Homozygous Type Research Articles

Genotyping of a p73 Dinucleotide Polymorphism in Human Cancer Cell Lines

The p73 gene is a member of the p53 tumor suppressor family. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14‐to‐A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and DNp73 isoforms, respectively. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI =0.31‐0.99] for aggressive prostate cancer (PCa) and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. We hypothesize the presence of the p73 DNP allele causes altered p73 promoter usage, ultimately resulting in increased TAp73/DNp73 protein isoform ratios, which could explain the observed decreased risk for PCa aggressiveness. Our ultimate goal is to assess the potential of p73 DNP as a biomarker for lower aggressive PCa risk. Therefore, our initial goal in this study was to determine the p73 DNP genotype in four human cancer cell lines (DU 145, MDA‐MB‐468, PC‐3 and SW48) not yet characterized for p73 DNP status. We previously characterized the p73 DNP genotype of three human cancer cell lines that we used as genotyping controls: Caco‐2 (homozygous polymorphic), NCI‐H1299 (homozygous wild type), and HepG2 (heterozygous). These seven cell lines were cultured and genomic DNA was isolated and quantitated. Cell line genomic DNA samples were used in TaqMan Real Time‐PCR allelic discrimination assays to determine the p73 DNP genotypes. Our data conclusively determined that DU 145, MDA‐MB‐468, and SW48 were p73 DNP homozygous wild type, and PC‐3 was p73 DNP heterozygous.

250 Large Deletion in the EPCAM Gene Responsible for the Milder Phenotype of Congenital Tufting Enteropathy

Introduction: A number of point mutations within the EPCAM gene have been found to be responsible for congenital tufting enteropathy (CTE). We previously described a milder phenotype of this condition in a cohort of patients in the Maltese Islands1. EpCAM staining was negative in all patients in this cohort1, suggesting a defective EPCAM gene in the milder phenotype as well. Aims: To identify the underlying genetic abnormality within the EPCAM gene responsible for the milder phenotype of CTE. Methods: In the period 1985 2012, eight Maltese patients with CTE from six unrelated families were retrospectively identified and enrolled. Genomic DNA was extracted from peripheral blood. Primers for all nine exons within the EPCAM gene were designed and optimized. PCR products were amplified, checked on a gel, purified and sequenced. To sequence exon 4 exon 6 region, the PCR product was purified from the gel and ligated in a TA Vector. The ligated products were transformed into DH5α bacteria and cultured on ampicillin-containing agar plates. The cultured DNA was extracted, sequenced and analysed. All DNA sequences were compared with controls. Unaffected family members (parents) and healthy controls were screened for any mutation identified in the affected patients. Results: Genetic analysis of the EPCAM gene in Maltese CTE patients revealed a novel homozygous 1773bp deletion in seven out of eight patients, starting from 1170bp downstream of exon 4, up to 721bp upstream of exon 6, resulting in complete deletion of exon 5 and partial deletion of Intron 4-5 and Intron 5-6. The remaining patient was a heterozygote for the deletion. The mutant homozygous variant resulted in a frameshift, introducing 23 novel amino acids, followed by the formation of a premature stop codon, p.Ala164Metfs*24. This resulted in a predicted truncated protein with the loss of the transmembrane domain and thus the complete lack of EpCAM protein within the intestinal epithelium. Three sets of parents of 3 affected patients were all heterozygous for this deletion. Out of 100 healthy controls tested, 98 were homozygous wild type and two (2%) heterozygous for the deletion. Conclusions: This novel large deletion within the EPCAM gene was found to be responsible for the milder phenotype of CTE in seven Maltese patients. The heterozygous mutation in the remaining patient is likely to be compounded by a second mutation, which is as yet unidentified. The heterozygous mutation of the patients' parents confirms the autosomal recessive pattern of inheritance of CTE. This genetic deletion present in all patients implies a founder effect and we propose this as the first genotype-phenotype correlation in isolated intestinal TE patients.

Does the Naked Neck Meat Type Chicken Yield Lower Methionine Requirement Data?

Simple SummaryIt was hypothesized that naked neck chickens could have a lower methionine requirement according to their reduced feather coverage. The hypothesis was examined by nitrogen balance studies and non-linear model application for estimating methionine requirement data of naked neck chickens. It was concluded that naked neck birds do not require less methionine than normally-feathered birds.Methionine (Met) requirement studies with homozygous (Na/Na) and heterozygous (Na/na) naked neck meat type chicken utilized 144 birds of average weight (50% each genotype and sex) within two N balance experiments involving both the starter (d10–20) and grower period (d25–35). The birds were randomly allotted to five experimental diets with graded protein supply and Met as the limiting amino acid. The proportion of native feed protein sources (soy protein concentrate, maize, wheat, fishmeal and wheat gluten) was kept constant to ensure a uniform protein quality in all diets. The Met requirement depending on genotype, sex, age period and growth performance (protein deposition) was estimated using a non-linear modeling procedure of N utilization in monogastric animals. On average, 0.47% (Na/Na) and 0.45% (Na/na) dietary Met was established as adequate in the starter diet, as well as 0.37% (Na/Na) and 0.36% (Na/na) Met in the grower diet for both of the sexes. In conclusion, the Met requirement of the naked neck chicken is not significantly different from its normally-feathered counterparts. In addition, the low feather production was not reflected by reduced requirement for Met in naked neck birds. However, these conclusions are valid only at the given Met:Cys ratio (1:1) in the experimental diets.

Slow R-warfarin 7-hydroxylation mediated by P450 2C19 genetic variants in cynomolgus monkeys in vivo

Cynomolgus monkeys are widely used as non-human primate species in preclinical studies, due to their close evolutionary relationship to humans. Monkey cytochrome P450 2C19 (formerly known as P450 2C75), highly homologous to human P450 2C19, has been identified to be R-warfarin 7-hydroxylase in cynomolgus monkeys. In the present study, the in vivo pharmacokinetics of stereoselective warfarin and metabolites at a dose of 1.0mg/kg were investigated after oral and intravenous administration of racemic warfarin to fasted male cynomolgus monkeys (n=11, from Indochina, 4–8 years of age, 3.5–7.4kg of body weight), which had been genotyped for P450 2C19 [c.298TT>AA; c.308C>T; and c.334ATC>CTT]. Kinetic parameters for S-warfarin were not different among the homozygous mutant, heterozygous mutant, and wild type groups; however, values of elimination half-lives, area under the curves, and total body clearance of R-warfarin in the homozygous mutant group showed one-order differences from those values in the wild type group after oral or intravenous administration. R-Warfarin 7-hydroxylations in vivo in homozygous mutant groups were slow compared to wild type or heterozygous mutant groups. These results demonstrate that inter-animal variations of R-warfarin clearance in cynomolgus monkeys are associated with P450 2C19 genetic variants [p.Phe100Asn, p.Ala103Val, and p.Ile112Leu]. Because some interindividual variability of P450 2C-dependent drug metabolism in cynomolgus monkeys, similarly in humans, is accounted for by polymorphic P450 2C19 variants, genotyping of drug metabolism enzymes should be considered before and after P450-dependent drug metabolism testing and evaluations in cynomolgus monkeys.

Endothelial Nitric Oxide Synthase Gene Polymorphism Relationship With Semen Parameters and Oxidative Stress in Infertile Oligoasthenoteratozoospermic Men

To assess endothelial nitric oxide synthase (eNOS) gene polymorphism relationship with semen parameters and oxidative stress in infertile oligoasthenoteratozoospermic (OAT) men. Three hundred subjects were divided into healthy fertile men (n = 80) and infertile OAT men (n = 220). They were subjected to history taking, clinical examination, and semen analysis in addition to malondialdehyde and glutathione peroxidase estimation in seminal plasma. Polymorphisms of eNOS G894T and T786C genotypes in peripheral blood were identified by the polymerase chain reaction-restriction fragment length polymorphism analysis. Comparing infertile OAT men with fertile controls, eNOS genotype G894T demonstrated prevalence of 36.8% vs 50% for wild type (GG), 35.0% vs 47.5% for heterozygous type (GT), and 28.2% vs 2.5% for mutant homozygous type (TT). Compared with GG homozygotes, carriers with A allele exhibited >1.716-fold increased risk of OAT occurrence. Comparing infertile OAT men with fertile controls, eNOS genotype T786C demonstrated prevalence of 37.3% vs 51.3% for wild type (TT), 32.7% vs 45% for heterozygous type (TC), and 30% vs 3.7% for mutant type (CC) with significant differences. Compared with TT homozygotes, carriers with C allele exhibited >1.769-fold increased risk of OAT occurrence. G894T and T786C genotypes demonstrated significant negative correlation with sperm count, total sperm motility, sperm normal forms, and seminal glutathione peroxidase, and significant positive correlation with seminal malondialdehyde. G894T genotype demonstrated significant positive correlation with T786C genotype. There is a significant relationship between eNOS genotypes T786C, G894T polymorphisms with decreased sperm parameters and increased seminal oxidative stress.

Preemptive Genotyping of CYP2C8 and CYP2C9 Allelic Variants Involved in NSAIDs Metabolism for Sickle Cell Disease Pain Management.

Interindividual variability in analgesic effects of nonsteroidal anti-inflammatory drugs prescribed for sickle cell disease (SCD) pain is attributed to polymorphisms in the CYP2C8 and CYP2C9 enzymes. We described CYP2C8 and CYP2C9 genotype/phenotype profiles and frequency of emergency department (ED) visits for pain management in an African American SCD patient cohort. DNA from 165 unrelated patients was genotyped for seven CYP2C8 and 15 CYP2C9 alleles using the iPLEX ADME PGx multiplexed panel. CYP2C8*1 (0.806),*2 (0.164), *3 (0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%), respectively. CYP2C9*1 (0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%), respectively. Fifty-two and 55 subjects, respectively had at least one variant CYP2C8 or CYP2C9 allele. Although the distribution of the CYP2C9 (p = 0.0515) phenotypes was marginally significantly in high and low ED users; some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure. CYP2C8 and CYP2C9 preemptive genotyping could potentially enable clinicians to identify patients with impaired metabolic phenotypes.

