Prediction of the antigenic regions in eight RhD variants identified by computational biology.

BACKGROUND AND OBJECTIVES: Changes in RHD generate variations in protein structure that lead to antigenic variants. The classical model divides them into quantitative (weak and Del) and qualitative (partial D). There are two types of protein antigens: linear and conformational. Computational biology analyses the theoretical assembly of tertiary protein structures and allows us to identify the 'topological' differences between isoforms. Our aim was to determine the theoretical antigenic differences between weak RhD variants compared with normal RhD based on structural analysis using bioinformatic techniques. MATERIALS AND METHODS: We analysed the variations in secondary structures and hydrophobicity of RHD*01, RHD*01W.1, W2, W3, RHD*09.03.01, RHD*09.04, RHD*11, RHD*15 and RHD*21. We then modelled the tertiary structure and calculated their probable antigenic regions, intra-protein interactions, displacement and membrane width and compared them with Rhce. RESULTS: The 10 proteins are similar in their secondary structure and hydrophobicity, with the main differences observed in the exofacial coils. We identified six potential antigenic regions: one that is unique to RhD (R3), one that is common to all D (R6), three that are highly variable among RhD isoforms (R1, R2 and R4), one that they share with Rhce (R5) and two that are unique to Rhce (Ra and Rbc). CONCLUSION: The alloimmunization capacity of these subjects could be explained by the variability of the antigen pattern, which is not necessarily recognized or recognized with lower intensity by the commercially available antibodies, and not because they have a lower protein concentration in the membrane.

Use of computational biology to compare the theoretical tertiary structures of the most common forms of RhCE and RhD.

BACKGROUND AND OBJECTIVES: Computational biology analyses the theoretical tertiary structure of proteins and identifies the 'topological' differences between RhD and RhCE. Our aim was to identify the theoretical structural differences between the four isoforms of RhCE and RhD using computational biological tools. MATERIALS AND METHODS: Physicochemical profile was determined by hydrophobicity and electrostatic potential analysis. Secondary and tertiary structures were generated using computational biology tools. The structures were evaluated and validated using Ramachandran algorithm, which calculates the single score, p-value and root mean square deviation (RMSD). Structures were overlaid on local refinement of 'RhAG-RhCE-ANK' (PBDID 7uzq) and RhAG to compare their spatial distribution within the membrane. RESULTS: All proteins differed in surface area and electrostatic distance due to variations in hydrophobicity and electrostatic potential. The RMSD between RhD and RhCE was 0.46 ± 0.04 Å, and the comparison within RhCE was 0.57 ± 0.08 Å. The percentage of amino acids in the hydrophobic thickness was 50.24% for RhD while for RhCE it ranged between 73.08% and 76.68%. The RHAG hydrophobic thickness was 34.2 Å, and RhCE's hydrophobic thickness was 33.83 Å. We suggest that the C/c antigens differ exofacially at loops L1 and L2. For the E/e antigens, the difference lies in L6. By contrast, L4 is the same for all proteins except Rhce. CONCLUSION: The physicochemical properties of Rh proteins made them different, although their genes are homologous. Using computational biology, we model structures with sufficient precision, similar to those obtained experimentally. An amino acid variation alters the folding of the tertiary structure and the interactions with other proteins, modifying the electrostatic environment, the spatial conformations and therefore the antigenic recognition.

Analysis of a Novel Mexican Variant of the HBB Gene Associated with ß-Thalassemia Using Bioinformatic Tools.

