BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements.Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described.In conclusion: a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina.

No abstract

In the past, human endometrial receptivity has been investigated by chasing specific molecules throughout the menstrual cycle. Now the genomic approach allows us to investigate the hierarchical contribution of a high number of genes to a specific function. In this study, we analyzed differentially the gene expression pattern of 375 human cytokines, chemokines, and related factors, plus that of their receptors, in endometrial receptivity. To do this, we used a combined approach of human endometrium and cell lines. We have compared the gene expression pattern in receptive vs. prereceptive human endometria and contrasted the results with gene expression in the highly adhesive cell line (to JAR cells and mouse blastocysts) RL95-2 vs. HEC-1A, a cell line with markedly less adhesiveness. IGF-binding protein-related protein 1 (IGFBP-rP1), also known as IGFBP-7/mac 25, was the second most up-regulated gene in both of the investigated models. These results were corroborated by performing RT-PCR on the same RNA samples and validated by quantitative fluorescent RT-PCR and in situ hybridization in endometrium throughout the menstrual cycle. Interestingly, a 35-fold increase in expression during the receptive phase was compared with the prereceptive phase followed by a sharp increase in the late luteal. Further quantitative fluorescent RT-PCR experiments using the epithelial and stromal endometrial fraction throughout the menstrual cycle confirmed that IGFBP-rP1 expression was localized in the epithelial and stromal compartments and up-regulated mainly in the latter. In situ experiments confirmed the endometrial localization and regulation of IGFBP-rP1 mRNA. At the protein level, IGFBP-rP1 was localized by immunohistochemistry at the apical part of the luminal and glandular epithelium, stromal, and endothelial cells. In conclusion, using a genomic approach with a combined experimental design of receptivity in vivo and in vitro, we have discovered the implication of IGFBP-rP1 in endometrial physiology, which seems related to endometrial receptivity.

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.

Rotavirus is a leading cause of acute diarrhea in children worldwide. Costa Rica recently started universal rotavirus vaccinations for infants with a two-dose schedule in February 2019. We aimed to study the seasonality of rotavirus during the pre-vaccination era. We retrospectively studied a six-year period of hospital admissions due to rotavirus gastroenteritis. We estimated seasonal peak timing and relative intensities using trend-adjusted negative binomial regression models with the δ-method. We assessed the relationship between rotavirus cases and weather characteristics and estimated their effects for the current month, one-month prior and two months prior, by using Pearson correlation coefficients. A total of 798 cases were analyzed. Rotavirus cases predominated in the first five months of the year. On average, the peak of admissions occurred between late-February and early-March. During the seasonal peaks, the monthly count tended to increase 2.5–2.75 times above the seasonal nadir. We found the strongest negative association of monthly hospitalizations and joint percentiles of precipitation and minimal temperature at a lag of two months (R = −0.265, p = 0.027) and we detected correlations of −0.218, −0.223, and −0.226 (p < 0.05 for all three estimates) between monthly cases and the percentile of precipitation at lags 0, 1, and 2 months. In the warm tropical climate of Costa Rica, the increase in rotavirus hospitalizations coincided with dry and cold weather conditions with a two-month lag. The findings serve as the base for predictive modeling and estimation of the impact of a nation-wide vaccination campaign on pediatric rotaviral infection morbidity.

In humans, a germline mutation (c.309C>G) in the TWIST1 oncogene may predispose to breast cancer and its expression has been associated with tumour progression and metastasis. In this study, the feline TWIST1 gene was screened for sequence variations in 37 neoplastic and eight hyperplastic mammary gland lesions from cats. In addition, mRNA levels were examined in 15 mammary tumours and three cases of mammary hyperplasia by quantitative real-time reverse-transcriptase PCR. Feline mammary carcinomas had significantly lower levels of expression of TWIST1 mRNA than benign mammary tumours. No variations were identified in the TWIST1 coding region in feline mammary tumours and the mutation described in humans was not detected. However, two germline variants in the TWIST1 gene intron were identified in four and three carcinomas, respectively: GQ167299:g.535delG and GQ167299:g.460C>T. There was no association between these sequence alterations and TWIST1 mRNA levels.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH ), prolactin (PRL), thyroid-stimulating hormone (TSH ) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-β, with a 78 bp insert in the trans-activation domain; POU1F1-γ that lacks exon 3 and POU1F1-δ that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the β variant presented only 12% of the Wild-type trans-activation capacity. Variants γ and δ showed no capacity to trans-activate PRL promoter. Both γ and δ variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone ( p < 0.05). Concerning the GH promoter, the β variant presented a trans-activation capacity 10% higher than Wt. Wt and β variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants γ and δ showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly ( p < 0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.

Angiogenesis is a fundamental step in the transition of solid tumors from a dormant state to a malign one. Many of the low-molecular-weight anti-angiogenic drug candidates mimic the short peptide epitope Arg-Gly-Asp (RGD), [1] disrupting the extracellular matrix/integrin adhesion and, ultimately, leading to tumor cell apoptosis. In contrast to the detailed structural information available for the extracellular adhesion inhibition of endothelial cells through the recognition of integrins (typically a V b 3 ) by RGD peptidomimetics, [2] most aspects of possible intracellular angiogenic gene regulation caused by peptidomimetics remain unexplored.[3] Importantly, dysfunction of this signaling system is suspected to be behind the resistance phenomena developed in anti-angiogenic therapies. [4] Inside the endothelial cell, dozens of proteins mediate or control the signaling pathways of angiogenesis after integrin activation, but only a couple of kinases (JNK, ERK) and transcription factors (NFkB, FoxO) are able to promote gene regulation [5] (Figure 1). In addition, large environmental ligands, such as vascular endothelial growth factors (VEGFs) [6] or peptide hormones, [7] are required to elicit proangiogenic gene regulation. In this context, we set out to design alternative lowmolecular-weight RGD probes for interaction with a V b 3 integrin and gene regulation in HUVECs. Ideally, these peptidomimetics should: 1) contain a minimal scaffold to prevent undesired scaffold/integrin interactions, 2) have a uniform and predictable scaffold conformation, 3) bear a maximum of recognition groups, including hydrophobic or hydrophilic ones, and 4) permit the deletion of selected residues from the RGD triad without global shape change. We selected five-membered and four-membered small cyclic peptides for development. The residual flexibility of these cyclopeptides can be further constrained by incorporating lactam bridges between neighboring amino acids to stabilize protein secondary structure motifs characterized by combinations of b-turns and/or g-turns.[8] Several lactam pseudopeptides resulting from such an extension of Freidinger's concept [9] (Scheme 1; 1!2) have been explored with the goals of mimicking receptor-bound conformations of bioactive peptides and of providing pharmacophore information for nonpeptide drug design.[10] However, despite its apparent simplicity, this design is not always reliable for the precise positioning of a maximum number of recognition groups around the pseudopeptide cyclic core. Because the interresidual lactam bridge created by modification of the side chain (R 1 ) shares recognition and constraint functions, the design of mimetic libraries becomes difficult and non-general, when synthetically achievable. An alternative way to constrain cyclic peptides is based on the incorporation of a d-amino acid and an N-alkyl-amino acid into the macrocycle, as illustrated by the remarkable a V b 3 antagonist cilengitide (3, cyclo-[Arg-Gly-Asp-d-Phe-N(Me)Val]) developed by Kessler et al. [11] Here we re...