which the S2 subdomain of the extracellular domain is missing (ΔS2). Our preliminary data showed that ΔS2 PRLR was able to dimerize and to constitutively activate the ß-casein promoter (in the absence of its ligand, PRL) in breast and prostate epithelial cells. Enhancer of zeste homologue 2 (EZH2), an important histone-modifying enzyme, is able to trimethylate histone 3 on lysine 27 (H3K27Me3), consequently leading to gene silencing, especially silencing of tumor suppressor genes such as p53. We hypothesized that ΔS2 PRLR played an important pathogenic role in prostate cancer through, at least partly, alterations in the expression of EZH2 and the trimethylation of histone 3 on lysine 27. In the present study, overexpression of ΔS2 PRLR in prostate epithelial cells was achieved by infection with an adenoviral vector carrying the cDNA. The viable cell number overexpressing ΔS2 PRLR was assessed using MTS reagent. Western blot, chromatin immunoprecipitation (ChIP) assay and acid histone extraction were applied to detect expression of EZH2 as well as trimethylation of histone 3, respectively. In prostate epithelial cells, overexpression of ΔS2 PRLR increased the levels of EZH2 methyltransferase mRNA and protein, induced EZH2 methyltransferase recruitment to chromatin, increased the trimethylation of histone 3 on lysine 27, and decreased expression of the p53 gene. We concluded that ΔS2 PRLR plays an important pathogenic role in prostate cancer through epigenetic covalent modification leading to chromatin remodeling. Hypertrimethylation on H3K27 of the p53 gene promoter region due to elevated expression of ΔS2 PRLR by alternative splicing of the pre-mRNA in its full-length form might serve as a new mechanism underlying human prostate cancer.

Efficacy and safety of neoadjuvant pertuzumab plus trastuzumab in combination with chemotherapy regimen in Chinese patients with HER2-positive early breast cancer: a real-world retrospective multi-center cohort study.

Background: Pertuzumab plus trastuzumab combined with chemotherapy has become a standard neoadjuvant therapy option for patients with high-risk human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC). There is still not enough evidence for the efficacy and safety of neoadjuvant pertuzumab and trastuzumab plus chemotherapy in HER2-positive BC patients in China, both in clinical trials and real-world settings. This study aimed to assess the efficacy and safety of neoadjuvant pertuzumab plus trastuzumab in combination with chemotherapy in Chinese patients with HER2-positive BC in real-world clinical application. Methods: We retrospectively collected the data from the electronic medical records of HER2-positive patients treated with neoadjuvant trastuzumab and pertuzumab plus chemotherapy from December 2018 to May 2021 at 21 hospitals located in Hunan Province, China, including age, American Joint Committee on Cancer (AJCC) stage, clinical tumor size, clinical lymph node status, pathological characteristics (before neoadjuvant systemic therapy), treatment approach, adverse events to neoadjuvant therapy, and achievement of pathological complete response (pCR). The primary endpoint was the total rate of pCR, and the secondary endpoints were the rate of pCR of each subgroup and the safety of dual anti-HER2 therapy. Results: A total of 188 patients met the inclusion criteria and were included in the analysis. Of the 188 patients, 119 (63.3%) were diagnosed at stage II and 64 (34.0%) at stage III; 163 (86.7%) were cT2-3; 149 patients (79.3%) were ≥ cN1; 84 patients (44.7%) were hormone receptor (HR)-positive. pCR was observed in 88 of 188 patients (46.8%). The pCR rate of HR-negative patients (54.8%) was higher (P=0.014) than that of HR-positive patients (36.9%). Patients with Ki-67 <15% achieved a higher (P=0.033) pCR rate (68.2%) than those with Ki-67 ≥15% (44.0%). Anemia was the most common adverse event (63.4%), and the most common grade 3-4 adverse event was nausea and vomiting (8.5%). Conclusions: Our study confirmed the benefit of neoadjuvant pertuzumab plus trastuzumab in combination with chemotherapy on pCR with a tolerable safety profile in routine clinical practice in Chinese patients with HER2-positive BC. HR-negativity and Ki-67 <15% were associated with pCR in these patients.

Analyzing genetic polymorphism and mutation of 44 Y-STRs in a Chinese Han population of Southern China.