Evaluating the Age-Dependent Potential for Protein Deposition in Naked Neck Meat Type Chicken

Simple SummaryGrowth rates of fast-growing chickens are reduced by a higher ambient temperature (AT) because of difficulties in dissipating heat through the feather coverage. Naked neck meat type genotypes could be helpful in increasing the tolerance for high AT. However, basic model parameters of this genotype necessary to further assess amino acid requirements are as yet unavailable. The experiments were conducted to estimate both the daily nitrogen maintenance requirement (NMR) and the potential for daily nitrogen retention NRmaxT). These observed model parameters provide the basic information to characterize the growth potential of the genotype for further application in modeling of individual amino acid requirements of naked neck meat type chicken.The introduction of the naked neck gene (Na) into modern meat type chicken is known to be helpful in increasing the tolerance for a high ambient temperature (AT) by reducing the feather coverage which allows for a higher level of heat dissipation compared to normally feathered (na/na) birds. In addition, reduced feather coverage could affect requirements for sulfur containing amino acids. As a prerequisite for further modeling of individual amino acid requirements, the daily N maintenance requirement (NMR) and the threshold value of daily N retention (NRmaxT) were determined. This was carried out using graded dietary protein supply and exponential modeling between N intake (NI) and N excretion (NEX) or N deposition (ND), respectively. Studies with homozygous (Na/Na) and heterozygous (Na/na) naked neck meat type chicken utilized 144 birds of average weight (50% of each genotype and sex) within two N balance experiments during both the starter (days 10–20) and the grower period (days 25–35). Birds were randomly allotted to five diets with graded dietary protein supply but constant protein quality. The observed estimates depending on genotype, sex and age varied for NMR and NRmaxT from 224 to 395 and 2881 to 4049 mg N/BWkg0.67/day, respectively.

Metabolic and cardiac phenotype characterization in 37 atypical Dunnigan patients with nonfarnesylated mutated prelamin A

Laminopathies are associated with a broad spectrum of clinical manifestations, from lipodystrophy to cardiac diseases. The purpose of this study was to assess genotype-phenotype correlations in a lipodystrophic laminopathy caused by the Lamin A (LMNA) mutation T655fsX49. This mutation leads to synthesis of nonfarnesylated-mutated prelamin A that does not undergo the physiologic lamin A maturation process. We studied 35 patients originating from Reunion Island who carried the LMNA T655fsX49 mutation. Comparisons of cardiac and endocrinologic features were made between homozygous and heterozygous patients. Homozygous patients presented more overlapping syndromes with severe cardiac phenotypes, defined by cardiolaminopathy, early atheroma with coronary heart disease (CHD) and high-degree conduction disorder compared with heterozygous (40% vs 4%; P = .016). Moreover, homozygous patients had earlier onset (49.6 vs 66 years old; P = .0002). Left ventricle lowered ejection fraction associated with heart failure was more frequent in homozygous than in heterozygous patients (40% vs 0%, respectively). Lipodystrophic traits were more marked in the homozygous group but only reached statistical significance for L4 subcutaneous fat measurement (2.8 ± 2.16 vs 18.7 ± 8.9 mm; P = .008) and leptin levels (2.45 ± 1.6 vs 11.26 ± 7.2 ng/mL; P = .0001). Our results suggest that there is a relationship between mutated prelamin-A accumulation and the severity of the phenotypes in homozygous familial partial lipodystrophy type 2 patients who harbor the LMNA T655fsX49 mutation. A dose-dependent effect seems likely.

Evidence of Paternal N5, N10 - Methylenetetrahydrofolate Reductase (MTHFR) C677T Gene Polymorphism in Couples with Recurrent Spontaneous Abortions (RSAs) in Kolar District- A South West of India.

Recurrent spontaneous abortion (RSA) is a multifactorial clinical obstetrics complication commonly occurring in pregnancy. Many research studies have noted the mutations such as C677T in N5, N10 - Methylenetetrahydrofolate reductase (MTHFR)gene which is regarded as RSA risk factor. This study was carried out to determine the occurrence of frequency of C677T of the MTHFR gene mutations with RSA. The purpose of present study is to determine the frequency of MTHFR C677T polymorphisms in couples with recurrent pregnancy loss and the impact of paternal polymorphisms of MTHFR C677T in recurrent pregnancy loss in population of couples living in Kolar district of Karnataka with RSA. A total of 15 couples with a history of two or more unexplained RSA were enrolled as subjects in the study and a total of 15 couples with normal reproductive history, having two or more children and no history of miscarriages were enrolled as controls. DNA extraction from samples case and control group couples and its quantification by Agarose gel electrophoresis, assessment of DNA purity, MTHFR C 677T gene mutation detection by PCR-RFLP method. Carried out by web based online SPSS tool. The frequency of C677T genotype showed homozygous wild type CC (80%), heterozygous CT type (13.3%) and homozygous mutation TT type (6.67%) observed in males. Similarly from female's homozygous wild type CC (86.6%), heterozygous type (13.3%), and homozygous type mutations TT (0%) was recorded. In couple control groups, we observed homozygous wild type CC (86.6%), heterozygous CT type (13.3%) and homozygous type mutations TT type (0%). We noticed a high frequency of MTHFR specifically T allele associated with paternal side.Therefore, the present study indicated the impact of paternal gene polymorphism of MTHFR C677T on screening in couples with recurrent pregnancy loss.

Triallelic digenic mutation in the prokineticin 2 and GNRH receptor genes in two brothers with normosmic congenital hypogonadotropic hypogonadism

Purpose/aim of the study: To date, different genes have been identified as responsible for the presence of normosmic congenital hypogonadotropic hypogonadism (nCHH). Herein, we report the molecular findings regarding the analysis of PROK2, in two brothers with nCHH. Subjects and methods: Two siblings with nCHH, in whom mutations in GNRHR, PROKR2 and FGFR1 had been investigated previously, as well as their family were studied. DNA was amplified by PCR and sequenced for the PROK2 gene. Controls were analyzed by restriction fragment-length polymorphism. The structure of PROK2 and its mutant protein were compared using a protein molecular model. Results: Both affected siblings exhibited a heterozygous p.R117W mutation in PROK2, while their mother was a heterozygous carrier and their father, an unaffected brother and their sister were homozygous wild type. Besides, both patients presented a homozygous p.E90K mutation in GNRHR that had been previously reported. Conclusions: We found a novel mutation in PROK2 in two siblings in whom a mutation in the GNRHR gene had been previously reported.

Novel R41Q-PAR1-Modified Mice Enable Proof-of-Concept Studies for in Vivo Mechanisms of Action for Thrombin (IIa) and Activated Protein C (APC)

Introduction: Thrombin (IIa) and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses that affect many cell types in the body. IIa employs the GPCR, protease activated receptor 1 (PAR1), to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC requires PAR1 for its opposing actions to stabilize endothelial barriers and to provide anti-inflammatory and anti-apoptotic activities. Studies of murine in vivo injury models using PAR1 knockout mice show that APC requires PAR1 to reduce sepsis-induced mortality and to provide robust neuroprotection following ischemic stroke. Extensive in vitro studies support the hypothesis that IIa’s cleavage at R41 in PAR1 initiates its signaling. Although APC was long thought, paradoxically, to act also via cleavage at R41, we recently proposed that APC’s cleavage at R46 initiates its endothelial barrier-protective and cytoprotective signaling via biased signaling. Since PAR1 knock-out mice cannot provide mechanistic data for testing these hypotheses in vivo, mice carrying the point mutation of R41 to Q in PAR1 were generated to enable mechanistic studies to test whether or not IIa and APC require Arg41 for their PAR1-dependent effects.Methods: Using C57BL/6-derived embryonic stem cells and standard gene targeting methods, we prepared C57BL/6 mice carrying the PAR1 mutation of R41 to Q. IIa-induced and APC-induced signaling, detected as phosphorylation of ERK1/2 or Akt in Brain Microvascular Endothelial Cells (BECs), was quantified using immunoblotting. BECs were obtained from homozygous 41QQ-PAR1 mice and wild type 41RR-littermates. Endothelial barrier disruption of cultured BECs was assayed using Trans-Endothelial Resistance (TER) assays (iCelligence, Acea, San Diego). Mortality of wild type and 41QQ-PAR1 mutant mice that was caused by live E. coli-induced pneumonia and to endotoxin was determined using standard methods. The ability of a cytoprotective-selective murine APC mutant (5A-APC) to reduce mortality of E.coli-challenged wild type and homozygous mutant mice was determined.Results: Upon breeding of R41Q-PAR1 heterozygous mice, the progeny did not fit a Mendelian pattern and yielded only 14% rather than 25% homozygous 41QQ mice. This reduced yield of homozygous mutant mice was similar to the previously reported low yield of homozygous PAR1 knockout mice. Homozygous 41QQ-PAR1 mice showed normal protein expression in BECs for PAR1 and endothelial cell protein C receptor (EPCR) antigens. When BECs from homozygous mutant mice were compared to those from wild type littermates, the IIa-induced vascular disruption in TER assays was greatly reduced by the mutation. Intracellular Ca2+ release, a hallmark of IIa-induced signaling, was greatly impaired (>90%) in BECs from homozygous mutant mice compared to wild type controls. IIa-induced phosphorylation of ERK1/2 in BECs was also significantly reduced by the mutation whereas APC-induced phosphorylation of Akt was not significantly affected. In murine sepsis-induced mortality studies, homozygosity for the R41Q-PAR1 mutation conveyed considerable resistance to death induced by either E. coli pneumonia or endotoxin in female mice but not in male mice. Tests to determine whether 5A-APC rescued male mice from sepsis-induced lethality showed that homozygous 41QQ-PAR1 mice were entirely responsive to 5A-APC therapy because 5A-APC treatment reduced mortality from 50 % to 0 % (see Figure). Wild type control mice also showed a beneficial response with reduced mortality in response to 5A-APC therapy, as previously described.Conclusions: These studies show that mutation of Arg41 to Gln in murine PAR1 diminishes or eliminates signaling induced by IIa but not by APC. Moreover, the ability of cytoprotective-selective 5A-APC to reduce bacteria-induced septic mortality in 41QQ-PAR1 mutant mice provides strong in vivo proof-of-concept data for PAR1 activation caused by non-canonical cleavage by APC. In summary, the 41QQ-PAR1 mutant mouse provides a unique and powerful tool to define in vivo requirements for cleavage sites that enable PAR1 signaling activities induced by IIa, APC or other proteases. [Display omitted] DisclosuresMosnier:The Scripps Research Institute : The Scripps Research Institute Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties.

