The Reprimo-Like Gene Is an Epigenetic-Mediated Tumor Suppressor and a Candidate Biomarker for the Non-Invasive Detection of Gastric Cancer.

Reprimo-like (RPRML) is an uncharacterized member of the Reprimo gene family. Here, we evaluated the role of RPRML and whether its regulation by DNA methylation is a potential non-invasive biomarker of gastric cancer. RPRML expression was evaluated by immunohistochemistry in 90 patients with gastric cancer and associated with clinicopathologic characteristics and outcomes. The role of RPRML in cancer biology was investigated in vitro, through RPRML ectopic overexpression. Functional experiments included colony formation, soft agar, MTS, and Ki67 immunofluorescence assays. DNA methylation-mediated silencing was evaluated by the 5-azacytidine assay and direct bisulfite sequencing. Non-invasive detection of circulating methylated RPRML DNA was assessed in 25 gastric cancer cases and 25 age- and sex-balanced cancer-free controls by the MethyLight assay. Downregulation of RPRML protein expression was associated with poor overall survival in advanced gastric cancer. RPRML overexpression significantly inhibited clonogenic capacity, anchorage-independent growth, and proliferation in vitro. Circulating methylated RPRML DNA distinguished patients with gastric cancer from controls with an area under the curve of 0.726. The in vitro overexpression results and the poor patient survival associated with lower RPRML levels suggest that RPRML plays a tumor-suppressive role in the stomach. Circulating methylated RPRML DNA may serve as a biomarker for the non-invasive detection of gastric cancer.

Identification of c.1531C>T Pathogenic Variant in the CDH1 Gene as a Novel Germline Mutation of Hereditary Diffuse Gastric Cancer.

Germline pathogenic variants in the CDH1 gene are a well-established cause of hereditary diffuse gastric cancer (HDGC) syndrome. The aim of this study was to characterize CDH1 mutations associated with HDGC from Chile, a country with one of the highest incidence and mortality rates in the world for gastric cancer (GC). Here, we prospectively include probands with family history/early onset of diffuse-type of GC. The whole coding sequence of the CDH1 gene was sequenced from genomic DNA in all patients, and a multidisciplinary team managed each family member with a pathogenic sequence variant. Thirty-six cases were included (median age 44 years/male 50%). Twenty-seven (75%) patients had diffuse-type GC at ≤50 years of age and 19 (53%) had first or second-degree family members with a history of HDGC. Two cases (5.5%) carried a non-synonymous germline sequence variant in the CDH1 gene: (a) The c.88C>A missense variant was found in a family with three diffuse-type GC cases; and (b) c.1531C>T a nonsense pathogenic variant was identified in a 22-year-old proband with no previous family history of HDGC. Of note, six family members carry the same nonsense pathogenic variant. Prophylactic gastrectomy in the proband's sister revealed stage I signet-ring cell carcinoma. The finding of 1531C>T pathogenic variant in the CDH1 in proband with no previous family history of HDGC warrants further study to uncover familial clustering of disease in CDH1 negative patients. This finding may be particularly relevant in high incidence countries, such as the case in this report.

The Phylogeographic Diversity of EBV and Admixed Ancestry in the Americas⁻Another Model of Disrupted Human-Pathogen Co-Evolution.

Epstein-Barr virus (EBV) is an etiological agent for gastric cancer with significant worldwide variations. Molecular characterizations of EBV have shown phylogeographical variations among healthy populations and in EBV-associated diseases, particularly the cosegregated BamHI-I fragment and XhoI restriction site of exon 1 of the LMP-1 gene. In the Americas, both cosegregated variants are present in EBV carriers, which aligns with the history of Asian and European human migration to this continent. Furthermore, novel recombinant variants have been found, reflecting the genetic makeup of this continent. However, in the case of EBV-associated gastric cancer (EBV-associated GC), the cosegregated European BamHI-"i" fragment and XhoI restriction site strain prevails. Thus, we propose that a disrupted coevolution between viral phylogeographical strains and mixed human ancestry in the Americas might explain the high prevalence of this particular gastric cancer subtype. This cosegregated region contains two relevant transcripts for EBV-associated GC, the BARF-1 and miR-BARTs. Thus, genome-wide association studies (GWAS) or targeted sequencing of both transcripts may be required to clarify their role as a potential source of this disrupted coevolution.

Genetic Variants in pre-miR-146a, pre-miR-499, pre-miR-125a, pre-miR-605, and pri-miR-182 Are Associated with Breast Cancer Susceptibility in a South American Population.

Breast cancer (BC) is one of the most frequent tumors affecting women worldwide. microRNAs (miRNAs) single-nucleotide polymorphisms (SNPs) likely contribute to BC susceptibility. We evaluated the association of five SNPs with BC risk in non-carriers of the BRCA1/2-mutation from a South American population. The SNPs were genotyped in 440 Chilean BRCA1/2-negative BC cases and 1048 controls. Our data do not support an association between rs2910164:G>C or rs3746444:A>G and BC risk. The rs12975333:G>T is monomorphic in the Chilean population. The pre-miR-605 rs2043556-C allele was associated with a decreased risk of BC, both in patients with a strong family history of BC and in early-onset non-familial BC (Odds ratio (OR) = 0.5 [95% confidence interval (CI) 0.4⁻0.9] p = 0.006 and OR = 0.6 [95% CI 0.5⁻0.9] p = 0.02, respectively). The rs4541843-T allele is associated with increased risk of familial BC. This is the first association study on rs4541843 and BC risk. Previously, we showed that the TOX3-rs3803662:C>T was significantly associated with increased risk of familial BC. Given that TOX3 mRNA is a target of miR-182, and that both the TOX3 rs3803662-T and pri-miR-182 rs4541843-T alleles are associated with increased BC risk, we evaluated their combined effect. Risk of familial BC increased in a dose-dependent manner with the number of risk alleles (p-trend = 0.0005), indicating an additive effect.

