Consanguinidad no declarada en pacientes peruanos con trastorno del neurodesarrollo detectada a través de micromatrices

Introducción. La consanguinidad es la unión entre personas que comparten un ancestro en común, y cuya descendencia presenta un mayor riesgo de aparición de enfermedades autosómicas recesivas, manifestándose en algunos pacientes como trastornos del neurodesarrollo. Objetivos. Describir la consanguinidad parental no declarada en pacientes menores de 18 años con trastornos del neurodesarrollo, descubierta mediante el análisis cromosómico por micromatrices. Métodos. Se realizó el análisis cromosómico por micromatrices a 967 pacientes con trastorno del neurodesarrollo entre 2016 y 2021. Fueron seleccionados los pacientes con regiones de homocigosidad (ROH) con un valor superior a 0,5%. Resultados. Se evaluó a 288 pacientes, el 58,3% fueron varones y el 29,8% presentó una ROH mayor o igual a 0,5%. Se encontró que el 25,9% y el 0,83% de los pacientes tenían padres con un quinto y primer grado de consanguinidad no declarada, respectivamente. Los departamentos con mayor frecuencia relativa de consanguinidad no declarada por cada 10 000 habitantes fueron Huancavelica, Cajamarca y Apurímac. Conclusión. En Perú, existen regiones donde se evidencia uniones parentales consanguíneas, el cual es un factor de riesgo alto para la aparición de enfermedades recesivas autosómicas en su descendencia, como los trastornos del neurodesarrollo.

Introduction. Consanguinity is the union between people who share a common ancestor, and whose offspring have a higher risk of autosomal recessive diseases, manifesting in some patients as neurodevelopmental disorders. Objectives. To describe non-declared parental consanguinity of patients under 18 years of age with neurodevelopmental disorders, discovered by chromosomal microarray analysis. Methods. Chromosomal microarray analysis was performed on 967 patients with neurodevelopmental disorders between the years 2016-2021 and were selected to patients with regions of homozygosity (ROH) with a value greater than 0.5%. Results. 288 patients were evaluated, 58.3% of the patients were male and 29,8% presented an ROH greater than or equal to 0.5%. We found 25.9% and 0.83% of the patients had their parents of a fifth and first degree of consanguinity not previously declared, respectively. The most frequent neurodevelopmental disorder was delayed psychomotor development with 38.2%. The departments with the highest frequency relative of non declared consanguinity were Huancavelica, Cajamarca y Apurimac. Conclusions. In Peru, non-declared parental consanguinity is frequent, which is a high-risk factor for the appearance of autosomal recessive diseases in their offspring, how neurodevelopment disorders.

DNA rare copy number alterations in Reinke's Edema

Abstract Introduction: Reinke's Edema (RE) is a laryngeal lesion related to excessive tobacco smoking, voice overuse, and laryngopharyngeal reflux. Although the risk of malignancy has been considered low in literature, RE is classified among precancerous lesions. Objectives: We investigated DNA Copy Number Alterations (CNAs) in specimens of RE and its potential association with malignant progression. Methods: We used array-based comparative genomic hybridization (aCGH, Agilent 4 × 180 K platform) to study eight RE cases. All patients were heavy tobacco users for at least 30 years, and none of them progressed to cancer in the follow-up (>8 years). Two RE presented mild dysplasia, one moderate dysplasia, and no histological alterations were found in the remaining five cases. CNAs were compared with the Database of Genomic Variants (DGV) and genes mapped on altered regions had their functions annotated. Results: Six of eight patients showed different rare copy number alterations on chromosomes 2q37.3, 4q13.1, 4q13.3, 7q11.22, 10p14, and 13q34. A gain of the whole chromosome 8 were detected in one case. Of interest, four of eight RE cases showed copy number imbalances involving genes previously described in several tumor types (RASA3, COL6A3, LINC00707, LINP1, SMR3A, and SMR3B). Conclusion: The genomic imbalances herein found in RE have the potential to contribute to the phenotype but with limited or no risk of cancer. A long-term follow-up in a large series of patients could clarify the mechanisms involved in the malignant progression of RE. Level of evidence: 4.

