Recurrent Copy Number Variants Associated with Syndromic Short Stature of Unknown Cause.

BACKGROUND/AIMS: Genetic imbalances are responsible for many cases of short stature of unknown etiology. This study aims to identify recurrent pathogenic copy number variants (CNVs) in patients with syndromic short stature of unknown cause. METHODS: We selected 229 children with short stature and dysmorphic features, developmental delay, and/or intellectual disability, but without a recognized syndrome. All patients were evaluated by chromosomal microarray (array-based comparative genomic hybridization/single nucleotide polymorphism array). Additionally, we searched databases and previous studies to recover recurrent pathogenic CNVs associated with short stature. RESULTS: We identified 32 pathogenic/probably pathogenic CNVs in 229 patients. By reviewing the literature, we selected 4 previous studies which evaluated CNVs in cohorts of patients with short stature. Taken together, there were 671 patients with short stature of unknown cause evaluated by chromosomal microarray. Pathogenic/probably pathogenic CNVs were identified in 87 patients (13%). Seven recurrent CNVs, 22q11.21, 15q26, 1p36.33, Xp22.33, 17p13.3, 1q21.1, 2q24.2, were observed. They are responsible for about 40% of all pathogenic/probably pathogenic genomic imbalances found in short stature patients of unknown cause. CONCLUSION: CNVs seem to play a significant role in patients with short stature. Chromosomal microarray should be used as a diagnostic tool for evaluation of growth disorders, especially for syndromic short stature of unknown cause.

Melatonin Regulates Angiogenic and Inflammatory Proteins in MDA-MB-231 Cell Line and in Co-culture with Cancer-associated Fibroblasts.

BACKGROUND: Cancer-associated fibroblast (CAFs) are the most abundant cells in the tumor microenvironment, able to secrete growth factors and act on tumor progression. Melatonin is associated with several mechanisms of action with oncostatics and oncoprotectors effects, and also participate in the reduction of synthesis of surrounding fibroblasts and endothelial cells in breast cancer. OBJECTIVE: The objectives of this study were to determine the effectiveness of melatonin in cell viability and expression of proteins involved in angiogenesis and inflammation in triplenegative mammary tumor cell line (MDA-MB-231) and in co-culture with CAFs. METHOD: Cell viability was measured by MTT assay and the protein expression was evaluated by Membrane Antibody Array after melatonin treatment. RESULTS: Melatonin treatment (1 mM) for 48 hours reduced the cell viability of MDA-MB-231, CAFs and co-culture (p < 0.05). The semi-quantitative protein analysis showed that when monoculture of tumor cells were compared with co-culture of CAFs, there was a regulation of angiogenic and inflammatory proteins (p < 0.05). Melatonin treatment also leads a differential expression of angiogenic and inflammatory proteins in both monoculture and co-culture of tumor cells and CAFs (p < 0.05). CONCLUSION: The influence of CAFs under the tumor microenvironment was confirmed, increasing the malignancy of the tumor. In addition, melatonin is effective in both monoculture and co-culture, regulating angiogenic and inflammatory proteins that contribute to tumor progression. This study show an overview of melatonin ability in regulating angiogenic and inflammatory proteins, and opens the way for exploration of each individual protein in further studies.

Specific upregulation of RHOA and RAC1 in cancer-associated fibroblasts found at primary tumor and lymph node metastatic sites in breast cancer.

The importance of tumor-stromal cell interactions in breast tumor progression and invasion is well established. Here, an evaluation of differential genomic profiles of carcinoma-associated fibroblasts (CAFs) compared to fibroblasts derived from tissues adjacent to fibroadenomas (NAFs) revealed altered focal adhesion pathways. These data were validated through confocal assays. To verify the possible role of fibroblasts in lymph node invasion, we constructed a tissue microarray consisting of primary breast cancer samples and corresponding lymph node metastasis and compared the expression of adhesion markers RhoA and Rac1 in fibroblasts located at these different locations. Two distinct tissue microarrays were constructed from the stromal component of 43 primary tumors and matched lymph node samples, respectively. Fibroblasts were characterized for their expression of α-smooth muscle actin (α-SMA) and vimentin. Moreover, we verified the level of these proteins in the stromal compartment from normal adjacent tissue and in non-compromised lymph nodes. Our immunohistochemistry revealed that 59 % of fibroblasts associated with primary tumors and 41 % of the respective metastatic lymph nodes (p = 0.271) displayed positive staining for RhoA. In line with this, 57.1 % of fibroblasts associated with primary tumors presented Rac1-positive staining, and the frequency of co-positivity within the lymph nodes was 42.9 % (p = 0.16). Expression of RhoA and Rac1 was absent in fibroblasts of adjacent normal tissue and in compromised lymph nodes. Based on our findings that no significant changes were observed between primary and metastatic lymph nodes, we suggest that fibroblasts are active participants in the invasion of cancer cells to lymph nodes and support the hypothesis that metastatic tumor cells continue to depend on their microenvironment.