Identification of a Rare Germline Heterozygous Deletion Involving the Polycistronic miR-17-92 Cluster in Two First-Degree Relatives from a BRCA 1/2 Negative Chilean Family with Familial Breast Cancer: Possible Functional Implications.

Micro-RNAs (miRNAs) have emerged as novel gene expression regulators. Recent evidence strongly suggests a role for miRNAs in a large variety of cancer-related pathways. Different studies have shown that 18.7 to 37% of all human miRNA genes are clustered. miR-17-92 polycistronic cluster overexpression is associated with human hematolymphoid and solid malignancies including breast cancer (BC). Here, we report the identification of rs770419845, a rare 6 bp deletion located within the polycistronic miR-17-92 cluster, in two first-degree relatives from a Chilean family with familial BC and negative for point mutations in BRCA 1/2 genes. The deletion was identified by Sanger sequencing when 99 BRCA1/2 mutation-negative BC cases with a strong family history were initially screened. In silico analysis predicts that rs770419845 affects the secondary structure and stability of the pre-miR-17-pre-miR-18 region and the entire 17-92 cluster. The deletion was screened in 458 high-risk BRCA1/2-negative Chilean families and 480 controls. rs770419845 was not detected in any control but identified in a single family with two cases of BC and other cancers. Both BC cases, the mother and her daughter, carried the deletion. Based on bioinformatic analyses, the location of the deletion and its low frequency, we presume rs770419845 may be a pathogenic variant. Functional studies are needed to support this hypothesis.

Mutations in BRCA1, BRCA2 and other breast and ovarian cancer susceptibility genes in Central and South American populations.

Breast cancer (BC) is the most common malignancy among women worldwide. A major advance in the understanding of the genetic etiology of BC was the discovery of BRCA1 and BRCA2 (BRCA1/2) genes, which are considered high-penetrance BC genes. In non-carriers of BRCA1/2 mutations, disease susceptibility may be explained of a small number of mutations in BRCA1/2 and a much higher proportion of mutations in ethnicity-specific moderate- and/or low-penetrance genes. In Central and South American populations, studied have focused on analyzing the distribution and prevalence of BRCA1/2 mutations and other susceptibility genes that are scarce in Latin America as compared to North America, Europe, Australia, and Israel. Thus, the aim of this review is to present the current state of knowledge regarding pathogenic BRCA variants and other BC susceptibility genes. We conducted a comprehensive review of 47 studies from 12 countries in Central and South America published between 2002 and 2017 reporting the prevalence and/or spectrum of mutations and pathogenic variants in BRCA1/2 and other BC susceptibility genes. The studies on BRCA1/2 mutations screened a total of 5956 individuals, and studies on susceptibility genes analyzed a combined sample size of 11,578 individuals. To date, a total of 190 different BRCA1/2 pathogenic mutations in Central and South American populations have been reported in the literature. Pathogenic mutations or variants that increase BC risk have been reported in the following genes or genomic regions: ATM, BARD1, CHECK2, FGFR2, GSTM1, MAP3K1, MTHFR, PALB2, RAD51, TOX3, TP53, XRCC1, and 2q35.

The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice.

Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8-10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.

Variants in DNA double-strand break repair genes and risk of familial breast cancer in a South American population.

The double-strand break (DSB) DNA repair pathway has been implicated in breast cancer (BC). RAD51 and its paralogs XRCC3 and RAD51D play an important role in the repair of DSB through homologous recombination (HR). Some polymorphisms including XRCC3-Thr241Met, RAD51-135G>C, and RAD51D-E233G have been found to confer increased BC susceptibility. In order to detect novel mutations that may contribute to BC susceptibility, 150 patients belonging to 150 Chilean BRCA1/2-negative families were screened for mutations in XRCC3. No mutations were detected in the XRCC3 gene. In addition, using a case-control design we studied the XRCC3-Thr241Met, and RAD51D-E233G polymorphisms in 267 BC cases and 500 controls to evaluate their possible association with BC susceptibility. The XRCC3 Met/Met genotype was associated with an increased BC risk (P = 0.003, OR = 2.44 [95%CI 1.34-4.43]). We did not find an association between E233G polymorphism and BC risk. We also analyzed the effect of combined genotypes among RAD51-135G>C, Thr241Met, and E233G polymorphisms on BC risk. No interaction was observed between Thr241Met and 135G>C. The combined genotype Thr/Met-E/G was associated with an increased BC risk among women who (a) have a family history of BC, (b) are BRCA1/2-negative, and (c) were <50 years at onset (n = 195) (P = 0.037, OR = 10.5 [95%CI 1.16-94.5]). Our results suggested that the variability of the DNA HR repair genes XRCC3 and RAD51D may play a role in BC risk, but this role may be underlined by a mutual interaction between these genes. These findings should be confirmed in other populations.