Short tandem repeat on the non-recombining part of chromosome Y with paternally inheritable capability is a valuable tool in the studies of forensic genetics, population genetics and anthropology. The mutation rate of Y-STR is an important parameter in the applications. A total of 629 haplotypes at 44 Y-STR markers were found in 629 unrelated males of our population. Mutation rates at 44 Y-STR loci ranged from 0 (CI: 0-5.70 × 10-3) to 40.63 × 10-3 (25.90 × 10-3-57.2 × 10-3) in our population. A higher mutation rate was noted at DYS612, DYS449, DYS547, DYS518, DYS576, DYS627, DYF403S1b, DYF387S1, DYS385a/b, DYS527a/b, DYF404S1, DYF403S1a and DYF399S1 in this population. The Y-STR set showed a higher discrimination capacity in forensic applications, and the present study provided reference data for the application of forensic and population genetics.

Inhibitor of apoptosis protein­like protein­2: A novel growth accelerator for breast cancer cells.

Although the inhibitor of apoptosis protein­like protein­2 (ILP­2) has been shown as a serological biomarker for breast cancer, its effect on breast cancer cell growth remains elusive. The present study aimed to determine the role of ILP­2 in breast cancer cell growth. We used immunohistochemistry to analyze ILP­2 expression in 59 tissue paraffin­embedded blocks, which included 35 breast cancer tissues and 24 galactophore hyperplasia tissues. Western blot analysis was used to detect protein expression levels of ILP­2 in breast cancer cell lines such as HCC­1937, MX­1 and MCF­7 as well as breast gland cell line MCF 10A. ILP­2 was silenced by siRNA in HCC­1937, MX­1 and MCF­7 cell lines. MTT assays, scratch assays and AO­EB double staining analysis were conducted to evidence the role of ILP­2 on breast cancer cell growth. Results from this study showed increased ILP­2 expression in breast cancer tissues and breast cancer cell lines such as HCC­1937, MX­1 and MCF­7. Cell viability or rate of cell migration of HCC­1937, MX­1 and MCF­7 cell lines was significantly inhibited when ILP­2 was knocked down by siRNA. The apoptosis rate of HCC­1937, MX­1 and MCF­7 cell lines was increased when compared with that of the control group. Thus, ILP­2 plays an active role in the growth of breast cancer cells.

Expression of a constitutively active prolactin receptor causes histone trimethylation of the p53 gene in breast cancer.

BACKGROUND: Prolactin (PRL) is a pituitary polypeptide hormone characterized by multiple biological actions including stimulation of growth in the prostate and formation of secretory alveoli and stimulation of milk protein gene expression in the mammary gland. PRL exerts its effect by dimerizing its receptor (PRLR) on the plasma membrane and regulating gene expression through the JAK-Stat signal pathway. We have previously described a natural variant of the PRLR in which the S2 subdomain of the extracellular domain is missing (Delta S2). Delta S2 PRLRs are dimerized in the absence of PRL and have constitutive activity in the promotion of breast cancer cell growth. Enhancer of zeste homolog 2 (EZH2), as one of the histone-modifying enzymes, is a key factor regulating gene expression by epigenetic modification. We hypothesized that these constitutive activated Delta S2 PRLRs played a pathogenic role in breast cancer in part through alterations in the expression of EZH2 and the trimethylation of histone 3 on lysine 27 (H3K27Me3). METHODS: In order to verify the clinical significance and to establish the link between Delta S2 PRLR expression and epigenetic change, EZH2, H3K27Me3, and Delta S2 PRLR were detected in both normal and cancerous human breast tissues. Also, overexpression of Delta S2 PRLR in breast epithelial cells was achieved by infection with adenovirus carrying the cDNA. Western blotting and chromatin immunoprecipitation (ChIP assay) and acid histone extraction were applied to detect the expression of EZH2 and the trimethylation of histone 3, respectively. RESULTS: In breast tissue, higher EZH2 expression and higher H3K27Me3 were found associated with higher Delta S2 expression in breast cancer samples. In breast epithelial cells, overexpression of Delta S2 PRLR increased EZH2 methyltransferase mRNA and protein, induced EZH2 methyltransferase recruitment to chromatin, increased the trimethylation of H3K27Me3, and decreased the expression of p53 gene. CONCLUSIONS: Delta S2 PRLR plays an important pathogenic role in breast cancer through epigenetic modification. Elevated expression of Delta S2 PRLR, achieved by alternate splicing of the pre-mRNA of the full-length form, is a new mechanism contributing to human breast cancer.

An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma.

We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1-2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with ß-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma.