Homozygous Type Research Articles

Generation, Characterization and Genetic Modification of Human iPSCs Containing Calr, MPL and JAK2 Mutations Found in MPN Patients

Introduction: Myeloproliferative neoplasms (MPNs) that include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are associated with multiple somatic and myeloid-specific mutations. A major challenge to the study of these diseases has been the lack of model systems that can accurately represent their complexity; the clonal heterogeneity within the hematopoietic stem/progenitor cells (HSPC) population is confounding and it is technically challenging to isolate and expand patient HSPC clones in vitro. To overcome this hurdle, we have utilized induced pluripotent stem cell (iPSC) technology to generate disease and clone-specific iPSC lines that preserve the genetic identity of HSPC clones with the JAK2-V617F mutation (Ye et al., 2009 in Blood). Recent advances in MPN genetics have identified CALR and MPL mutations in the majority of JAK2-V617F negative MPN. We therefore created additional panels of iPSCs from patients carrying CALR and MPL mutations in addition to our JAK2-mutated iPSCs. In addition, we used CRISPR/Cas9 technology to study genetic lesions and their contribution the phenotypes by using iPSC in vitro hematopoietic differentiation assays (Ye, et al., 2015 in Molecular Therapy).Methods: We generated iPSC lines from MPN patients with JAK2V617F, CALR or MPL mutations using peripheral blood CD34+ cells by transient expression of reprogramming factors in plasmid vectors. We characterized the pluripotency and karyotype of the derived iPSC lines, and determined if they contained the same somatic mutation as found in blood samples of each MPN patient. Then the derived iPSC cell lines were differentiated to CD34+ HSPCs, erythroid or megakaryocytic cell lineages using defined in vitro culture systems. Erythroid differentiation was determined by flow cytometric analysis of CD235a (Glycophrin A)-positive and CD45-negative cells. Megakaryocytic differentiation was determined by the levels of CD41+CD42+ cells.Results: In vitro differentiation assays have shown increased erythroid differentiation of HSPCs derived from patient-specific iPSCs that carry the JAK2V617F mutation, recapitulating a major PV disease feature. Editing a PV-derived JAK2V617F/V617F iPSC line to JAK2V617F/WT abrogated endogenous erythroid colony (EEC) formation, erythroid expansion and altered erythropoietin dose-response curve characteristic of the parent line. Conversely, gene editing a JAK2V617F/WT to a JAK2V617F/V617F iPSC line from a different PV patient conferred EEC formation and expanded erythropoiesis. In addition, data generated from these isogenic iPSCs suggested the contribution of additional genetic lesions to MPN disease phenotypes in JAK2-V617F positive patients. We also generated iPSC lines from MPN patients with the CALR type II (+5 bp) mutation and the rare MPL-V501L mutation. We found that the iPSC line containing a heterozygous MPL-V501L mutation (derived from an ET patient) appeared normal with iPSC typical morphology and phenotypes (expressing OCT4 and TRA-1-60 pluripotency markers) as the wildtype and JAK2-V617F iPSCs. We also generated four iPSC lines from a MF patient: all the four clones contained the homozygous CALR type II (+5 bp) mutation as found in the blood cells of the MF patient. Two iPSC clones we examined had a normal karyoptype. However, the iPSC lines showed signs of increased spontaneous differentiation when being cultured under the standard culture condition, based on cell morphology and reduction of OCT4 & TRA-1-60 expression. We are currently testing the potential of these iPSC lines containing the CALR and MPLmutations in hematopoietic differentiation, focusing on erythrocytic and megakaryocytic differentiation activities.Conclusions: we believe that by comparative analysis of isogenic iPSCs containing the common and rare MPN driver mutations, both common and specific pathways essential for MPNs can be determined. Precise genome editing or surgery of these mutations by engineered targeting may also provide a novel approach to eliminate the malignant MPN clones.This project is supported in part by the MPN Research Foundation and LLS. DisclosuresNo relevant conflicts of interest to declare.

Identifying mutations in Tunisian families with retinal dystrophy.

Retinal dystrophies (RD) are a rare genetic disorder with high genetic heterogeneity. This study aimed at identifying disease-causing variants in fifteen consanguineous Tunisian families. Full ophthalmic examination was performed. Index patients were subjected to IROme analysis or whole exome sequencing followed by homozygosity mapping. All detected variations were confirmed by direct Sanger sequencing. Mutation analysis in our patients revealed two compound heterozygous mutations p.(R91W);(V172D) in RPE65, and five novel homozygous mutations: p.R765C in CNGB1, p.H337R in PDE6B, splice site variant c.1129-2A > G and c.678_681delGAAG in FAM161A and c.1133 + 3_1133 + 6delAAGT in CERKL. The latter mutation impacts pre-mRNA splicing of CERKL. The other changes detected were six previously reported mutations in CNGB3 (p.R203*), ABCA4 (p.W782*), NR2E3 (p.R311Q), RPE65 (p.H182Y), PROM1 (c.1354dupT) and EYS (c.5928-2A > G). Segregation analysis in each family showed that all affected individuals were homozygotes and unaffected individuals were either heterozygote carriers or homozygous wild type allele. These results confirm the involvement of a large number of genes in RD in the Tunisian population.