We present a study performed on 54 unrelated subjects, with and without thalassemic features. Two primer pairs were proposed to perform Sanger sequencing of the complete HBB gene. The bioinformatic analysis was performed taking advantage of the availability of free online tools. In the sample, we found 11 variants, 10 reported, and one novel. Among the variants found, six are clinically important: three encode a premature stop codon [codon 39 (C>T) (HBB: c.118C>T); IVS-II-1 (G>A) (HBB: c.315+1G>A), and one not reported], a double substitution within the same allele [Hb Borås (HBB: c.266T>G) and Hb Santa Giusta Sardegna (HBB: c.282T>C)], and one whose pathogenicity is not yet defined [Hb Fannin-Lubbock I (HBB: c.359G>A)]. Even though the variants Hb Borås and Hb Santa Giusta Sardegna have been described, there is no report of their combined occurrence on the same allele, which could cause hemolytic anemia. Although the p.Leu88Arg and p.Cys93Trp variants do not alter the final length of the protein, the bioinformatic results suggest that there are differences in the tertiary structure of ß-globin genes, mainly affecting helices E and F, being the motifs of interaction with the heme group. The novel variant is a 4 bp insertion that modifies the open reading frame, changing the last amino acid residue and causing a premature stop codon (HBB: c.291-294insGCAC). The variant was associated with ß-thalassemia (ß-thal). Bioinformatic analysis made it possible to predict the consequences that the new variant of the HBB gene caused on the ß-globin tertiary structure.

Hyperglycemia differentially affects proliferation, apoptosis, and BNIP3 and p53 mRNA expression of human umbilical cord Wharton's jelly cells from non-diabetic and diabetic pregnancies.

Diabetes in pregnancy constitutes an unfavorable environment for embryonic and fetal development, where the child has a higher risk of perinatal morbidity and mortality, with high incidence of congenital malformations and predisposition to long-term metabolic diseases that increase with a hypercaloric diet. To analyze whether hyperglycemia differentially affects proliferation, apoptosis, and mRNA expression in cells from children of normoglycemic pregnancies (NGPs) and diabetes mellitus pregnancies (DMPs), we used umbilical cord Wharton jelly cells as a research model. Proliferation assays were performed to analyze growth and determine the doubling time, and the rate of apoptosis was determined by flow cytometry-annexin-V assays. AMPK, BNIP3, HIF1α, and p53 mRNA gene expression was assessed by semi-quantitative RT-PCR. We found that hyperglycemia decreased proliferation in a statistically significant manner in NGP cells treated with 40 mM D-glucose and in DMP cells treated with 30 and 40 mM D-glucose. Apoptosis increased in hyperglycemic conditions in NGP and DMP cells. mRNA expression of BNIP3 and p53 was significantly increased in cells from DMPs but not in cells from NGPs. We found evidence that maternal irregular metabolic conditions, like diabetes with hyperglycemia in culture, affect biological properties of fetal cells. These observations could be a constituent of fetal programming.

A fetus with hemifacial microsomia and sirenomelia. The same mesodermal defect spectrum?

Sirenomelia is the most severe malformation complex affecting the human caudal pole, although its etiology is unclear, a primary defect of blastogenesis has been proposed. Studies consider sirenomelia as the most severe form of caudal dysgenesis, VACTERL association, or axial mesodermal dysplasia, although others still support the idea of a different pathologic entity. We report the prenatal, clinical, and pathologic features of a fetus with cleft lip and palate, microtia, cardiac, renal and intestinal malformations, radial aplasia, and sirenomelia. Karyotype, chromosomal breakage studies, and SHH sequence analysis were normal. The occurrence of cephalic, midline-paramedial, and caudal malformations in the same patient imply the diagnosis of hemifacial microsomia and sirenomelia. These entities are part of the same mesodermal malformation spectrum and the clinical presentation depends on environmental and genetic interactions in embrionic development. Future clinical and genome wide studies will help to better delineate this spectrum.

Tissue-specific IL-10 secretion profile from term human fetal membranes stimulated with pathogenic microorganisms associated with preterm labor in a two-compartment tissue culture system.

