Molecular Analysis Of Mutations Research Articles

FOXL2 Mutation Status in Sex Cord-stromal Tumors Cannot be Predicted by Morphology.

Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The histopathologic diagnosis of these tumors can be challenging. A recurrent somatic mutation of the forkhead box L2 (FOXL2) gene has been identified in adult GCT. In this retrospective single-center study of 44 SCST, a morphologic review together with analysis of FOXL2 C134W was evaluated in relation to tumor morphology. In addition, TERT promoter mutation testing was performed. Twelve of 36 cases got an altered diagnosis based on morphology alone. The overarching architectural growth pattern in 32/44 (72.7%) tumors was diffuse/solid with several tumors showing markedly heterogeneous architecture. In correlation to FOXL2 C134W mutation status, cytoplasmic color, and nuclear shape, differed between the FOXL2 C134W positive and FOXL2 C134 W negative groups, but these differences were not significant when comparing them separately. Nineteen of 44 cases underwent TERT promoter sequencing with a positive result in 3 cases; 2 adult GCTs and 1 cellular fibroma. Three patients developed a recurrence of which 2 were FOXL2 C134W positive adult GCTs and the third was an unclassified SCST. In conclusion, the morphologic and immunohistochemical diagnosis of different SCSTs is challenging and one cannot reliably identify FOXL2 mutation-positive tumors solely by morphologic features. Therefore, broad use of molecular analysis of the FOXL2 C134W mutation is suggested for SCSTs, and further studies are needed to evaluate the clinical outcome of these tumors as well as the diagnostic and prognostic implications of TERT promoter mutations.

Molecular characterization and mutational analysis of beta-globin gene among Kodagu population of Karnataka, India

Introduction and Aim: Beta-thalassemia is a hereditary blood disorder caused by genotypic alterations in the HBB gene. Over 400 mutations have been identified, which are known to be responsible for this disease. In this study, we investigated the mutations for the globin gene prevalent among adult beta-thalassemia trait positive individuals and their distribution within the Kodagu community of Karnataka, India. Materials and Methods: This study included 100 individuals (81 Females and 19 males) in the age group of 18-28 years previously tested as carriers for beta-thalassemia trait. Blood collected from each individual was subjected to genomic DNA extraction and PCR amplification of a 610 bp fragment of the beta-globin gene. The PCR product was purified and sequenced. The gene sequences were subjected to mutation analyses and characterization using bioinformatics tools. Results: The study identified 13 known and 27 novel mutations for this study population. Among the known mutations observed 2 were of type synonymous (HBB:c.9T>C and HBB:c.18T>G), 5 missense (HBB:c.146T>G; HBB:c.149C>T; HBB:c.151A>T; HBB:c.203T>G; HBB:c.269G >A) and 6 intronic (HBB:c.92+2T >G; HBB:c.92+5G>C; HBB:c.315+16G>C; HBB:c.315+74T>G and HBB:c.315+81C>T). HBB:c.-6G>C at the 5'UTR region and IVS-II-16 G>C, an intronic variant, was observed to be the most common mutation showing highest frequency of 82.1%. Several novel mutation types were identified in Exon 2 (3 missense; 2 synonymous), Intron I and Intron II. Conclusion: This study is the first of its kind for the Kodagu population, which identified several previously known and novel mutations in the HBB gene within this population. Since, our study subjects were carriers of very mild or silent beta-thalassemia trait, the mutations observed particularly the novel mutations warrant further investigation for a better understanding and management of beta-thalassemia in this population.

A novel thermostable and salt-tolerant carboxylesterase involved in the initial aerobic degradation pathway for pyrethroids in Glycomyces salinus