The Influence of CYP2C9 and VKORC1 Gene Polymorphisms on the Response to Warfarin in Egyptians

Warfarin is the most commonly used drug for chronic prevention of thromboembolic events, it also ranks high among drugs that cause serious adverse events. The variability in dose requirements has been attributed to inter-individual differences in medical, personal, and genetic factor. Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin by catalyzing the conversion of the pharmacologically more potent S-enantiomer to its inactive metabolites. Warfarin exerts its effect by inhibition of vitamin K epoxide reductase. This protein is encoded by vitamin K epoxide reductase complex subunit 1 gene (VKORC1). The current study aimed to investigate the pharmacogenetic effect of CYP2C9 and VKORC1 gene polymorphisms on the patients response to warfarin. One hundred cases starting warfarin treatment and 20 healthy controls were enrolled. The mean daily dose of warfarin was calculated from patient's medical records. For each patient, less than 10% variability in warfarin dose and a target international normalized ratio (INR) within the therapeutic target range were required for at least 3months for one of the following indications (deep vein thrombosis, pulmonary embolism, cerebrovascular stroke and myocardial infarction) prior to inclusion in the study. Tetraprimer amplification refractory mutation system PCR was performed to determine CYP2C9*2, CYP2C9*3, and the VKORC1 1639 G>A genetic polymorphisms. Plasma warfarin determination was performed using rapid fluorometric assay. Plasma warfarin concentration ranged from 2.19 to 10.98μg/ml with a median 3.52μg/ml. Supratherpeutic INR was observed in 11% of the cases. Thromboembolic complications occurred in 7% of the cases and 8% of cases experienced major bleeding. High maintenance dose (>7mg/day) was associated with the combined non VKORC1*2 and homozygous wild type CYP2C9 (CYP2C9*1*1) alleles, while low maintenance dose was associated with the Variant (AG+AA)/Wild (*1/*1). (p value <0.001). CYP2C9 variant was a risk factor for supratherapeutic INR in the multivariate logistic regression model. Thromboembolic complication and incidence of supratherapeutic INR were observed in patients carrying combined VKORC1 Variant (AG+AA) and CYP2C9 Variant (*2/*3). Data from our study suggest that together with clinical factors, VKORC1 and CYP2C9 polymorphisms are important contributors to warfarin dosing and may help predict adverse effects in Egyptian patients.

MSX1 gene polymorphisms in Mexican patients with non-syndromic cleft lip/palate

ObjectiveNon-syndromic cleft lip/palate malformation (CL/P) is one of the most common birth defects in humans and has a complex etiology involving genetic and environmental factors. Mutations in the MSX1 gene are critical during craniofacial development. The purpose of this study was to investigate the contribution of MSX1 gene polymorphisms to the risk of developing CL/P in a sample of Mexican patients. MethodsThe sample consisted of 282 subjects (69 cases and 213 relatives). Four single-nucleotide polymorphisms (SNP1, P147Q, SNP5 and P278S) were tested for association with CL/P in triad and case-pseudo-control analyses. Polymorphism typing was performed by restriction fragment length polymorphism and dot-blot techniques. Allele and genotype frequencies were calculated between patients and pseudo-controls and compared using the Chi square test with Yates correction. Odds ratios and 95% confidence intervals were obtained using SPSS software (v19). Triad analysis was also performed using the program HAPLIN (v5.3). ResultsIn the cases and pseudo-controls, an association was found between CL/P and the SNP1-G allele (P = 0.031) and the SNP1-G/G genotype (P = 0.032), a polymorphism located near MSX1. Triad analysis showed a tendency toward CL/P susceptibility for the genotype SNP1-G/G (P = 0.075) and an association between CL/P and the haplotype GCTC (P = 0.037). No associated haplotype was found in the cases and pseudo-controls. Two partial haplotypes, GT (SNP1-SNP5) (P = 0.032) and GC (SNP1-P278S) (P = 0.033), were associated with susceptibility in the heterozygous and homozygous types, respectively. In contrast, haplotype AT (SNP1-SNP5) was associated with protection (P = 0.012) in the homozygous type. ConclusionsThe results of this study suggest an association between CL/P susceptibility and SNP1, located near the MSX1 gene, in the Mexican population.

NUDT15 and TPMT genetic polymorphisms are related to 6-mercaptopurine intolerance in children treated for acute lymphoblastic leukemia at the Children's Cancer Center of Lebanon.