Microarray analysis reveals distinct immune signatures in BCR?ABL positive and negative myeloproliferative neoplasms

Background: BCR?ABL mutation on the Philadelphia chromosome is the key driver of chronic myeloid leukemia (CML) pathogenesis. However, there are certain cases of myeloproliferative neoplasms (MPN) wherein no inherent driver mutation is detected resulting in clinical phenotype. It is important to identify key genes and pathways in driving the disease. The aim of the study was to use a gene-based omics approach to molecularly characterize these mutation-positive and negative cases to further strengthen diagnostics and precision medicine. Methods: A microarray profiling was done on CD34 positive cells isolated from two BCR?ABL positive and five BCR?ABL negative samples. JAK2V617F mutation testing was also done to rule out the presence of any other mutation in the latter group. The fold change cut-off was taken as �5 with p?0.5 for significant genes. The gene network and pathway analysis were done using DAVID and STRING software. Results: The genes upregulated in BCR?ABL negative samples were shown to be involved in immune regulation, signal transduction and T- and B-cell signalling. The protein-protein interaction network of upregulated genes in these samples were enriched for various immunomodulatory genes such as HLADP, HLADQ , IL7R, CCR7, CD3 subtypes. These genes further formed a network with signal transduction genes such as LCK, FYN, RAG1, DOCK1, AKT3, SMAD3, LEF1. Conclusion: The results suggested a modulation of immune response genes and its subsequent effect on oncogenic signalling in BCR?ABL negative samples as compared to BCR?ABL positive samples. The protein network analysis was enriched for genes involved in Src, TGF-beta and PI3K-AKT pathway contributing to the proliferation of neoplastic clone.

Circular RNA hsa_circ_0087893 participates in intraventricular hemorrhage occurrence and progression possibly as a competitive endogenous RNA in preterm infants / 南方医科大学学报

OBJECTIVE@#To screen for differentially expressed circular RNAs (circRNAs) in the serum of preterm infants with intraventricular hemorrhage (IVH) and explore the competitive endogenous RNA (ceRNA) mechanism of circRNAs in IVH in these infants.@*METHODS@#Fifty preterm infants (gestational age of 28 to 34 weeks) admitted in our department between January, 2019 and January, 2020 were enrolled in this study, including 25 with a MRI diagnosis of IVH and 25 without IVH. Serum samples were collected from 3 randomly selected infants from each group for profiling differentially expressed circRNAs using circRNA array technique. Gene ontology (GO) and pathway analyses were performed to reveal the function of the identified circRNAs. The circRNA-miRNA-mRNA network was constructed to identify the co-expression network of hsa_circ_ 0087893.@*RESULTS@#A total of 121 differentially expressed circRNAs were identified in the infants with IVH, including 62 up-regulated and 59 down-regulated circRNAs. GO and pathway analyses showed that these circRNAs were involved in multiple biological processes and pathways, including cell proliferation, activation and death, DNA damage and repair, retinol metabolism, sphingolipid metabolism, cell adhesion molecules. Among these circRNAs, hsa_circ_0087893 was found to have significant down-regulation in IVH group and co-express with 41 miRNAs and 15 mRNAs (such as miR-214-3p, miR-761, miR-183-5p, AKR1B1, KRT34, PPP2CB, and HPRT1).@*CONCLUSION@#The circRNA hsa_circ_0087893 may function as a ceRNA and play an important role in the occurrence and progression of IVH in preterm infants.

Cytogenomic Profiling of Chronic Lymphocytic Leukaemia Patients Using DNA Microarray

@#Introduction: Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. The clinical presentation varies greatly, from very indolent cases to those with aggressive and fast advancing disease. This variation has significant implications for clinical approaches, therapeutic tactics, and, ultimately, survival durations from diagnosis. Acquired chromosomal aberrations play a key role in CLL aetiology. Due to difficulty to obtain abnormal metaphases for analysis, few methods such as fluorescence in-situ hybridization (FISH) and multiplex ligation-dependent probe assay (MLPA) were employed to detect chromosomal aberration however the methods are limited to specific locus only. Thus, this study is aimed to detect the chromosomal aberrations using DNA microarray platform. Methods: In this retrospective study, DNA archive obtained from 7 CLL patients which collected at diagnosis and subjected to Affymetrix CytoScan® 750K single nucleotide polymorphism (SNP) array following the manufacture procedure. The raw data obtained were analysed using the Chromosome Analysis Suite (ChAS) software (Affymetrix) using annotations of genome version GRCh38 (hg38). Result: Out of 7 patients, 4 of them showing deletion of 13q while 3 of them showing deletion of 14q in various region . Some of the deleted loci were too small (0.42-0.6Mb) to be detected by conventional cytogenetic analysis (CCA). There was also the presence of additional chromosomal aberrations that could be missed by CCA, FISH, or MLPA due to cryptic deletion or duplication that was as small as 0.4MB in size. Conclusion: The present study showed that low resolution chromosomal aberration was able to be detected using DNA microarray platform in comparison to CCA, FISH and MLPA.

Prenatal diagnosis and pregnancy outcome of fetuses with isolated echogenic bowel / 中华围产医学杂志

Objective:To investigate the clinical value of isolated fetal echogenic bowel (FEB) as an indicator for invasive prenatal diagnosis.Methods:This retrospective study enrolled 183 pregnant women who were diagnosed with isolated FEB and underwent invasive prenatal diagnosis in Fujian Maternity and Child Health Hospital from August 2013 to January 2021. Clinical data including the results of conventional karyotyping and chromosomal microarray analysis (CMA), cytomegalovirus (CMV) DNA loads in amniotic fluid and pregnancy outcomes were reviewed analyzed. Chi-square test was used for statistical analysis Results:Karyotyping was performed on all of the 183 fetuses and three (1.64%) aneuploidies (one case of trisomy 21, one trisomy 18 and one 47,XYY syndrome) were detected. One trisomy 21 and four pathogenic (P)/likely pathogenic (LP) copy number variation (CNV) were detected among 108 fetuses who received CMA. The detection rate of P/LP chromosomal abnormalities by CMA was higher than that by karyotyping, but there was no significant difference between them [4.63% (5/108) vs 0.93% (1/108), χ 2=1.54, P>0.05]. In addition, three cases of variants of uncertain significance (VOUS) were detected by CMA. CMV DNA loads of fetal cells in the amniotic fluid samples of the 166 cases were determined, and only one (0.6%) was positive (CMV DNA up to 7.01×10 6 copies/ml), and no abnormalities were found in karyotype analysis and CMA detection. A total of 176 cases were followed up, and among them only one case of intrauterine infection and seven cases (three aneuploidies and four P/LP CNV) of chromosomal abnormalities were terminated after genetic counseling. Three fetuses with VOUS and other 165 fetuses without chromosomal abnormalities had a good prognosis after birth. Conclusions:Isolated FEB may be the abnormal ultrasound finding in fetuses with chromosomal abnormalities or CMV infection. Prenatal genetic testing and the exclusion of intrauterine infection are important for management during pregnancy and prognosis assessment of FEB.

Clinical value of different genetic testing methods for detection of true fetal chromosome mosaicism / 中华围产医学杂志

Objective:To investigate the performance of chromosome karyotype, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) in prenatal diagnosis of true fetal chromosome mosaicism. Methods:This retrospective study enrolled 40 women with true fetal chromosome mosaicism from 4 071 singleton pregnant women who were indicated for and underwent amniocentesis or/and cordocentesis in the the First Affiliated Hospital of Sun Yat-sen University from April 2018 to August 2021. The results of chromosome karyotyping, CMA and FISH, the types of chromosomal mosaicism, mosaicism ratio and pregnancy outcomes were analyzed using Chi-square test. Results:(1) The detection rate of true fetal mosaicism was 0.98% (40/4 071). (2) Sex chromosome mosaicism accounted for 42.5% (17/40). Other chromosomal mosaicism involved chromosomes 21, 22, 18, 16, 7, 12, 15, 17 and 20, as well as balanced chromosomal translocation. (3) The detection rate of true fetal mosaicism by chromosome karyotyping was 77.4% (24/31) from amniotic fluid samples and 10/19 from umbilical cord blood samples, while that data by CMA was 76.7% (23/30) and 7/11,respectively. (4) Of the 40 pregnant women with fetal chromosome mosaicism, FISH test was performed on 20 cases (14 cases were verified with both amniotic fluid and umbilical cord blood samples, five with amniotic fluid samples and one with umbilical cord blood sample), and all of the diagnosis of mosaicism were confirmed. For those with mosaicism ratio <30%, the detection rate by FISH was higher than that by CMA among amniotic fluid samples [14/19 vs 43.5% (10/23), χ2=3.88, P=0.049]. (5) Among the 40 pregnant women, five were lost to follow-up; 18 chose to terminate the pregnancy; and 17 continued the pregnancy to delivery. No abnormalities in mental or physical development were reported in the 17 neonates after birth or during on-line follow-up between 6 to 24 months old. Of the 14 pregnant women with mosaicism ratio <30% which confirmed by FISH, eight chose to continue the pregnancy, and no abnormalities in mental development or growth were found in the neonates. Conclusions:In prenatal diagnosis of true fetal choromosome mosaicism, the incidence of sex chromosome mosaicism is the highest. FISH may improve the prenatal diagnosis rate of mosaicism and is more accurate in determining the mosaicism ratio. The combination of FISH, CMA and chromosome karyotyping would significantly improve the detection rate of chromosomal mosaicism and assess the mosaicism ratio more accurately, which is of great value in clinical consultation and evaluation of fetal prognosis.

Prenatal phenotypic analysis and genetic counseling for 22q11.21 microdeletion and microduplication syndrome / 中华围产医学杂志

Objective:To analyze the prenatal clinical phenotypes and pregnancy outcomes of fetuses with 22q11.21 microdeletion and microduplication syndrome to provide a basis for clinical genetic counseling.Methods:This retrospective study involved the cases diagnosed with 22q11.21 microdeletion or microduplication by chromosomal microarray analysis (CMA) due to abnormal ultrasound findings, advanced maternal age, or high-risk pregnancies indicated by serum screening in the Prenatal Diagnosis Center of the First Affiliated Hospital of Air Force Medical University from January 2015 to January 2022. Clinical phenotypes and pregnancy outcomes of the fetuses were analyzed and described.Results:Among 9 141 cases referred for CMA during the study period, 77 cases (0.8%) were diagnosed as 22q11.21 microdeletion or microduplication, including 62 (80.5%) with 22q11.21 microdeletion and 15 (19.5%) with microduplication. In the 22q11.21 microdeletion cases, 58 had typical deletion, and four had atypical deletions, but all fetuses carried TBX1 gene that was clearly associated with congenital heart disease. The 15 fetuses with 22q11.21 microduplication including 14 in the typical region and one in the atypical region. Forty-eight (77.4%) out of the 62 fetuses with 22q11.21 microdeletion were complicated by congenital heart defects, including 28 with conotruncal defects. Five of the 15 fetuses with 22q11.21 microduplication were complicated by congenital heart defects. The cases were followed up on telephone at three to six months after the expected date of delivery. Among the 62 cases with 22q11.21 microdeletion, 52 terminated pregnancies, five were lost to follow-up, and five were delivered (one died after one month of premature delivery, one was born with anal advancement and growth retardation, and three were followed up without obvious abnormality). Among the 15 cases with 22q11.21 microduplication, four terminated pregnancies, two were lost to follow-up, and nine gave birth (eight were followed up without obvious abnormality, one grew slowly). Conclusions:The application of CMA in the prenatal diagnosis of 22q11.21 microdeletion and microduplication fetuses, and the comprehensive analysis of clinical manifestations and pregnancy outcome combined with ultrasonic diagnosis are of great significance in guiding the treatment and rehabilitation after birth of an affected child. Genetic counseling for cases with 22q11.21 microdeletion and microduplication syndrome should be cautious and consider ultrasound findings.

Analysis of whole genome expression profile for the effect of melatonin on radiation-induced intestinal injury in mice / 中华放射医学与防护杂志

Objective:To elucidate the change of whole genome expression profile for the effect of melatonin on radiation-induced intestinal injury in mice.Methods:C57BL/6J male mice were administrated with melatonin at 10 mg/kg body weight by intraperitoneal injection once a day for five consecutive days before abdominal irradiation with 14 Gy of γ-rays. Small intestines were harvested 3 d after radiation. GO annotation and KEGG pathway of the differential genes involved in small intestine were explored by DNA microarray analysis.Results:Compared with the control group, 584 differential genes were up-regulated and 538 differential genes were down-regulated for administration group pre-irradiation. The overlapping differential genes were selected from the irradiated mice and the administrated mice pre-irradiation. There were 324 up-regulated genes and 246 down-regulated genes unique to the administrated mice pre-irradiation. GO annotation analysis of the differential genes indicated that the top 15 significantly enriched biological processes for the administrated mice pre-irradiation mainly included autophagosome assembly (GO: 0000045), autophagosome organization (GO: 1905037) and regulation of acute inflammatory response (GO: 0002673). The genes ATG12, ATG16L2 and AMBRA1 were involved in autophagosome assembly and autophagosome organization. The genes C3, CPN1, CD55, CFP, CNR1, C1QA, C2 and CREB3L3 were involved in the regulation of acute inflammation response. KEGG pathway analysis of the differential genes involved indicated that the top 15 significantly enriched pathways for the administrated mice pre-irradiation mainly included O-glycan biosynthesis (hsa00512), glycosphingolipid biosynthesis (hsa00603), ECM-receptor interaction (hsa04512) and biosynthesis of unsaturated fatty acids (hsa01040). qRT-PCR verification showed that the expressions of ATG12 and ATG16L2 genes involved in autophagy for the administrated mice pre-irradiation increased significantly compared with the irradiated mice ( t=2.40, 4.35, P<0.05). Conclusions:The differential genes related with the biological process of autophagy, acute inflammatory response and the pathway of unsaturated fatty acid biosynthesis might be involved in the effect of melatonin on radiation-induced intestinal injury.