JAK2V617F Complete Molecular Remission in Long-Term Follow-up of Patients with Polycythemia Vera and Essential Thrombocythemia Treated with Ruxolitinib

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by the presence of JAK2V617F mutation in >95% and 60% of patients (pts), respectively. This mutation usually affects one allele in ET while most PV pts are homozygous due to mitotic recombination. Acquisition of the JAK2V617F mutation is strongly associated with the germline 46/1 predisposition haplotype. Ruxolitinib is a JAK1/JAK2 inhibitor recently approved for myelofibrosis (MF) and under investigation in PV and ET pts intolerant or resistant to hydroxyurea. We enrolled 24 pts, 11 with PV and 13 with ET, in the phase II INCB18424-256 trial that overall included 34 PV and 39 ET pts. 21/24 pts were still on treatment at 5 years (yr), of which 19 JAK2V617F mutated. Results of the PV cohort have been reported recently (Verstovsek et al. Cancer, 2014): with a median follow up of 35 months (mo), the JAK2V617F allele burden decreased by a mean of 8%, 14%, and 22%, respectively, after 12, 24 and 36 mo. The proportion of pts who achieved a reduction ≥50 % at any time during the 1st yr, 2nd yr, and 3rd yr were 5.9%, 14.7%, and 23.5%, respectively, but no patients achieved a complete remission. In our series of pts we evaluated the JAK2V617F allele burden by two RTQ-PCR methods, according to Lippert (sensitivity, 0.8%) and to Larsen (sensitivity, 0.08%) method. We also analysed by next generation sequencing (NGS; Ion Torrent platform) a series of MPN-associated mutations including TET2, ASXL1, IDH1/2, LNK, CBL, SRSF2, EZH2 and MPL at baseline and at 5 yr of treatment in ruxolitinib treated pts who achieved a >25% JAK2V617F allele burden reduction at 5 yr (n=13/19). JAK2V617F allele burden decreased by a mean of 7%, 11%, and 19% at 12, 24 and 36 mo, and decreased further by a mean of 28% after 60 mo. Three (1 PV, 2 ET) of 19 pts (16%) achieved a 50% or greater allele burden reduction after 2 yr; no additional pts achieved this degree of allele burden reduction even in prolonged follow up. These 3 pts further improved their molecular response to a complete molecular response (CMR) after 5 yr of treatment. Their mean JAK2V617F allele burden was 46.6% at baseline, 28.3%, 16.3%, 8.7% and 0% after 1 yr, 2 yr, 3 yr and 5 yr, respectively. The JAK2 CMR was confirmed in at least one independent sample at 3 mo after first discovery. At this last timepoint, the PV pt was in complete haematological remission according to ELN criteria, the 2 ET pts were in partial remission due to platelet count still >400x109/L: 422x109/L and 812x109/L, respectively. BM histopathology in the 2 ET pts at 5 yr, while they were in CMR, showed still evidence of megakaryocyte hyperplasia. In the PV pt, histopathology at 5 yr is pending; evaluation at 3 yr, a time when she was in complete hematologic remission and JAK allele burden had decreased from 69 to 8%, showed normalization of cellularity, megakaryocyte and myeloid lineage compared to baseline but still slight erythroid hyperplasia. All 3 pts had normal karyotype at baseline that remained unchanged thereafter. CMR for JAK2V617F was confirmed by NGS. The 2 ET pts achieving CMR did not show any additional mutations, while the PV pts presented a TET2 Y867H mutation with an allele burden of 48.9% and 52%, respectively at baseline and 5 yr. No recurrent mutations in genes other than JAK2 were found in all other examined cases at baseline or at 5 yr. In 3 informative pts, we also analysed the proportion of JAK2V617F homozygous, heterozygous and wild type clones by the method of Hasan et al (Leukemia 2013) based on allelic discrimination of 46/1 haplotype and JAK2. We found that JAK2V617F/V617F clones were reduced by a mean of 95.5%, JAK2V617F/WT showed an uneven trend with a mean reduction of 45.54% while JAK2WT/WT conversely increased (mean 61.43%) at 5 yr, suggesting that in a subset a patients who present significant reduction of VF allele burden ruxolitinib may preferentially target the homozygous clones. Until now, complete molecular remission in PV pts has been described only in patients treated with interferon. Our data suggest that a subset of pts who present a rapid and sustained reduction of the JAK2V617F allele burden under ruxolitinib may eventually reach a condition of CMR with prolonged treatment. However, similar to findings with interferon, mutations establishing clonality, such as in TET2, may still persist in patients who eventually show the disappearance of JAK2V617F mutated subclones. DisclosuresVerstovsek:Incyte: Research Funding. Vannucchi:Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Susceptibility to SLE in South Indian Tamils may be influenced by genetic selection pressure on TLR2 and TLR9 genes

IntroductionSystemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with complex etiology. Genetics plays an important role in lupus pathogenesis through its influence on clinical and autoantibody phenotype of the disease. Toll like receptors (TLR) recognize molecular patterns of pathogens and activate the innate immune system. Their ability to identify nucleic acids makes them suitable candidates for investigation of their role in lupus pathogenesis. Hence, this study was carried out to analyze the G to A and C to T transitions in TLR2 and TLR9 genes respectively and to test their association with lupus susceptibility, clinical and autoantibody phenotypes in South Indian Tamils. MethodThree hundred SLE patients fulfilling ACR 2012 criteria for SLE and 460 age, sex similar, ethnicity matched controls were recruited as cases and controls. TLR2 (R753Q) and TLR9 (−1237C/T) polymorphisms were analyzed by real time PCR. ResultsThe TLR2 gene remained monomorphic in patients and controls, the frequency of the homozygous wild type allele being 100% and 99.6% respectively. Hence, it did not confer susceptibility to SLE. The more frequent T allele of TLR9 gene conferred a significant risk to develop SLE (p=0.011, OR 1.69, 95% CI 1.1–2.6). Both the polymorphisms did not influence clinical or autoantibody phenotype of the disease. ConclusionPrevailing endemic infections in the Indian subcontinent may have exerted a selection pressure resulting in TLR2 gene remaining monomorphic and the TLR9 adapting to a mutation for its increased expression. These may have an additive effect in the presence of other genetic and environmental risk factors to confer susceptibility to SLE in South Indian Tamils.

The relationship of Bradykinin B₂ receptor gene variation with obesity, hypertension and lipid variables in obese patients.

This study examined the association of C-58T genotypes with obesity/hypertension related parameters and serum lipids in obese (n=108) and non-obese (n=80) patients. Bradykinin receptor (B₂R) C-58T genotypes were determined by PCR-RFLP. B₂R gene C-58T frequencies for T/T (homozygous wild type), T/C (heterozygous) and C/C (homozygous polymorphic) genotypes for obese and non-obese patients were respectively: 36.1%, 37.5%; 45.4%, 52.5% and 18.5%, 10%. Obese patients using diuretic medication had lower C/C genotype frequency compared to T/T and T/C genotypes. Total cholesterol (T-Chol) (p=0.035) levels were found to be associated with B₂R C-58T polymorphism, where the T/T genotype had higher total cholesterol levels compared to the T/C genotype in obese patients. Non-obese patients using oral antidiabetic medication had higher C/C genotype frequency than that of T/T and T/C genotypes. Waist circumference (p=0.016) and diastolic blood pressure (p=0.01) levels were elevated in the non-obese subjects with the C/C genotype compared to T/C and T/T. Although B₂R C-58T gene polymorphism was not found to be effective on obesity with logistic regression analysis in the whole study population in obese subjects, the T-Chol decreasing effect of the B₂R gene C allele and the higher waist circumference measurements in the non-obese subjects may indicate there may be a link between B₂R gene C-58T polymorphism and obesity in study populations of higher numbers.

Label-free efficient and accurate detection of cystic fibrosis causing mutations using an azimuthally rotated GC-SPR platform.

Plasmonic nanosensors are candidates for the development of new sensors with low detection limits, high sensitivity, and specificity for target detection: these characteristics are of critical importance in the screening of mutations responsible for inherited diseases. In this work, we focused our study on the detection of some of the most frequent mutations responsible for cystic fibrosis (CF) among the Italian population. For the detection of the CF mutations we adopted a recently developed and highly sensitive Grating Coupled-Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method for label-free molecular identification exploiting a conical illumination configuration. Gold sinusoidal gratings functionalized with heterobifunctional PEG were used as sensing surfaces, and the specific biodetection was achieved through the coupling with DNA hairpin probes designed for single nucleotide discrimination. Such substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples, deriving from clinical samples, for the screened mutations. Hybridization conditions were optimized to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridizing wild type and heterozygous samples show DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the concomitant discrimination of several alleles also scalable for a high throughput screening setting.