Interindividual variability in thiopurine-related toxicity could not be completely explained by thiopurine S-methyltransferase (TPMT) polymorphisms, as a number of patients who are homozygous wild type or normal for TPMT still develop toxicity that necessitates 6-mercaptopurine (MP) dose reduction or protocol interruption. Recently, few studies reported on an inherited nucleoside diphosphate-linked moiety X motif 15 (NUDT15) c.415C>T low-function variant that is associated with decreased thiopurine metabolism and leukopenia in childhood acute lymphoblastic leukemia (ALL) and other diseases. The aim of this study is to measure the frequency of TPMT and NUDT15 polymorphisms and assess whether they are predictors of MP intolerance in children treated for ALL. One hundred thirty-seven patients with ALL of whom 121 were Lebanese were evaluated. MP dose intensity was calculated as the ratio of the tolerated MP dose to planned dose during continuation phase to maintain an absolute neutrophil count (ANC) dose above 300 per μl. One patient was NUDT15 heterozygous TC and tolerated only 33.33% of the planned MP dose, which was statistically significantly different from the median-tolerated MP dose intensity of the rest of the cohort (76.00%). Three patients had the TPMT*3A haplotype and tolerated 40.00-66.66% of the planned MP dose, which was also statistically significantly different from the rest of the cohort. This is the first report on the association of TPMT and NUDT15 polymorphisms with MP dose intolerance in Arab patients with ALL. Genotyping for additional polymorphisms may be warranted for potential gene/allele-dose effect.

Associations Between Newly Discovered Polymorphisms of the CEBPD GENE LOCUS and Body Parameters in Sheep

ABSTRACTAn understanding of what effects particular genes can have on body parameters in productive animals is particularly significant for the process of marker-assisted selection. The gene of transcriptional factor CCAAT/enhancer-binding protein delta (CEBPD gene) is involved in the process of growth in animals and is known to be a promising candidate for use as a genomic marker. The structure of the CEBPD gene locus was determined using NimbleGen sequencing technology (Roche, USA). The effect of polymorphisms, which were identified using the aforementioned technology, was investigated in 30 rams of the Manych Merino sheep breed. Twenty-two single nucleotide polymorphisms (SNP) were detected in the CEBPD gene locus. Significantly, two SNPs, namely, g.315T>G and g.327C>T, have been identified for the first time. It was demonstrated that the complex of linked SNPs, consisting of g.301A>T, g.426T>C, and g.1226T>C, had a negligible effect on body parameters in Manych Merino sheep. Animals with the heterozygous type of SNP g.1142C>T exhibited changes solely in the chest and croup width. The newly discovered SNP g.327C>T was proven to have a negative effect on live weight and body size (p < 0.05) in Manych Merino sheep. Sheep with the heterozygous type of g.562G>A and g.3112C>G SNP complex showed an increase in live weight and dimensions (p < 0.05) compared with those of wild homozygous type. Consequently, SNPs g.327C>T, g.562G>A, and g.3112C>G in the CEBPD gene locus can be successfully used as markers in sheep breeding. Future research will evaluate the influence of the aforementioned SNPs on slaughter indicators for sheep meat production.

A Tetra-Primer Amplification Refractory System Technique for the Cost-Effective and Novel Genotyping of Eight Single-Nucleotide Polymorphisms of the Catechol-O-Methyltransferase Gene

Catechol-O-methyltransferase (COMT) is an enzyme involved in the degradation of catecholamine neurotransmitters. Due to its role in neurotransmitter flux, multiple COMT variants have been associated with the development of psychiatric disorders. Notably, select single-nucleotide polymorphisms (SNPs) of the COMT gene have been implicated in schizophrenia risk, severity, and treatment response. In recognition of the value of a streamlined genotyping method for COMT SNP detection, this study was designed to develop a simple and economical tetra-primer amplification refractory mutation system (T-ARMS) assay for the concurrent detection of eight COMT SNPs: rs4680, rs737865, rs165599, rs2075507, rs4633, rs4818, rs6269, and rs165774. T-ARMS is a genotyping method that uses polymerase chain reaction (PCR) to amplify a multiplex reaction consisting of two primer pairs. T-ARMS primers are customized to each SNP and designed to generate different-sized allele-specific amplicons. This assay was applied to a total of 39 genomic DNA samples. Genotypic designations across the panel of SNPs were subsequently validated by Sanger sequencing. T-ARMS reliably and unambiguously detected all three genotypes (homozygous wild type, heterozygous, and homozygous mutant) for each of the eight COMT SNPs. Compared to traditional low-throughput methods that require post-PCR modification or high-throughput technologies that require sophisticated equipment, T-ARMS is a cost-effective and efficient assay that can be easily adapted by any standard molecular diagnostics laboratory. This T-ARMS assay provides a practical and robust method for COMT SNP detection.