Genetic diagnosis of microcephaly / 中华妇产科杂志

Objective:To explore the diagnostic value of chromosome karyotype analysis, chromosomal microarray analysis (CMA) and whole exome sequencing (WES) in microcephaly.Methods:A total of 9 cases of microcephaly fetuses diagnosed by prenatal ultrasound or children with microcephaly diagnosed after birth were selected from the Sixth Affiliated Hospital of Guangzhou Medical University from January 2014 to August 2022.Karyotype analysis and/or CMA were used to detect. The cases with negative karyotype analysis and CMA results were further sequenced by trio-based WES (Trio-WES). Then the coding genes contained in the pathogenic copy number variation (CNV) fragments were analyzed by gene ontology (GO) enrichment. The genes related to the development of the central nervous system contained in the pathogenic CNV and the pathogenic genes found by Trio-WES were combined for gene interaction network analysis.Results:In this study, 9 cases of microcephaly were recruited, with the time of diagnosis ranged from 23 weeks of gestation to 7 years after birth, and the head circumference of fetus or children ranged from 18.3 to 42.5 cm (-7SD to -2SD). Karyotype analysis was detected in all 9 cases and no abnormality result was found. Eight cases were detected by CMA, and one abnormal was found. Five cases were detected by Trio-WES, and two cases were detected with likely pathogenic genes. The GO enrichment analysis of the coding gene in the 4p16.3 microdeletion (pathogenic CNV) region showed that: in biological process, it was mainly concentrated in phototransduction, visible light; in terms of molecular function, it was mainly concentrated in fibroblast growth factor binding; in terms of cell components, it was mainly concentrated in rough endoplasmic reticulum. Gene interaction network analysis suggested that CDC42 gene could interact with CTBP1, HTT and ASPM gene.Conclusions:CMA could be used as a first-line detection technique for microcephaly. When the results of chromosome karyotype analysis and/or CMA are negative, Trio-WES could improve the detection rate of pathogenicity of microcephaly.

Mechanism of Polygonum capitatum in treatment of Helicobacter Pylori associated gastritis by network pharmacology, microarray data analysis and molecular docking / 中国药理学通报

Aim To explore the mechanism of Polygonum capitatum(PC)in the treatment of Helicobacter Pylori associated gastritis(HAG). Methods The databases were used to identify the target of PC active compounds and HAG-related genes,and the intersection was taken to obtain the potential targets of PC treatment of HAG. The interaction network diagram of “drug-active compound-target-disease” and the protein-protein interaction(PPI)network of potential target protein interaction in HAG treated by PC were constructed by software Cytoscape 3.6.0. The important nodes in the network were screened by several topological indexes,and the GO and KEGG enrichment were analyzed by STRING database to obtain the potential signaling pathway of PC in the treatment of HAG. The binding ability of PC active components with key target proteins was observed by molecular docking method. On this basis,the related targets of PC in the treatment of HAG were verified in vivo and in vitro experiments. Results The PC active compounds and targets were identified through the database,and the “drug-active compound-target-disease” network diagram and the PPI network of potential target proteins were constructed. Combined with several topological indexes,the PPI network of potential target-protein interaction was analyzed,and 52 hub genes were screened. Further bioinformatics analysis and high-throughput sequencing revealed that PC exerted an effect on HAG through the Akt/NF-κB/NLRP3 pathway. Based on this,it was found that PC could reduce IL-18 and IL-1β in HAG GES-1 cells and HAG SD rats,up-regulate Akt and its phosphorylation level and reduce NF-κB expression,inhibit the activation of NLRP3 inflammatory body,so as to improve HAG inflammatory response. Conclusions PC could exert a therapeutic effect on HAG by activating Akt and its phosphorylation level,and inhibiting the expression of NF-κB and NLRP3 inflammasome related factors. This study provides a theoretical basis for explaining the mechanism of PC in the treatment of HAG.

Clinical and Genetic Profile of Children With Short Stature Presenting to a Genetic Clinic in Northern India

Objective: To define the spectrum of genetic disorders in patients with short stature visiting the genetic out-patient department in a tertiary care hospital. Methods: A chart review was done for 455 individuals (10 months-16 yrs) with short stature, who were evaluated at the genetic clinic from 1 January, 2017 upto 31 October, 2018. 226 patients who needed detailed evaluation, the spectrum of genetic diagnosis is presented. Results: Proportionate short stature was identified in 63% individuals (n=142) of which 93 (65%) were recognizable syndromes such as Turner syndrome, and William syndrome, and RASopathies. In clinically undefined syndromes (39, 27%), a diagnosis could be made by karyotype (n=3/10), chromosomal microarray (6/12) and exome sequencing (1/6). In the 84 children in the disproportionate short stature group (37%), lysosomal storage disorders (LSDs) (45%, n=38) were identified by enzyme analysis in 86.8% and skeletal dysplasias (44%, n=37) identified by skeletal survey in 89% cases. Conclusions: In undefined syndromic short stature, chromosomal microarray may be the first investigation of choice if phenotyping is not suggestive of a specific genetic syndrome. Exome sequencing can be useful in identifying newer genes among idiopathic and familial short stature cohorts.