The long-term and excessive use of pyrethroid pesticides poses substantial health risks and ecosystem concerns. Several bacteria and fungi have been reported that could degrade pyrethroids. The ester-bond hydrolysis using hydrolases is the initial regulatory metabolic reaction of pyrethroids. However, the thoroughly biochemical characterization of hydrolases involved in this process is limited. Here, a novel carboxylesterase, designated as EstGS1 that could hydrolyze pyrethroid pesticides was characterized. EstGS1 showed low sequence identity (<27.03%) compared to other reported pyrethroid hydrolases and belonged to the hydroxynitrile lyase family that preferred short short-chain acyl esters (C2 to C8). EstGS1 displayed the maximal activity of 213.38 U/mg at 60 °C and pH 8.5 using pNPC2 as substrate, with Km and Vmax were 2.21 ± 0.72 mM and 212.90 ± 41.78 µM/min, respectively. EstGS1 is a halotolerant esterase and remains stable in 5.1 M NaCl. Based on molecular docking and mutational analysis, the catalytic triad of S74-D181-H212 and three other substrate-binding residues I108, S159, and G75 are critical for the enzymatic activity of EstGS1. Additionally, 61 and 40 mg/L of deltamethrin and λ-cyhalothrin were hydrolyzed by 20 U of EstGS1 in 4 h. This work presents the first report on a pyrethroid pesticide hydrolase characterized from a halophilic actinobacteria.

Fine-needle biopsy of thyroid nodules and the contribution of molecular analysis of BRAF and RAS mutations.

Thyroid cancer is the most common endocrine malignancy. There is a significant overdiagnosis of thyroid carcinomas that would never clinically manifest, with consequent unnecessary surgical treatment. The fine-needle biopsy and subsequent cytologic examination is of crucial importance in the differential diagnosis of thyroid nodules. On the other hand, a significant portion of the results are indeterminate. To assess the relationship of BRAF/RAS mutations in biopsy specimens to histological characteristics of thyroid nodules in individuals who undergone fine-needle biopsy and surgery. This cross-sectional study involves 170 subjects with indeterminate cytology analyzed for BRAF/RAS mutations. Of all 170 patients with indeterminate cytological finding, 103 were indicated for surgery. Of these, 31 were BRAF and 25 RAS positive. Thyroid cancer was diagnosed in 59 patients, while 44 patients had non-malignant thyroid lesions. The BRAF V600E mutation was detected in 30 patients, and the RAS (K-RAS, N-RAS, and H-RAS) mutation in 13 patients with thyroid cancer. In all BRAF-positive nodules, thyroid cancer was histologically confirmed. This means a 100 % positive predictive value of BRAF testing in our study. Stratification of thyroid lesions with uncertain results of fine-needle cytology using genetic markers can help to deliver more tailored medical treatment (Tab. 6, Ref. 19).

Molecular dynamic simulation and functional analysis of pathogenic PTEN mutations in glioblastoma

PTEN, a dual-phosphatase and scaffold protein, is one of the most commonly mutated tumour suppressor gene across various cancer types in human. The aim of this study therefore was to investigate the stability, structural and functional effects, and pathogenicity of 12 missense PTEN mutations (R15S, E18G, G36R, N49I, Y68H, I101T, C105F, D109N, V133I, C136Y, R173C and N276S) found by next generation sequencing of the PTEN gene in tissue samples obtained from glioblastoma patients. Computational tools and molecular dynamic simulation programs were used to identify the deleterious effects of these mutations. Furthermore, PTEN mRNA and protein expression levels were evaluated by qRT-PCR, Western Blot, and immunohistochemistry staining methods. Various computational tools predicted strong deleterious effects for the G36R, C105F, C136Y and N276S mutations. Molecular dynamic simulation revealed a significant decrease in protein stability for the Y68H and N276S mutations when compared with the wild type protein; whereas, C105F, D109N, V133I and R173C showed partial stability reduction. Significant residual fluctuations were observed in the R15S, N49I and C136Y mutations and radius of gyration graphs revealed the most compact structure for D109N and least for C136Y. In summary, our study is the first one to show the presence of PTEN E18G, N49I, D109N and N276S mutations in glioblastoma patients; where, D109N is neutral and N276S is a damaging and disease-associated mutation. Communicated by Ramaswamy H. Sarma

Targeted Identification of Rice Grain-Associated Gene Allelic Variation Through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

BackgroundThe mining of new allelic variation and the induction of new genetic variability are the basis for improving breeding efficiency.ResultsIn this study, in total, 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected. The grain length was between 8 and 10.22 mm. The grain width was between 1.54 and 2.87 mm. The results showed that there was extensive variation in granulotype. The allelic variation in GS3 and GW5 was detected in 484 mixed samples (8:1) using targeted sequencing technology, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; combined with Sanger sequencing and phenotype data, 13 key mutants and their corresponding SNPs were obtained; protein structural and functional analysis of key mutants screened out 6 allelic variants leading to altered grain shape, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; whole genome sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3.ConclusionBased on the above studies, we found 6 granulotype mutants and 9 granulotype-related allelic variants, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis. We propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. The method has the characteristics of low detection cost, short detection period, and flexible detection of traits and genes.

128. Clinical testing of mismatch repair in neoplasms using multiple laboratory methods

<h3>Background</h3> Deficiency of DNA mismatch repair function can be assessed by immunohistochemical (IHC) analysis for loss of expression of the mismatch repair (MMR) proteins MLH1, MSH2, MSH6 and PMS2 or microsatellite instability (MSI) analysis. In some cases, however, there is a discrepancy between IHC and MSI analysis. Our aim was to explain with molecular mutation analysis these discordant cases between IHC and MSI results. <h3>Methods</h3> We reviewed 706 neoplasms accessioned between 2015-2021 (345 gastrointestinal, 142 genitourinary, 192 gynecologic, 27 miscellaneous). All neoplasms were assessed by IHC testing, MSI analysis and tumor-normal paired next generation sequencing (NGS) analysis, and 80% of cases with MLH1 loss had concurrent MLH1 methylation analysis. <h3>Results</h3> Fifty-eight (8.2%) of neoplasms had deficient MMR. Forty-four (76%) of these patients had both dMMR and MSI-high status. Ten of 14 discordant cases had a major discordance between IHC and MSI analysis: [MSI-High/intact MMR expression (n=3), microsatellite stable (MSS)/loss of MLH1&PMS2 (n=1), MSS/loss of MSH2&MSH6 (n=3), MSS/loss of MSH6 (n=3), MSS/loss of MSH1 &MSH2 (n=1)]. Remaining 4 neoplasms are either MSI-low or with equivocal IHC results. Methylation and NGS analysis revealed a molecular etiology in 3 of 10 (30%) neoplasms with major discordance, whereas in neoplasms without major discordance, this figure was 77%. (p=0.007) <h3>Conclusion</h3> For most neoplasms, IHC and MSI testing are concordant, and a molecular etiology that explains dMMR or MSI-High results can be identified. For neoplasms with major discordance, the addition and integration of NGS results and MLH1 methylation analysis can be beneficial for resolving borderline cases.

Development of a 4-miRNA prognostic signature for endometrial cancer.

To develop an effective uterine corpus endometrial carcinoma (UCEC) risk assessment tool to monitor treatment outcomes. Limma package was used to analyze differentially expressed microRNAs (miRNAs) between UCEC tissues and normal tissues in the TCGA database. According to univariate Cox risk regression, least absolute shrinkage, and selection operator (LASSO) Cox analysis were performed to screen prognostic miRNAs and construct a risk scoring model. The prognostic performance of signature was evaluated by Kaplan–Meier and receiver operating characteristic. Multivariate Cox regression analysis was used to determine the independent prognostic factors of UCEC. Nomogram was constructed according to age, clinical stage, and risk score. A 4-miRNA signature based on miR-31-5p, miR-34a-5p, miR-26a-1-3p and miR-4772-3p was established. Risk scores of each patient were calculated by the 4-miRNA signature. After z-score, the patients were divided into high- and low-risk groups. The overall survival of high-risk patients was significantly shorter than that of low-risk patients, pointing to the high performance and independence of the 4-miRNA signature in predicting UCEC prognosis. The nomogram showed a high accuracy in predicting overall survival of UCEC patients. We developed a 4-miRNA signature that could effectively predict the prognosis of UCEC.

Molecular and Structural Analysis of Specific Mutations from Saudi Isolates of SARS-CoV-2 RNA-Dependent RNA Polymerase and their Implications on Protein Structure and Drug-Protein Binding.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has stressed the global health system to a significant level, which has not only resulted in high morbidity and mortality but also poses a threat for future pandemics. This situation warrants efforts to develop novel therapeutics to manage SARS-CoV-2 in specific and other emerging viruses in general. This study focuses on SARS-CoV2 RNA-dependent RNA polymerase (RdRp) mutations collected from Saudi Arabia and their impact on protein structure and function. The Saudi SARS-CoV-2 RdRp sequences were compared with the reference Wuhan, China RdRp using a variety of computational and biophysics-based approaches. The results revealed that three mutations—A97V, P323I and Y606C—may affect protein stability, and hence the relationship of protein structure to function. The apo wild RdRp is more dynamically stable with compact secondary structure elements compared to the mutants. Further, the wild type showed stable conformational dynamics and interaction network to remdesivir. The net binding energy of wild-type RdRp with remdesivir is -50.76 kcal/mol, which is more stable than the mutants. The findings of the current study might deliver useful information regarding therapeutic development against the mutant RdRp, which may further furnish our understanding of SARS-CoV-2 biology.

КЛИНИКО-ГЕНЕТИЧЕСКАЯ ХАРАКТЕРИСТИКА БОЛЬНЫХ С TRAPS (ПЕРИОДИЧЕСКИЙ СИНДРОМ, АССОЦИИРОВАННЫЙ С РЕЦЕПТОРОМ ФАКТОРА НЕКРОЗА ОПУХОЛИ) ПО ДАННЫМ ФЕДЕРАЛЬНОГО РЕВМАТОЛОГИЧЕСКОГО ЦЕНТРА

The tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a rare autosomal dominant disease associated with a mutation in the TNFRSF1A gene. It belongs to the group of monogenic autoinflammatory diseases (mAIDs) characterized by the repeated and prolonged episodes of fever, skin rashes, musculoskeletal, ophthalmic symptoms and an increase in the level of acute phase markers. The main targeted therapy is inhibitors of interleukin-1 (IL-1). The purpose of the study was to present the clinical and genetic characteristics of patients with TRAPS according to the data from the V.A. Nasonova Research Institute of Rheumatology (Moscow, Russia). The study included 28 patients from 2013 till 2021 with a clinical diagnosis of TRAPS from a selection of 162 (both pediatric and adult) patients with mAIDs: 17 females/11 males aged 2.5 to 65 years old (Me 9.0 y/o [7.9; 14.5]); 7 adults/21 children. All of them have underwent molecular genetic analysis for mutations in the TNFRSF1A gene. Results: the TRAPS was diagnosed in 28 (17.3%) among 162 patients with mAIDs. The age of onset ranged from 1 month till 28 years old (Me 4.0 y/o [1.0; 7.1]). Most patients fell ill prior to the age of 10 years old (89.3%), of which 8 (28.6%) before 1 year old, 7 (25%) from 1 to 5 years old, 1 (3.6%) from 5 to 10 years old, 10 (35.7%) from 10 to 18 years old and 2 (7.1%) from 21 to 28 years old. The duration of the disease and the delay in diagnosis at the time of inclusion ranged from 3 months till 59 years old. The diagnosis was made within the first year of illness in 4 (14.3%) of observed cases; within 1 to 10 years in 18 (64.3%); in 8 (28.6%) there was a delay in diagnosis of over 10 years; and in 4 (14.3%) the delay was more than 20 years of illness. The duration of attacks varied from 10 to 30 days, the intervals between the attacks ranged from 2 weeks till 6 months. The clinical manifestations were as follows: fever in 96.4% of cases, skin rashes in 71.4%, musculoskeletal symptoms in 18 (64.3%) cases, oligoarthritis in 9 (32.1%), polyarthritis in 2 (7.1% ), gastrointestinal manifestations in 18 (64.3%), lymphadenopathy of various groups in 10 (35.7%), ophthalmological manifestations in 8 (28.6%). The less often were the different nervous system manifestations, which accounted for 6% to 21% of cases, pharyngitis in 4 (14.3%) and stomatitis in 2 (7.1%). An increase in the sedimentation rate of erythrocytes was noted in 23 (82.1%), C-reactive protein in 20 (71.4%), leukocytosis in 11 (39.3%). The diagnosis in all patients was confirmed genetically. 10 patients had a low-penetrance mutation of р.Arg121Gln (р.Arg92Gln according to the old nomenclature). 5 familial cases were identified, the total number of sick family members was 12, from 2 to 4 in each family; 4 cases of amyloidosis (the all four were adults). Treatment: glucocorticoids in 19 (67.9%) cases, Cyclosporine A in 1, Colchicine in 5. 15 patients (53.6%) received genetically engineered biological preparations, the IL-1 (Canakinumab) was prescribed in 10 (35.7%) patients with the duration of admission from 1 to 9 years, Etanercept in 1, Adalimumab in 1 and Tocilizumab in 3. Conclusion: due to the similarity of clinical and laboratory symptoms with manifestations of rheumatic diseases, patients with TRAPS can be encountered in the rheumatologists’ practice. Most patients in the studied cohort had an early onset of the disease (prior to 10 years old), and the diagnosis delay of 10+ years was noted in 1/3 of patients. All patients had a specialized inflammatory phenotype coupled with genetic mutations. About half had required the appointment of biological drugs, IL-1 mainly. A complete response to the therapy in most patients and a good tolerability of treatment were noted, which allows the authors to consider this as the basis in the treatment and management of patients with TRAPS.