Characterization of miR-206 Promoter and Its Association with Birthweight in Chicken

miRNAs have been widely investigated in terms of cell proliferation and differentiation. However, little is known about their effects on bird growth. Here we characterized the promoter of miR-206 in chicken and found that the preferable promoter was located in 1200 bp upstream of pri-miR-206. In this region, many key transcription factors, including MyoD, c-Myb, CEBPα/β, AP-4, RAP1, Brn2, GATA-1/2/3, E47, Sn, upstream stimulatory factor (USF) and CdxA, were predicted to bind and interact with miR-206 promoter. Overexpression of MyoD sharply increased miR-206 expression in both fibroblast and myoblast cells, and also the regulation in the myoblast cells was much stronger, indicating that miR-206 was regulated by MyoD combined with other muscle specific transcriptional factors. Aiming to further investigate the relationship between miR-206 mutation and transcriptional expression, total of 23 SNPs were identified in the two distinct bird lines by sequencing. Interestingly, the motif bound by MyoD was individually destroyed by G-to-C mutation located at 419 bp upstream of miR-206 precursor. Co-transfecting MyoD and miR-206 promoter in DF-1 cells, the luciferase activity of promoter containing homozygous GG types was significantly higher than CC ones (p < 0.05). Thus, this mutation caused low expression of miR-206. Consistently, eight variants including G-419C mutation exhibited a great effect on birthweight through maker-trait association analysis in F2 population (p < 0.05). Additionally, the regulation of miR-206 on embryo muscle mass mainly by increasing MyoG and muscle creatine kinase (MCK) expression (p < 0.05) with little change in MyoD, TMEM8C and myosin heavy chain (MHC). In conclusion, our findings provide a novel mutation destroying the promoter activity of miR-206 in birds and shed new light to understand the regulation mechanism of miR-206 on the embryonic muscle growth.

Pharmacokinetics and pharmacogenomics of β-lactam-induced neutropenia.

Determine if individuals with β-lactam induced neutropenia have polymorphisms that impair function of MRP4 or OAT1/OAT3. Subjects with β-lactam induced neutropenia were compared to controls for the presence of MRP4 and OAT1/OAT3 polymorphisms, estimated plasma trough concentrations and area under the curve. Subjects with a homozygous polymorphism at MRP4 3348 A to G were 5.3 times more likely to develop neutropenia (p = 0.171). No statistical differences were noted in pharmacokinetic parameters. Contingency analysis of children greater than 5 years of age showed neutropenia in subjects who were homozygous wild type at MRP4 3348 A to G was significantly associated with standard or high dosing (p = 0.03). MRP4 3348 A to G should be further studied for potential contribution to the development of β-lactam induced neutropenia.

Mechanism of TLR-4/NF-κB pathway in myocardial ischemia reperfusion injury of mouse.

To detect the expression of Toll-like receptor 4 (TLR-4) and NF-κB and to discuss the mechanism of TLR-4/NF-κB pathway in the myocardial ischemia reperfusion injury of mouse. TLR-4 mutant mice and wild homozygous mice were divided into the model group and sham group. Mice in the model group were given the ligation of left anterior descending coronary artery for the modeling, while mice in the sham group were not given the ligation after threading. The cardiac muscle tissues were collected for the morphological observation. The immunohistochemistry was employed to detect the expression of NF-κB, Western blot was used to detect the expression of TLR-4 and ELISA to detect the expression of serum inflammatory factors. The expression of NF-κB in TLR-4 null mice after the myocardial ischemia reperfusion was significantly lower than that in wild homozygous mice. For the model group and sham group, the expression of TLR-4 in wild homozygous mice was all significantly higher than that in TLR-4 null mice, while the expression of TLR-4 in TLR-4 null mice in the model group was significantly higher than that in sham group, with the statistical difference (P<0.05). The expression of inflammatory factors in TLR-4 null mice and wild homozygous mice in the model group was significantly higher than that in sham group. The expression of all factors in group A with TLR-4 null was significantly lower than that in group B with wild homozygous type, with the statistical difference (P<0.05). TLR-4/NF-κB pathway is closely related to the myocardial ischemia reperfusion injury, which plays its role through the release of inflammatory cytokines.

Establishment of a quenching probe method for detection of NPM1 mutations in acute myeloid leukemia cells.

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

Association of the C3435T Multi-Drug Resistance Gene-1 (MDR-1) Polymorphism with Clopidogrel Resistance among Moroccan Acute Coronary Syndromes (ACS) Patients

Background: Antiplatelet drugs are recommended as one of the main medications used in Acute Coronary Syndromes (ACS) patients. However, an inter-individual variability in platelet response to Clopidogrel has been found in a substantial group of them. Genetic is known be the dominant influencing factor for individual and interethnic variations in drug responses. Polymorphisms in the genes involved in Clopidogrel absorption, biotransformation to the active metabolite, or platelet response to adenosine diphosphate (ADP) have been associated to this impaired response. Interestingly, Multi-Drug Resistance gene-1 (MDR-1) polymorphism influences oral bioavailability of clopidogrel and prognosis of ACS patients. The objectives of our study are, first, to determine the frequency of the C3435T MDR1 polymorphism among Moroccan ACS patients compared to healthy subjects; and second, to assess its effect on Clopidogrel response in a sample of Moroccan ASC patients. Methods and results: 40 ACS patients were recruited and compared to 99 healthy controls. Extracted DNA samples were genotyped by PCR-RFLP method using MboI restriction enzyme. The VerifyNow assay was used to evaluate platelet function among ACS patients. Our results showed that HTA, Smoking, Creatinine and Sex were statistically associated to Clopidogrel resistance (P=0.05; P=0.05; P=0.05 and P=0.04 respectively). 63.64% of the ST (+) patients were carrying the mutant allele, 54.5% of them having the heterozygous genotype and 36.4% the homozygous mutant one, compared to 9.1% having the homozygous wild type genotype. 62.5% of the resistant group was carrying the mutant allele (50% of them had the TT mutant genotype, 25% CT and 25% CC profiles). Among cases, 42.5% were homozygous mutant TT, 35% CC and 22.5% CT, compared to 39.4% CC, 51.5% CT and 9.1% TT among healthy controls. This polymorphism was positively correlated to ACS risk of development in the CT genotype and the additive transmission model (OR [95% CI]=0.49 [0.16-0.99], P=0.002; OR [95% CI]=2.17 [0.94-2.72], P=0.02), increasing-thus- the association of this polymorphism with the risk of pathology occurrence. Conclusion: To the best of our knowledge, our study is the first in Morocco to assess the effect of the C3435T MDR1 polymorphism on Clopidogrel response in a sample of Moroccan ACS patients; we also tried to explore the frequency of this polymorphism among Moroccan ACS patients and compare them to healthy individuals. The distribution of the mutant allele in Clopidogrel resistance groups, among ACS sub-types, and also in cases compared to controls and there correlations, suggest a potential association of this variant with Clopidogrel resistance and ACS occurrence risk in our population. Understanding the functional and clinical consequences of this MDR1 variant and others may provide a basis for treating patients more effectively. If this variability in response could be assigned to a mutation in the MDR1 gene, patients could be screened and appropriate dose adjustments could be made on the basis of their MDR1 genotype.

Association of tumor necrosis factor-alpha 308G/A polymorphism with recurrent miscarriages in women.

BACKGROUND:Recurrent miscarriage (RM) is the most common pregnancy loss in the first trimester affecting approximately 0.5–2% of women. It is a heterogeneous condition and remains an enigma as the underlying cause is still difficult to track down.AIM:This study was aimed to investigate the distribution of tumor necrosis factor-alpha (TNF-α) 308G/A polymorphism and its association with RM in females. The comparative picture was also demonstrated by comparing genotyping results with healthy control women having no history of miscarriage.METHODS:This clinical study was conducted among 115 women aged 21–44 years with history of recurrence of miscarriage. The samples were collected from women attending the outpatient departments of various hospitals, nursing homes, and infertility clinics of this region. In the present study, 111 fertile healthy women aged 24–46 years with at least one live birth and no history of miscarriage were also included.RESULTS:Mean age of women with RM was found to be 28 ± 5.6 years by recall method, whereas it was found to be 30 ± 7.4 in context to healthy women with no history of pregnancy loss. In the present study, 66% of women with RM had homozygous wild type genotype (GG) while 30% and 4% of women had heterozygous (GA) and homozygous mutant genotype (AA), respectively. Among control group, 79%, 16%, and 5% of women showed GG, GA, and AA genotype, respectively.CONCLUSION:The current study supports the concept of TNF-α 308G/A variant in particular with reproductive failure, GG and GA alleles showing 1-fold risk association with RM (odds ratio: 1.86 and 1.43, respectively).

Does 24bp Duplication of Human CHIT1 Gene (Chitotriosidase1) Predispose to Filarial Chyluria? A Case-Control Study.