Outcomes of Very Preterm Neonates Born by Assisted Reproductive Techniques (ART): A Propensity Score Matched Retrospective Cohort Study

Objective: To compare outcomes of preterm neonates born through assisted reproduction techniques (ART) and non-ART conception. Methods: This retrospective cohort study included very preterm neonates (26 weeks to 31 weeks) admitted to our neonatal unit over a six year period from 2014 to 2019. The primary outcome was composite adverse outcome of mortality or any of the major morbidities i.e., intraventricular hemorrhage (IVH) grade ?3, periventricular leukomalacia (PVL) grade ?2, bronchopulmonary dysplasia (BPD) at 36 weeks, and retinopathy of prematurity (ROP) requiring treatment. Results: Total of 759 neonates (253 in ART group, 506 in non-ART group) were included after propensity score matching for gestational age, sex, and small for gestational age (SGA). Neonates in ART group had similar rates of composite adverse outcome [aOR (95% CI) 0.86 (0.55 – 1.36)], mortality [0.93, (0.53- 1.64)] BPD [1.18, (0.37 – 3.76)]; ROP requiring treatment [ 0.49 (0.14-1.71], and other morbidities. Conclusion: Very preterm neonates born through ART were not at increased risk of adverse neonatal outcomes.

Detection of MDM2 gene amplification on tissue microarray-based Fluorescence In-Situ Hybridization (FISH) in well-differentiated and dedifferentiated liposarcomas, displaying a wide morphological spectrum: A validation study at a tertiary cancer referral centre

Background: Liposarcomas including atypical lipomatous tumors (ALT)/well-differentiated liposarcomas (WDLPS) and dedifferentiated liposarcomas (DDLPSs) display a histomorphological spectrum with their several diagnostic mimics. Murine double minute 2(MDM2) gene amplification characterizes ALT/WDLPS and DDLPS. Presently, there is no documented study from our subcontinent on the validation of MDM2 gene testing in these tumors. Material and Methods: Twenty-eight cases, diagnosed as ALT/WDLPS (n = 5) and DDLPSs (n = 23), along with 10 other tumors were tested for MDM2 gene amplification, using fluorescence in situ hybridization (FISH) on tissue microarrays (TMAs). Fourteen cases, diagnosed as ALT/WDLPS and DDLPS, along with 49 other tumors were tested for MDM2 immunostaining. Twenty tumors were tested for p16INK4a immunostaining. Results: FISH was interpretable in 25 (89.2%) cases. Among the 20 cases diagnosed as DDLPSs, 19 displayed MDM2 gene amplification. Among the 5 cases diagnosed as ALT/WDLPS, four showed MDM2 gene amplification. Finally, 19 cases were confirmed as DDLPS and 4 as ALT/WDLPS. Furthermore, 7/19 cases confirmed as DDLPS and all 4 cases as ALT/WDLPS tested for MDM2 immunostaining, displayed its diffuse immunoexpression, while a single case of DDLPS showed its focal immunostaining. None of the 49 control cases displayed diffuse MDM2 immunoexpression. ALL 16 DDLPSs and 4 cases of ALT/WDLPS displayed p16INK4a immunostaining. The sensitivity for diffuse MDM2 immunostaining was 87.5% in cases of DDLPS, 100% in ALT/WDLPS, and specificity was 100%. The sensitivity for MDM2 gene amplification was 94.7% in cases of DDLPS and 100% in cases of ALT/WDLPS. The sensitivity for p16INK4a was 100%. Conclusion: This constitutes the first sizable study on MDM2 testing in ALT/WDLPS and DDLPS from our subcontinent using TMAs. MDM2 gene amplification testing continues as the diagnostic gold standard for ALTs/WDLPSs and DDLPSs and is useful in cases of diagnostic dilemmas. Diffuse MDM2 (IF2 clone) and p16INK4a immunostaining, together seem useful for triaging cases for FISH.

Prediction of protein-protein interaction networks and druggable genes associated with parkinson’s disease-Supplementary data

Parkinson’s disease (PD) affects about 2-3% of the global population over 65 years of age and hence, it is the second most common neurodegenerative disorder in the world. This study explored the key genes and miRNA involved in PD. Microarray dataset (accession number GSE19587) comprising of two regions of medulla: dorsal motor nucleus of vagus (DMNV) and inferior olivary nucleus (ION) was downloaded from Gene Expression Omnibus (GEO) database. A total of 697 DEGs from ION (605 up-regulated genes and 92 down-regulated genes) and 663 DEGs from DMNV (638 up-regulated genes and 25 down-regulated genes) were screened. These DEGs were found to be enriched in 46 (DMNV) and 24 (ION) pathways common in DAVID and Comparative Toxicogenomics Database. In PPI network analysis, IGF1 and CD44 were identified as hub genes in DMNV whereas for ION, the hub genes identified were CSF2 and CD44. In TF-miRNA-target gene networks, an aggregate of 11 transcription factors and 46 miRNA were observed to influence the target genes. In drug-gene interaction studies, CYP3A5 and ESR1 had higher connective degrees and hence, they might be novel druggable targets for Parkinson’s disease.