Molecular analysis for the OprD gene among Pseudomonas aueroginosa clinical isolates obtained from hospitals in Jordan.

Pseudomonas aeruginosa has increasingly been associated with the emergence of antibiotic resistance. Antibiotic resistance among P. aeruginosa isolates is an ambiguous and complicated mechanism utilizing several enzymes and structural proteins. This study was conducted to investigate the prevalence of mutations in the chromosomal OprD gene that show resistance to carbapenems among clinical isolates of P. aeruginosa. Sixty-three clinical isolates of P. aeruginosa resistant to meropenem were collected from public hospitals in Irbid city, north of Jordan. Analysis of antimicrobial susceptibility was carried out and their susceptibility was categorized. Molecular analysis of mutations in the OprD gene was performed using restriction fragment length polymorphism (RFLP) and DNA sequencing. Molecular analysis of P. aeruginosa isolates showed 52% of the common molecular modifications among the collected isolates. These alterations could be associated and affect meropenem-susceptibility rather than imipenem. The most frequent molecular changes among the resistant isolates were the F170L substitution mutation. This was detected in 22 (35%) of the isolates with an unusual insertion sequence (IS) of 100 bp within the 590 bp DNA segment downstream of the restriction site. The divergent sequence of 10 amino acids 372(VDSSSSYAGL)383 was detected in 7 (11%) of the isolates. A significant alteration in the OprD gene in P. aeruginosa clinical isolates was found. Alterations in the OprD gene could be linked to protein permeability of the outer membrane of P. aeruginosa associated with meropenem resistance. Further investigations with a larger number of bacterial isolates are needed to validate the proposed association.

Diagnosing gastrointestinal stromal tumors: The utility of fine-needle aspiration cytology versus biopsy.

BackgroundGastrointestinal stromal tumors (GIST) are mesenchymal tumors in the intestinal tract originating from a precursor to the interstitial cells of Cajal, which plays a role in gastric motility. The preoperative diagnosis of GIST may be very important for the surgical approach or the need for neoadjuvant treatment and is often done in conjunction with molecular testing.DesignGISTs diagnosed in Stockholm between 1999 and 2019 with biopsy and/or fine‐needle aspiration (FNA) material were included. Clinical and tumor characteristics, as well as sample representability, ancillary techniques, diagnostic accuracy, and time to diagnosis, were categorized and compared.ResultsWe identified 460 diagnostic samples from 347 patients, consisting of 212 biopsies and 248 FNAs. FNA cytology had a significantly (p < 0.05) better sample representability (92% vs. 77%), diagnostic accuracy (84% vs. 76%), and shorter time to diagnosis (4.5 vs. 12.3 days on average) in comparison with biopsies. In addition, ancillary techniques such as immunochemistry and molecular analysis for KIT and PDGFRA mutations could satisfactorily be performed on FNA materials.ConclusionsThere are advantages to both biopsy and FNA cytology in diagnosing GISTs. While the significantly shorter time to diagnosis for FNA cytology can be due to institutional differences, its many strengths make it both an accurate and time‐efficient method for preoperative diagnosis of GIST.