Chyluria which is endemic in many parts of the world is mainly caused by Wuchereria bancrofti. CHIT1 (chitotriosidase) is produced by macrophages and plays an important role in the defense against chitin containing pathogen such as filarial parasite. Variation in the coding region with 24 bp duplication allele results in reduced CHIT1 activity that enhance the survival of parasite which may play a role in the occurrence of disease. To examine the role of 24bp duplication of CHIT1 gene in patients of filarial chyluria (FC). A case-control study was carried out where 155 confirmed FC patients and equal number of age-, sex- and residence-matched controls without any symptoms or signs of lymphatic filariasis, confirmed by negative immunochromatographic card test (ICT) and IgG/IgM combo rapid antibody test, from a hospital-based population were enrolled. Filarial aetiology was confirmed on the basis of DEC-provocative test (Giemsa staining), ICT and IgG/IgM- antifiarial antibody test. The patients positive by either of these tests were enrolled as FC cases. 24bp duplication in CHIT1 gene in FC was detected by the product size 99bp of amplified gene using polymerase chain reaction. The mean ages of patients and controls were 38.25±12.09 and 35.45±12.53 years, respectively while male: female ratio was 2.4:1. The mean duration of illness in chyluria patients was 62.81±60.83 months and mean number of episodes was 2.54±1.11. Homozygous wild type, heterozygous and homozygous mutant frequencies were 10.3%, 81.3% and 8.4% in FC patients and 18.7%, 75.5%, and 5.8% in controls, respectively. The 24bp duplication in CHIT1 gene showed a significant association in Heterozygous (HT) genotype with Odd Ratio (OR) of 1.95, 95% Confidence Interval (CI) (1.01-3.77); p=0.04. However, the homozygous mutant genotype (TT) was found to be non-significant with OR of 2.61, 95% CI (0.91-7.45); p=0.07. The combination of both HT+TT was also found to be significant with OR of 2.00, 95% CI (1.03-3.85); p=0.03. In this study from Northern India, CHIT1 gene polymorphism showed an influence as a possible risk factor for susceptibility to FC. Further studies need to be done on a larger number of FC patients in different regions of the country.

Deletion of WISP1 leads to higher sensitivity to carbon tetrachloride-induced liver damage

WISP1 (Wnt-induced secreted protein-1) is a member of the CCN family of matricellular proteins. These highly conserved and secreted proteins can interact with integrins and modulate many biological processes like migration, proliferation, differentiation and apoptosis. CCN proteins have been show to play divergent roles in liver pathophysiology, in spite of having very similar structural domains. Importantly, little is known about WISP1 in this context. Gene array analysis of mouse liver after acute injury by intraperitoneal injection of CCl4 in C56BL/6N mice showed WISP1 mRNA upregulated during early injury phase, between 2h and 24h after CCl4 injection. These results were confirmed by real time PCR, showing a maximal induction of 40 fold over healthy liver at 24h. To establish the role of WISP1 in acute liver damage we used a knockout mouse model, in which WISP1 expression is disrupted by replacing Exon 2 with a resistance cassette and a stop codon. Heterozygous mice were bred to establish homozygous knockout and wild type lines which were validated by genotyping. Real time PCR analysis confirmed the absence of WISP1 mRNA expression in KO mice challenged with CCl4. Remarkably, deletion of WISP1 led to a significant increase of necrotic area at 24h. This was show by hematoxylin & eosin staining reaching 49% in KO vs. 27% in WT mice (p < 0.0001; n = 13 wild type, n = 11 KO) and serum transaminase activity (GPT: 8230 U/l in WT, 12300 U/l in KO). Importantly, immunofluorescence and real time PCR showed that WT and WISP1 KO mice have no significant differences in CYP2E1 expression, suggesting that the difference in tissue injury does not depend on alterations of CCl4 metabolism in KO mice. To further characterize this different phenotype, we performed a time course after CCl4 intoxication (460 mg/kg, i.p.) by collecting liver tissue at 30 min, 2h, 8h, 18h, and 24h. Interestingly, already at 18h KO mice showed a clear necrotic area around the central veins whereas WT mice showed ballooned hepatocytes with cytoplasmic translocation of high-mobility group protein B1 (HGMB1), indicating that WISP1 KO mice had a more advanced stage of liver injury at this time point than the WT counterparts. This phenotype was also observed upon intoxication with lower doses of CCl4 (132.4 mg/kg and 76 mg/kg). Analysis of cytokine expression by real time PCR showed a stronger induction of TNFα in WISP1 KO mice (7-fold v/s 10-fold at 18h, and 3-fold vs. 6-fold at 24h in WT vs. WISP1 KO respectively), and IL6 (5-fold vs. 10-fold at 18h, and no induction vs. 3-fold at 24h in WT v/s WISP1 KO respectively). Furthermore, western blot analysis showed marked alterations in signal transduction pathways in liver tissue of WISP1 KO mice, with stronger activations of p-JNK at 2h and p-STAT3 at 18h and 24h. Currently we are investigating the mechanism through which WISP1 exerts a protective role upon CCl4 stimulation in liver damage and in the subsequent regeneration process. In conclusion we have strong evidence that WISP1 plays an important role in acute liver damage.