Prediction of protein-protein interaction networks and druggable genes associated with parkinson’s disease

Parkinson’s disease (PD) affects about 2-3% of the global population over 65 years of age and hence, it is the second most common neurodegenerative disorder in the world. This study explored the key genes and miRNA involved in PD. Microarray dataset (accession number GSE19587) comprising of two regions of medulla: dorsal motor nucleus of vagus (DMNV) and inferior olivary nucleus (ION) was downloaded from Gene Expression Omnibus (GEO) database. A total of 697 DEGs from ION (605 up-regulated genes and 92 down-regulated genes) and 663 DEGs from DMNV (638 up-regulated genes and 25 down-regulated genes) were screened. These DEGs were found to be enriched in 46 (DMNV) and 24 (ION) pathways common in DAVID and Comparative Toxicogenomics Database. In PPI network analysis, IGF1 and CD44 were identified as hub genes in DMNV whereas for ION, the hub genes identified were CSF2 and CD44. In TF-miRNA-target gene networks, an aggregate of 11 transcription factors and 46 miRNA were observed to influence the target genes. In drug-gene interaction studies, CYP3A5 and ESR1 had higher connective degrees and hence, they might be novel druggable targets for Parkinson’s disease.

Novel rearrangements between different chromosomes with direct impact on the diagnosis of 5p- syndrome

Abstract Objectives Copy Number Variations (CNVs) in the human genome account for common populational variations but can also be responsible for genetic syndromes depending on the affected region. Although a deletion in 5p is responsible for a syndrome with highly recognizable phenotypical features, other chromosomal abnormalities might overlap phenotypes, especially considering that most studies in 5p use traditional cytogenetic techniques and not molecular techniques. Methods The authors have investigated 29 patients with clinical suspicion of 5p- syndrome using Chromosomal Microarray (CMA), and have gathered information on previous tests, clinical signs, symptoms, and development of the patients. Results The results showed 23 pure terminal deletions, one interstitial deletion, one deletion followed by a 3 Mb duplication in 5p, three cases of 5p deletion concomitant to duplications larger than 20 Mb in chromosomes 2, 9, and 18, and one 5p deletion with a chromosome Y deletion. CMA showed relevant CNVs not typically associated with 5p- that may have contributed to the final phenotype in these patients. Conclusions The authors have identified three novel rearrangements between chromosomes 5 and 2 (Patient 27), 5 and 18 (Patient 11), and 5 and Y (Patient 22), with breakpoints and overlapped phenotypes that were not previously described. The authors also highlight the need for further molecular investigation using CMA, in different chromosomes beyond chromosome 5 (since those cases did not show only the typical deletion expected for the 5p- syndrome) to explain discordant chromosomal features and overlapped phenotypes to unravel the cause of the syndrome in atypical cases. HIGHLIGHTS The authors The authors have described three novel rearrangements between chromosomes 5 and 2, 5 and 18, and 5 and Y with chromosomal breakpoints and overlapped phenotypes that were not previously described. One of the main atypical features for 5p- syndrome that the authors report was the presence of seizures that was found in the three patients with rearrangements between different chromosomes and in a patient with a deletion followed by duplication in 5p. The authors suggest physicians conduct further molecular investigation in the presence of atypical clinical features for patients with 5p- syndrome suspicion.

Clonagem, expressão recombinante e validação por microarray de proteínas alimentares potencialmente alergênicas / Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins

A maioria das respostas alérgicas a alimentos é mediada por IgE, que pode ser detectada para fins de diagnóstico da alergia alimentar. No entanto, para isso é necessário que alérgenos purificados estejam disponíveis para a elaboração dos diferentes formatos de ensaio, inclusive por microarray, que se constitui em uma ferramenta bastante útil para análise simultânea, e também para a identificação de reatividade cruzada. A esse respeito, é imprescindível ampliar a plataforma de alérgenos que possam ser empregados para a confecção de microarrays. Atualmente, alguns alimentos que constituem objeto de interesse na clínica em função do número de casos de alergia, e sobre os quais as informações a respeito dos alérgenos são escassas, são: abacaxi, mamão, mandioca e manga. Assim, o objetivo desse trabalho foi clonar, expressar e purificar proteínas potencialmente alergênicas de alimentos de importância regional. Após confirmadas por ensaios imunológicos, essas proteínas foram utilizadas na construção e validação de um microarray através de ensaios com os soros de pacientes alérgicos aos alimentos selecionados. Para atingir esse objetivo, foram selecionadas proteínas potencialmente alergênicas coincidentes, apontadas tanto pela similaridade com espécies taxonomicamente mais próximas, quanto pela técnica 2D Western Blotting acoplada à espectrometria de massas. Dezenove proteínas, sendo 4 de abacaxi, 5 de mamão, 6 de mandioca e 4 de manga, foram expressas em Pichia pastoris, purificadas e impressas em um microarray. Após incubar essas proteínas com os soros dos pacientes alérgicos aos alimentos estudados, 18 proteínas mostraram-se potencialmente alergênicas. Além disso, foi observada reatividade cruzada entre proteínas dos alimentos estudados e também em relação ao látex e outros frutos