Analysis of TP53 Polymorphisms in North Indian Sporadic Esophageal Cancer Patients

To investigate the relationship of five TP53 polymorphisms (p.P47S, p.R72P, PIN3 ins16bp, p.R213R and r.13494g>a) with the esophageal cancer (EC) risk in North Indians. Genotyping of p.P47S, p.R72P, PIN3 ins16bp, p.R213R and r.13494g>a polymorphisms of TP53 in 136 sporadic EC patients and 136 controls using polymerase chain reaction and PCR-RFLP. The frequencies of genotype RR, RP and PP of p.R72P polymorphism were 16.91 vs 26.47%, 58.82 vs 49.27% and 24.27 vs 24.27% among patients and controls respectively. We observed significantly increased frequency of RP genotype in cases as compared to controls (OR=1.87, 95% CI, 1.01-3.46, p=0.05). The frequencies of genotype A1A1, A1A2 and A2A2 of PIN3 ins16bp polymorphism were 69.12 vs 70.59%, 27.20 vs 25% and 3.68 vs 4.41% among patients and controls. There was no significant difference among genotype and allele distribution between patients and controls. The frequencies of genotype GG, GA and AA of r.13494g>a polymorphism were 62.50 vs 64.70%, 34.56 vs 30.15% and 2.94 vs 5.15% among patients and controls respectively. No significant difference between genotype and allele frequency was observed in the patients and controls. For p.P47S and p.R213R polymorphisms, all the cases and controls had homozygous wild type genotype. The RP-A1A1-GG genotype combination shows significant risk for EC (OR=2.01, 95%CI: 1.01-3.99, p=0.05). Among the five TP53 polymorphisms investigated, only p.R72P polymorphism may contributes to EC susceptibility.

Abstract 2156: Childhood exposure to secondhand smoke, nicotine dependence, and DRD1 are associated with lung cancer risk

Abstract Children who are exposed to secondhand smoke (SHS) are faced with an increased risk of nicotine dependence later in life. We have previously shown that children exposed to SHS have an increased risk of lung cancer as adults. Furthermore, genetic variation plays a role in the susceptibility of individuals to lung cancer. The gene DRD1 mediates dopamine signaling following nicotine exposure, thus we reasoned that DRD1 may be related to lung cancer via modulation of the nicotine reward pathway. Our previous studies identified 4 SNPs (rs4809294, rs2292975, rs4861327, rs686) that were predicted to modulate a microRNA binding site and we assessed the association of these SNPs with lung cancer risk. Our current study investigated whether these SNPs were associated with nicotine dependence and whether this had a subsequent effect on lung cancer risk. We used the National Cancer Institute-Maryland lung cancer case-control study. Cases and controls were recruited from the greater Baltimore metropolitan area. We used ordinal logistic regression to examine the association between SNPs and nicotine dependence. Logistic regression adjusted for age, gender and smoking was used to assess the association between SNPs and lung cancer risk. No significant associations were found between genotypes in the 4 SNPs and nicotine dependence levels (n=1,450). Individuals with the heterozygous allele (AG) exposed to SHS during childhood had increased odds of lung cancer compared to those with the homozygous wild type allele (AA) for rs686 (OR=1.52, CI= 1.12, 2.07). Individuals with the homozygous variant allele (GG) exposed to childhood SHS had increased odds of lung cancer compared to individuals with the homozygous wild type allele (AA) (OR=2.11, CI= 1.45, 3.10). Individuals with the heterozygous allele (GA) and the highest levels of nicotine dependence who were also exposed to childhood SHS, had higher odds of lung cancer than those with the homozygous recessive allele (AA) (OR=3.50, CI=1.37, 8.96). Consistent with earlier findings, heterozygotes (AG) had increased odds of lung cancer but this was not statistically significant (OR=1.90, CI= 0.82, 4.40). To further explore this association, we examined the effect of race on this relationship. Caucasians with the heterozygous allele (AG), the highest levels of nicotine dependence and childhood SHS, had increased odds of lung cancer compared to those with the homozygous recessive allele (AA) for rs686 (OR=1.47, CI=1.06, 2.05). Caucasians with the homozygous allele (GG) who were exposed to childhood SHS, had higher odds of lung cancer compared to individuals with the homozygous recessive allele (AA) for rs686 (OR=1.84, CI=1.15, 2.98). No association was found among African Americans. Although none of the SNPs examined were found to be associated with nicotine dependence, it appears that variations in rs686 may modify an individual's susceptibility to lung cancer due to SHS exposure. Citation Format: Victoria A. Zigmont, Brid M. Ryan, Jin Jen, Ana I. Robles, Cain McClary, Kara Calhoun, Elise D. Bowman, Kirsi Vähäkangas, K. Leigh Greathouse, Wang Yi, Angela S. Wenzlaff, Bo Deng, Ping Yang, Ann G. Schwartz, Curtis C. Harris, Susan Olivo Marston. Childhood exposure to secondhand smoke, nicotine dependence, and DRD1 are associated with lung cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2156. doi:10.1158/1538-7445.AM2014-2156

P.1.g.094 Influence of stress and ketamine in immediate early gene activation in serotonin transporter knockout rats