OBJECTIVE: Interleukin (IL)-10 is a cytokine with anti-inflammatory properties that plays pivotal roles in immune recognition and maintenance of pregnancy, limiting the harmful effects of pro-inflammatory modulators. The aim of this work was to characterize the contribution of amnion and choriodecidua regions of the human fetal membranes in the production of IL-10 after selective stimulation with Candida albicans, Gardnerella vaginalis and Streptococcus agalactiae. METHODS: Pre-labor human fetal membranes were cultured in a two-compartment tissue culture system and stimulated with 1 × 10(6) CFU/ml of each pathogen added to either the amniotic or choriodecidual region or both. RESULTS: Candida albicans and G. vaginalis were the pathogens most effective in inducing IL-10 secretion, increasing 20 and 10 times, respectively, the levels of this cytokine in the choriodecidual compartment. Stimulation with S. agalactiae was effective only in the choriodecidual region, increasing two times IL-10 concentration. CONCLUSIONS: Synthesis and secretion of IL-10 in response to three different pathogens associated with intrauterine infection and preterm birth are differential and depend on the nature of the microorganism and initial contact region.

MLPA (amplificación de sondas dependiente de ligandos múltiples) en el diagnóstico perinatal rápido de las principales aneuploidías / MLPA (multiplex ligation-dependent probe amplification) in perinatal rapid diagnosis of major aneuploidies

Introducción: El MLPA (amplificación de sondas dependiente de ligandos múltiples) es un método reciente, basado en la reacción en cadena de la polimerasa (RCP), de cuantificación relativa del número de copias normales y anormales de ácido desoxirribonucleico (ADN) de hasta 40 secuencias genómicas diferentes. Su uso se ha difundido tanto en la investigación como en el diagnóstico clínico. Recientemente, el MLPA se ha utilizado en diagnóstico prenatal y en el estudio de abortos. Objetivo: Presentar los primeros resultados obtenidos con MLPA en el diagnóstico perinatal de las principales aneuploidías de los cromosomas 13, 18, 21, X y Y. Material y métodos: Se obtuvo ácido desoxirribonucleico genómico de tejidos sin cultivar de aborto, biopsia de vellosidades coriales (VC), líquido amniótico (LA), cordón umbilical (CU) de óbitos o sangre periférica (SP) de recién nacidos, en 13 casos con patología perinatal. Para el MLPA se utilizó el kit p 290 para diagnóstico prenatal y sus productos se cuantificaron en un secuenciador ABI Prism 3130. Todos los casos se procesaron para cariotipo en forma paralela. Resultados: Se obtuvieron resultados con MLPA entre dos y tres días en las 13 muestras estudiadas. Seis de ellas mostraron aneuploidías. El cariotipo se obtuvo entre 15 y 21 días en 11 casos, cinco fueron aneuploides y dos fallaron. Los resultados entre MLPA y cariotipo fueron concordantes en las fallas del cultivo celular, el diagnóstico se obtuvo por MLPA. Conclusiones: El MLPA es un método útil y rápido en el diagnóstico perinatal de las principales aneuploidías; además, tiene la ventaja de permitir estudiar tejidos con baja viabilidad celular como ocurre en algunos casos de aborto u óbito.

Introduction: The MLPA (multiplex ligation-dependent probe amplification) is a new method based on polimerase chain reaction (PCR) for relative quantification of normal and abnormal numbers of copies of desoxyribonucleic (DNA), up to 40 different genomic sequences. Its use is widespread in both, research and clinical diagnosis. MLPA recently began to be used in prenatal diagnosis and study of pregnancy losses. Objective: Present the first results obtained with MLPA in perinatal diagnosis of major aneuploidies of chromosomes 13, 18, 21, X and Y. Methods: Genomic DNA was obtained from different uncultivated tissues: abortion, chorionic villus, amniotic fluid, umbilical cord blood or peripheral blood in 13 cases with perinatal pathology. MLPA kit p290 for prenatal diagnosis was used and its products were quantified in ABI Prism 3130 sequencer. All cases were processed in parallel for karyotype. Results: MLPA results were obtained in 2 to 3 days in the 13 samples studied. Six of them showed aneuploidy. Karyotype was obtained between 15 and 21 days in 11 cases, five were aneuploidy and two failed. The results obtained with MLPA and karyotypes were concordant and in cases where the cell culture failed, the diagnosis was obtained by MLPA. Conclusions: MLPA is a rapid and useful method in the perinatal diagnosis of major aneuploidies, it also has the advantage of allowing the study of tissues with low cell viability, as in some cases of abortion and fetal death.