The majority of allergic reactions to foods is IgE-mediated, which can be detected for the diagnosis of food allergy. However, purified allergens are necessary to produce different kinds of allergy tests, including microarray, which is a useful tool for simultaneous analysis, as well as for the identification of cross-reactivity. In this respect, it is essential to expand the platform of allergens to include them on microarrays. Nowadays, some foods that are object of interest in the clinical area in Brazil and it is necessary a further evaluation about their potential allergens, since there is a limited information about them, are: pineapple, papaya, cassava and mango. Therefore, the aim of this study was cloning, expressing and purifying potentially allergenic proteins of important Brazilian foods. After confirmed by immunological tests, these proteins were used in microarray production and validation by assays with sera from allergic patients to the selected foods. Achieving this goal, matching potentially allergenic proteins were selected, which were identified by comparison among taxonomically closer species (in silico) and 2D Western Blotting coupled with Mass Spectrometry. Nineteen proteins: 4 from pineapple, 5 from papaya, 6 from cassava and 4 from mango were expressed in Pichia pastoris, purified and printed on a microarray. After incubating those proteins with sera from allergic patients to the selected foods, 18 proteins were detected as potentially allergenic. In addition, cross-reactivity was observed among the proteins from the studied foods, and also regarding to the latex and other fruits

Identification of human LDHC4 as a potential target for anticancer drug discovery

One of the distinct hallmarks of cancer cells is aerobic glycolysis (Warburg effect). Lactate dehydrogenase A (LDHA) is thought to play a key role in aerobic glycolysis and has been extensively studied, while lactate dehydrogenase C (LDHC), an isoform of LDHA, has received much less attention. Here we showed that human LDHC was significantly expressed in lung cancer tissues, overexpression of Ldhc in mice could promote tumor growth, and knock-down of LDHC could inhibit the proliferation of lung cancer A549 cells. We solved the first crystal structure of human LDHC4 and found that the active-site loop of LDHC4 adopted a distinct conformation compared to LDHA4 and lactate dehydrogenase B4 (LDHB4). Moreover, we found that (ethylamino) (oxo)acetic acid shows about 10 times selective inhibition against LDHC4 over LDHA4 and LDHB4. Our studies suggest that LDHC4 is a potential target for anticancer drug discovery and (ethylamino) (oxo)acetic acid provides a good start to develop lead compounds for selective drugs targeting LDHC4.

SMARCA2 deficiency in NSCLC: a clinicopathologic and immunohistochemical analysis of a large series from a single institution

BACKGROUND@#SMARCA2 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, Subfamily A, Member 2) is an important ATPase catalytic subunit in the switch-sucrose nonfermenting (SWI/SNF) complex. However, its relationship with the pathological features of NSCLC and its prognosis remain unclear.@*METHODS@#We retrospectively reviewed 2390 patients with surgically resected NSCLC, constructed tissue microarrays (TMAs) and performed immunohistochemical assays. We analyzed the correlation of SAMRCA2 with clinicopathological features and evaluated its prognostic value.@*RESULTS@#Among 2390 NSCLC cases, the negative expression ratios of SAMRCA2, SMARCA4, ARID1A, ARID1B and INI1 were 9.3%, 1.8%, 1.2%, 0.4% and 0%, respectively. In NSCLC, male sex, T3 and T4 stage, moderate and poor differentiation, tumor ≥ 2 cm, Ki67 ≥ 15%, SOX-2 negative expression, middle lobe lesion and adenocarcinoma were relative risk factors affecting SMARCA2-negative expression. In lung adenocarcinomas, high-grade nuclei, histological morphology of acinar and papillary, solid and micropapillary and TTF-1-negative expression were relative risk factors affecting SMARCA2-negative expression. Kaplan-Meier survival analysis showed that the OS was shorter in the SMARCA2-negative group. Multivariate survival analysis revealed that SMARCA2-negative expression was an independent factor correlated with a poor prognosis in NSCLC.@*CONCLUSION@#In conclusion, SMARCA2-negative expression is an independent predictor of a poor outcome of NSCLC and is a potential target for NSCLC treatment.