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular Analysis of G1691A Mutation in Factor 5 Leiden and its relation with mtDNA common deletion in Human Recurrent Pregnancy Loss

Molecular modeling, mutational analysis and steroid specificity of the ligand binding pocket of mPRα (PAQR7): Shared ligand binding with AdipoR1 and its structural basis

The 7-transmembrane architecture of adiponectin receptors (AdipoRs), determined from their X-ray crystal structures, was used for homology modeling of another progesterone and adipoQ receptor (PAQR) family member, membrane progesterone receptor alpha (mPRα). The mPRα model identified excess positively charged residues on the cytosolic side, suggesting it has the same membrane orientation as AdipoRs with an intracellular N-terminus. The homology model showed identical amino acid residues to those forming the zinc binding pocket in AdipoRs, which strongly implies that zinc is also present in mPRα. The homology model showed a critical H-bond interaction between the glutamine (Q) residue at 206 in the binding pocket and the 20-carbonyl of progesterone. Mutational analysis showed no progesterone binding to the arginine (R) 206 mutant and modeling predicted this was due to the strong positive charge of arginine stabilizing the presence of an oleic acid (C18:1) molecule in the binding pocket, as observed in the X-rays of AdipoRs. High Zn2+ concentrations are predicted to form a salt with the carboxylate group of the oleic acid, thereby eliminating its binding to the free fatty acid (FFA) binding pocket, and allowing progesterone to bind. This is supported by experiments showing 100 µM Zn2+ addition restored [3H]-progesterone binding of the Q206R mutant to levels in WT mPRα and increased [3H]-progesterone binding to mPRγ and AdipoR1 which have arginine residues in this region. The model predicts hydrophobic interactions of progesterone with amino acid residues surrounding the binding pocket, including valine 146 in TM3, which when mutated into a polar serine resulted in a complete loss of [3H]-progesterone binding. The mPRα model showed there is no hydrogen bond donor in the vicinity of the 3-keto group of progesterone and ligand structure-activity studies with 3-deoxy steroids revealed that, unlike the nuclear progesterone receptor, the 3-carbonyl oxygen is not essential for binding to mPRα. Interestingly, the small synthetic AdipoR agonist, AdipoRon, displayed binding affinity for mPRα and mimicked progesterone signaling, whereas D-e-MAPP, a ceramidase inhibitor, blocked progesterone signaling. Thus, critical residues around the binding pocket and steroid structures that bind mPRα, as well as similarities with AdipoRs, can be predicted from the homology model.

Metanephric adenoma: molecular study and review of the literature.

Introduction: Metanephric adenoma (MA) is an uncommon benign tumor accounting for 0.2–0.7% of adult renal epithelial neoplasms. The clinical course is often indolent, but diagnosis should not be delayed since clinical symptoms (hematuria, fever, palpable abdominal mass, and flank pain) may be non-specific and overlap with those of a malign renal neoplasm. We report on 4 cases of AM, for which morphological and mutational analysis were performed.Material and Methods: Immunohistochemical staining was performed on sections cut from paraffin blocks to assess expression of WT1, vimentin, racemase, CK7, CD10 and RCC. Testing for the BRAF gene mutation V600 was carried out using real-time PCR (Cobas® 4800).Results: In all four cases, tumors were visible as well-circumscribed, non-encapsulated masses located in the renal cortex and extending towards the medulla. At immunohistochemical examination, tumor cells stained negative for CK7, CD10 and RCC and positive for both WT1 (nuclear, intense) and vimentin (cytoplasmic, intense, and diffuse). Molecular analysis revealed the BRAF gene mutation V600E in three cases and wild-type BRAF in the fourth.Conclusions: BRAF molecular mutation analysis may aid diagnosis in cases with atypical histological features, especially in small incisional biopsies when reassessment of surgical treatment may be considered.