Exploring the distribution of genetic markers of pharmacogenomics relevance in Brazilian and Mexican populations.

Studies of pharmacogenomics-related traits are increasingly being performed to identify loci that affect either drug response or susceptibility to adverse drug reactions. However, the effect of the polymorphisms can differ in magnitude or be absent depending on the population being assessed. We used the Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus array to characterize the distribution of polymorphisms of pharmacogenetics and pharmacogenomics (PGx) relevance in two samples from the most populous Latin American countries, Brazil and Mexico. The sample from Brazil included 268 individuals from the southeastern state of Rio de Janeiro, and was stratified into census categories. The sample from Mexico comprised 45 Native American Zapotecas and 224 self-identified Mestizo individuals from 5 states located in geographically distant regions in Mexico. We evaluated the admixture proportions in the Brazilian and Mexican samples using a panel of Ancestry Informative Markers extracted from the DMET array, which was validated with genome-wide data. A substantial variation in ancestral proportions across census categories in Brazil, and geographic regions in Mexico was identified. We evaluated the extent of genetic differentiation (measured as FST values) of the genetic markers of the DMET Plus array between the relevant parental populations. Although the average levels of genetic differentiation are low, there is a long tail of markers showing large frequency differences, including markers located in genes belonging to the Cytochrome P450, Solute Carrier (SLC) and UDP-glucuronyltransferase (UGT) families as well as other genes of PGx relevance such as ABCC8, ADH1A, CHST3, PON1, PPARD, PPARG, and VKORC1. We show how differences in admixture history may have an important impact in the distribution of allele and genotype frequencies at the population level.

The role of p38 mitogen-activated protein kinase in serum-induced leukemia inhibitory factor secretion by bone marrow stromal cells from pediatric myelodysplastic syndromes.

Stromal cells from pediatric myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) associated with MDS (MDS-AML) present high expression of leukemia inhibitor factor (LIF). We demonstrated using mitogen-activated protein kinase (MAPK) inhibitors that in stromal cells from pediatric MDS and MDS-AML, p38MAPK was critical in serum-induced secretion of LIF. The serum induction of phosphorylated p38MAPK form was observed only in stromal cells from healthy children, whereas in MDS and MDS-AML basal levels were maintained suggesting constitutive p38MAPK activation. Our study suggested the possible importance in pediatric MDS of p38MAPK signaling pathway which may be a future therapeutic target.

Gene stage-specific expression in the microenvironment of pediatric myelodysplastic syndromes.

Using cDNA microarray assays we have observed a clear difference in the gene expression pattern between bone marrow stromal cells obtained from healthy children (CT) and from pediatric patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) associated with MDS (MDS-AML). The global gene function profiling analysis indicated that in the pediatric MDS microenvironment the disease stages may be characterized mainly by underexpression of genes associated with biological processes such as transport. Furthermore, a subset of downregulated genes related to endocytosis and protein secretion was able to discriminate MDS from MDS-AML.

Bone marrow stroma in childhood myelodysplastic syndrome: composition, ability to sustain hematopoiesis in vitro, and altered gene expression.

We studied bone marrow stromal cell cultures from patients with childhood myelodysplastic syndromes (MDS, refractory anemia with excess of blasts, RAEB) and from matched normal donors. Stromal cell monolayers were characterized as myofibroblasts by the expression of smooth muscle alpha-actin, collagen IV, laminin and fibronectin. When normal cord blood cells were plated onto myelodysplastic stromas, a pathologic cell differentiation was observed, indicating altered myelosupportive properties. cDNA array analysis showed that patient stromas expressed increased levels of thrombospondin-1, collagen-I alpha2-chain, osteoblast-specific factor-2 and osteonectin, indicating the presence of increased osteoblast content, as confirmed by enhanced alkaline phosphatase synthesis. Alterations in the myelodysplastic stroma environment might contribute to abnormal hematopoiesis in this pathology.

Flow-cytometric DNA analysis and hormonal receptor status in breast carcinoma

The prognostic value of DNA ploidy in breast cancer relative to other prognostic factors is unsettled. We studied the nuclear DNA content and hormonal receptor levels of 57 frozen operable breast carcinomas using flow cytometry and a radiolabeled hormone binding assay. Tumor ploidy and S-phase fraction (SPF) was calculated from the DNA histogram. We found a statistically nonsignificant predominance of diploid cancer (65 per cent) in the ER+PR+ subgroup as compared to the ER-PR- subgroup (33 per cent). The high percentage of hypodiploid tumors (l2 per cent) compared to the literature (2 per cent), might be reflecting regional differences.Lower SPF were significantly correlated with features determining good prognosis like hormone receptor positivity or nodal status. Ploidy and DNA index presented a poor degree of correlation with these variables. We conclude that analysis of SPF and ploidy could be useful in the prognostic assessment of these cancer patients.