JAK2V617F Clonal Architecture in MPNs during JAK2 Inhibitor Treatment

IntroductionThe Myeloproliferative Neoplasms (MPNs) are characterized by a recurrent point mutation of JAK2 gene (JAK2 V617F). This mutation, which usually affects only one of the JAK2 gene alleles in Essential Thrombocythemia (ET), frequently becomes homozygous in Polycythemia Vera (PV) and Myelofibrosis (MF) due to homologous mitotic recombination. A JAK2 V617F-mutated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1(GGCC), (Nat Genet 2009; 41:446) with complete linkage disequilibrium although the risk of developing MPNs is independent of the 46/1 haplotype. Furthermore, JAK2 V617F specifically arises on the 46/1 allele in most cases, thus predisposes to the development of MPN (Leukemia 2010; 24:1533; Nat Genet 2009; 41:446). These observations suggest that it is possible to define the JAK 2V617F clonal architecture starting from 46/1 SNP allele burden and the homologous recombination frequency.Aim and Methods The aim of the study was to investigate changes in the JAK2 V617F clonal structure in patients affected by PV, ET or MF and treated with ruxolitinib, a JAK1/JAK2 inhibitor recently approved for MF and PV and under investigation in ET pts intolerant of or resistant to hydroxyurea. We used a recently described methodology (Leukemia 2014;28:460) combining allele burden evaluations of both 46/1 and JAK2 and the frequency of mitotic recombination to derive the percentages of JAK2 V617F clones in MPN patients in 46/1 heterozygous patients for rs12343867 polymorphism (C/T). The JAK2 allele burden values were confirmed independently by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method.Results We analysed 40 patients (pts) affected by MPNs (12 PV, 1 ET and 27 MF) treated with ruxolitinib over 1 to 5 years (yrs) of follow up (FU); 13 patients (32.5%) presented a reduction of at least 10% of JAK2 V617F allele burden values at latest FU: 7/12 PV (58.3%), 1/1 ET (100%), 6/27 PMF (22.2%). Among these, 9 pts with at least two years of FU were eligible for the study. A quantification of JAK2 homozygous (V617F/V617F), heterozygous (JAK2 V617F/wt) and wild type (wt/wt) clones was obtained using the methodology described above. We found a median reduction of JAK2 V617F/V617F and JAK2 V617F/wt clones of 32.31 % and 8.82 %, respectively, in PV (n.4); 12.22% and 17.86%, respectively, in MF (n.4); 35.99% and 100%, respectively, in ET (n.1). Furthermore, an almost complete molecular remission (CMR) was seen in two PV patients with 4 and 5 yrs of FU respectively and in one ET patient after 5 years of treatment. In these patients we observed reduction of homozygous clones of 99.60%, 86.91% and 100%, respectively, and the residual JAK2 allele burden was due to the persistence of JAK2 V617F/wt clones (Figure). Conversely, in one patients with an increase in JAK2 allele burden from 39% to 45% after 1,5 yrs under ruxolitinib, we observed an increase in JAK2V617F/wt but not in JAK2 V617F/V617F clones.Conclusion. Taken together, these results showed that ruxolitinib may preferentially target the homozygous clones inducing in some cases an almost complete molecular remission with prolonged treatment. Future analysis will be performed to study the clonal architecture in an increased number of patients treated with JAK inhibitors as single agent or in combination with other drugs. [Display omitted] DisclosuresVannucchi:Shire: Speakers Bureau; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

H2-EB1 Molecule Alleviates Allergic Rhinitis Symptoms of H2-Eb1 Knockout Mice.

H2-EB1 molecule which is the homolog of Human HLA-DRB1 is proposed to be associated with allergic rhinitis (AR). Construction of H2-Eb1 knockout animal models provides a tool to elucidate the role of H2-EB1 and AR pathogenesis. To establish the H2-Eb1 knockout model and investigate the H2-EB1 functions in H2-Eb1 knockout mice as a model of AR. The Cre/LoxP system and ES gene knockout technology were applied to create heterozygous H2-Eb1 (+/-) knockout mice and their offspring of knockout homozygous(-/-), heterozygous (+/-) and wild type (+/+) H2-Eb1 mice. After identification, offspring of heterozygous (+/-) and homozygous (-/-) H2-Eb1 knockout mice were randomly selected to establish AR models to demonstrate the role of H2-Eb1 in AR pathogenesis. The H2-Eb1 knockout mice model was successfully established. The reproduction and feeding of the homozygous (-/-) H2-Eb1 knockout mice were successful. Compared with the control group, the serum OVA-IgE and IL-4 levels significantly increased, while IFN-γ levels significantly dropped (p<0.05) in the experimental groups. For the two experimental groups, the homozygous (-/-) mice group had lower serum OVA-IgE and IL-4 levels, and higher IFN-γ levels than their heterozygous (+/-) counterparts (p<0.05), concomitant with slighter allergic symptoms (gentle behavior and less eosinophils in nasal mucosa). Our study demonstrated that knockout of H2-Eb1 gene could alleviate mouse AR Symptoms, indicating H2-Eb1 may play an important role in regulating Th1/Th2 balance during the pathogenesis of AR.