Этапная диагностика патологического гастроэзофагеального рефлюкса у детей

Цель работы: разработать и обосновать алгоритм этапной диагностики патологического гастроэзофагеального рефлюкса (ГЭР) в зависимости от тяжести течения и возраста ребенка.Материалы и методы. Под наблюдением были 172 ребенка в возрасте от 3 месяцев до 3 лет, находившихся на амбулаторном и стационарном лечении в детской городской больнице № 3 и городской клинической больнице № 9 им. проф. А.И. Минакова г. Одессы в период с июня 2012 г. по февраль 2014 г. Для скринингового обследования детей использовался рефлюкс-опросник. Также проводилось эндоскопическое исследование пищевода, позволяющее подтвердить наличие рефлюкс-эзофагита и оценить степень его тяжести. На следующем этапе проводился молекулярно-генетический анализ мутаций гена коллагена А3 на основе исследования ДНК клеток слизистой оболочки ротовой полости. Для подтверждения диагноза выполнялось рН-мониторирование пищевода. Статистический анализ полученных данных осуществлялся с использованием пакетов Statistica 7.0 и Microsoft Excel 2010 с интеграцией AtteStat 12.5, интернет-калькулятора SISA (Simple Interactive Statistical Analysis).Результаты и выводы. Этапная диагностика патологического ГЭР позволяет установить и подтвердить диагноз с минимальным использованием инвазивных методов. Использование рефлюкс-опросника способствует ранней диагностике патологического ГЭР и гастроэзофагеальной рефлюксной болезни у детей, дифференцированному подходу к использованию инвазивных методов исследования.

Subfertility, use of fertility treatments and BRCA mutation status and the risk of ovarian cancer

The objective of the study is to evaluate the possible association between history of subfertility, fertility treatments, BRCA mutations and the risk of ovarian cancer. This Israeli National Case-Control study included 1269 consecutive ovarian cancer cases and 2111 individually matched healthy controls. All participants were interviewed and molecular analysis of BRCA mutations were performed to 896 cases. The main outcome measure was reported history of subfertility and exposure to fertility treatments. The rate of reported subfertility was 15.1% and 14.3% in ovarian cancer cases and controls, respectively. However, subfertility was more prevalent in cases with borderline ovarian cancer (but not for invasive ovarian cancer cases) than controls. Multivariate conditional logistic regression revealed that the risk of borderline ovarian cancer was elevated in both women treated for subfertility and those that were not treated for subfertility, (OR = 1.74; 95% CI 0.9-3.36 and OR = 1.79; 95% CI 0.98-3.26, respectively). In non-carriers of BRCA1/2 mutations, fertility treatments were associated with a decreased risk of invasive ovarian cancer while a significant increased risk of borderline ovarian cancer was observed (OR = 2.92, 95%CI 1.67-5.10). Reported subfertility and exposure to fertility treatments were associated with borderline but not with invasive ovarian tumors. This association was more prominent in women who are non-carriers of a BRCA mutation.

Sulfonylurea-Insensitive Permanent Neonatal Diabetes Caused by a Severe Gain-of-Function Tyr330His Substitution in Kir6.2

Background/Aims: Mutations in KCNJ11, the gene encoding the Kir6.2 subunit of pancreatic and neuronal K_ATP channels, are associated with a spectrum of neonatal diabetes diseases. Methods: Variant screening was used to identify the cause of neonatal diabetes, and continuous glucose monitoring was used to assess effectiveness of sulfonylurea treatment. Electrophysiological analysis of variant K_ATP channel function was used to determine molecular basis. Results: We identified a previously uncharacterized KCNJ11 mutation, c.988T>C [p.Tyr330His], in an Italian child diagnosed with sulfonylurea-resistant permanent neonatal diabetes and developmental delay (intermediate DEND). Functional analysis of recombinant K_ATP channels reveals that this mutation causes a drastic gain-of-function, due to a reduction in ATP inhibition. Further, we demonstrate that the Tyr330His substitution causes a significant decrease in sensitivity to the sulfonylurea, glibenclamide. Conclusions: In this subject, the KCNJ11 (c.988T>C) mutation provoked neonatal diabetes, with mild developmental delay, which was insensitive to correction by sulfonylurea therapy. This is explained by the molecular loss of sulfonylurea sensitivity conferred by the Tyr330His substitution and highlights the need for molecular analysis of such mutations.