The Cellular Response to Oxidatively Induced DNA Damage and Polymorphism of Some DNA Repair Genes Associated with Clinicopathological Features of Bladder Cancer

Genome instability and impaired DNA repair are hallmarks of carcinogenesis. The study was aimed at evaluating the DNA damage response in H2O2-treated lymphocytes using the alkaline comet assay in bladder cancer (BC) patients as compared to clinically healthy controls, elderly persons, and individuals with chronic inflammations. Polymorphism in DNA repair genes involved in nucleotide excision repair (NER) and base excision repair (BER) was studied using the PCR-RFLP method in the Belarusian population to elucidate the possible association of their variations with both bladder cancer risk and clinicopathological features of tumors. The increased level of H2O2-induced DNA damage and a higher proportion of individuals sensitive to oxidative stress were found among BC patients as compared to other groups under study. Heterozygosity in the XPD gene (codon 751) increased cancer risk: OR (95% CI) = 1.36 (1.03–1.81), p = 0.031. The frequency of the XPD 312Asn allele was significantly higher in T ≥ 2 high grade than in T ≥ 2 low grade tumors (p = 0.036); the ERCC6 1097Val/Val genotype was strongly associated with muscle-invasive tumors. Combinations of homozygous wild type alleles occurred with the increased frequency in patients with non-muscle-invasive tumors suggesting that the maintenance of normal DNA repair activity may prevent cancer progression.

Pentraxin 3 genotyping in relation to serum levels of pentraxin 3 in patients with acute ST-segment elevation myocardial infarction

ObjectiveThe aim of the study was to investigate the association of serum Pentraxin 3 and genotyping with the risk of developing AMI and its severity. Patients and methodsFifty patients admitted to the coronary care unit presented with STEMI (acute ST segment myocardial infarction) at the Cardiology Department, Menoufia University Hospital in the period from October 2014 to April 2015 and another 20 subjects age- and gender-matched were taken as the control group. All patients and control groups were subjected to the following: Full history taking, complete clinical examination. ECG and echocardiography and Laboratory investigation including: estimation of lipid profile, urea and creatinine, CKMB, troponin I, serum pentraxin 3 and Genotyping of pentraxin 3 A/G SNP (rs2305619). ResultsThe patients with myocardial infarction had significantly higher levels of pentraxin 3 than the controls. The cut-off values for PTX3 and troponin I were 4.35ng/ml and 0.34μg/l respectively. Pentraxin 3 showed the highest diagnostic accuracy of coronary artery disease (96%), with sensitivity (96%) and specificity (95%). The highest serum pentraxin 3 levels were in the AA mutant homozygous type. ConclusionPTX3 is one of the earliest biomarkers for detecting acute coronary syndrome. rs2305619 AA genotyping of the pentraxin 3 gene might be a candidate risk factor for development of coronary artery disease, presumably by increased pentraxin 3 levels.

Prospective associations between vitamin D status, vitamin D–related gene polymorphisms, and risk of tobacco-related cancers

Experimental evidence has suggested that vitamin D may be protective against tobacco-related cancers through the inhibition of the formation of tumors induced by tobacco carcinogens. To our knowledge, only one previous epidemiologic study investigated the association between vitamin D status and tobacco-related cancer risk, and no study has focused on vitamin D-related gene polymorphisms. Our objective was to prospectively study the association between plasma 25-hydroxyvitamin D [25(OH)D] concentrations, vitamin D-related gene polymorphisms, and risk of tobacco-related cancers. A total of 209 tobacco-related cancers were diagnosed within the SU.VI.MAX (Supplémentation en vitamines et minéraux antioxydants) cohort (1994-2007) and were matched with 418 controls as part of a nested case-control study. Tobacco-related cancers (i.e., cancers for which tobacco is one of the risk factors) included several sites in the respiratory, digestive, reproductive, and urinary systems. Total plasma 25(OH)D was assessed with the use of an electrochemoluminescent assay. Polymorphisms were determined with the use of a TaqMan assay. Conditional logistic regression models were computed. A 25(OH)D concentration ≥30 ng/mL was associated with reduced risk of tobacco-related cancers (OR for ≥30 compared with <30 ng/mL: 0.59; 95% CI 0.35, 0.99; P = 0.046). This association was observed in former and current smokers (OR for ≥30 compared with <30 ng/mL: 0.43; 95% CI: 0.23, 0.84; P = 0.01) but not in never smokers (P = 0.8). The vitamin D receptor (VDR) FokI AA genotype and retinoid X receptor (RXR) rs7861779 TT genotype were associated with increased risk of tobacco-related cancers [OR for homozygous mutant type (MT) compared with wild type (WT): 1.87; 95% CI: 1.08, 3.23; P-trend = 0.02; OR for heterozygous type (HT) plus MT compared with WT: 1.60; 95% CI: 1.07, 2.38; P = 0.02]. In this prospective study, high vitamin D status [25(OH)D concentration ≥30 ng/mL] was associated with decreased risk of tobacco-related cancers, especially in smokers. These results, which are supported by mechanistic plausibility, suggest that vitamin D may contribute to the prevention of tobacco-induced cancers in smokers and deserve additional investigation. The SU.VI.MAX trial was registered at clinicaltrials.gov as NCT00272428.