Aim: Depression is one of the highest causes of ‘years of life lived with disability’ and the response of patients to antidepressant treatment isn’t optimal in 30−40% of the cases. Ketamine treatment has shown effectiveness in depressed patients refractory to monoaminergic antidepressants [1]. Animal models to induce depressive-like behavior are normally generated by exposure to stressors or through genetic modification. The serotonin transporter (Sert) is fundamental to the homeostasis of serotonin system. Studies in humans and animals have reported that alterations in this transporter lead to a depressive phenotype and Sert mutant animals (SertM) present a naturally depressive-like phenotype [2]. Exposure to different kinds of stress will trigger the activation of immediate early genes (IEGs) that are fundamental to proper response to stress stimuli and it can be used as a tool to define brain areas related with different stressors [3]. In the present study two IEGs (c-Fos and Arc) were analyzed after exposure to stress. Methods: Sert KO (homozygous, heterozygous and wild type) rats were used to perform this experiments. Male animals received ketamine (15mg/kg, intraperitoneally) or vehicle. Thirty minutes after injection, animals were tested in open field (OF, 5min) immediately followed by a 10min trial in the forced swim test (FST). Animals were euthanized 30min following FST, and their brains collected and frozen at −80oC until 14-mm sections were cut using a cryostat. In situ hybridization (ISH − to analyze the expression of Arc and c-Fos mRNA) and radioligand binding (to analyze Sert expression) were performed. 1 or 2-way ANOVA followed by Tukey pot-hoc test were used for statistical analysis. Results: No significant differences were observed in OF. In FST, Sert KO rats showed a slightly higher total immobility compared to wild type animals (which did reach statistical significance). Ketamine treatment markedly reduced immobility (p< 0.001) regardless of genotype. We found significant genotype differences in Sert expression in all the brain areas between genotypes (p< 0.001). Sert KO rats showed significantly lower levels of IEG expression: of c-Fos in the prefrontal cortex (PFC) and hippocampus (HP) (p< 0.05), and of Arc in the PFC, HP and Amg (p< 0.05). Ketamine treatment significantly reduced the expression of c-Fos in the HP and of Arc in the PFC, Striatum and Nacc. Conclusion: Since both IEGs are fundamental to promote neuronal plasticity and respond appropriately to stress, these findings can point towards possible neuronal mechanisms related with their depressive phenotype. Sert KO also showed low levels of Sert expression, which is linked with depressive phenotype and corroborate previous data. Analysis of locomotion in the OF showed that neither genotype nor treatment significantly affected motor activity. Nevertheless, acute ketamine treatment showed a robust anti-depressive effect in all genotypes, which may indicate that alterations in the serotonergic system, as modelled in the SertM rats, are not fundamental for ketamine to exert anti-depressive effects. Furthermore, the interesting ketamine-induced reduction in Arc levels in the striatum and Nacc may indicate key neuronal populations which are differentially affected by ketamine.

TP53 polymorphisms in sporadic North Indian breast cancer patients.

The purpose of this study was to evaluate the potential association of five (p.P47S, p.R72P, PIN3 Ins16bp, p.R213R and r.13494g>a) polymorphisms of TP53 with the risk of developing breast cancer in North Indian Punjabi population. We screened DNA samples of 200 sporadic breast cancer patients (197 females and 3 males) and 200 unrelated healthy, gender and age matched individuals for the polymorphisms. For the p.P47S polymorphism, we observed the PP genotype in 99.5% of the patients and PS genotype in only 1 patient. All the controls had the wild type PP genotype. The frequency of RR, RP and PP genotype of p.R72P was 23.5% vs 33.5%, 51.5% vs 45.5% and 25% vs 21% in patients and controls respectively. Heterozygous (RP) genotype was increased in breast cancer patients as compared to controls (51.5 vs 45.5%) and showed 1.61 fold significantly increased risk for breast cancer (OR=1.61, 95% CI, 1.01-2.58, p=0.04). In breast cancer patients the frequencies of A1A1, A1A2 and A2A2 genotypes of PIN3 Ins16bp polymorphism were 67%, 26% and 7% respectively whereas in controls the genotype frequencies were 68.5%, 27.5% and 4% respectively, with no significant difference. For p.R213R (c.639A>G), all individuals had homozygous wild type genotype. The frequencies of GG, GA and AA genotypes of TP53 r.13494g>a polymorphism were 62 vs 67.5%, 33 vs 28% and 5 vs 4.5% in patients and controls respectively, again without significant difference. We observed that RP- A1A1 genotype combination of p.R72P and PIN3 Ins16bp and RP-GG combination of p.R72P and r.13494g>a polymorphism showed significant risk of breast cancer (OR=1.65, 95%CI: 0.98-2.78, p=0.05; OR=1.72, 95%CI: 1.01-2.92, p=0.04). The results of present study indicated that among the five TP53 polymorphisms investigated, the p.R72P polymorphism, and the RP-A1A1 and RP-GG genotype combination contribute to breast cancer susceptibility in North Indians.

Effects of the FecL major gene in the Lacaune meat sheep population.

BackgroundThe major prolificacy gene FecL was first described in the Lacaune sheep meat breed Ovi-Test in 1998. A few studies estimated the effect of this gene on prolificacy but little data is available. In 2010, the Ovi-Test cooperative started genotyping FecL in all of their replacement ewe lambs. Thanks to the large amount of genotyping data that is available now, gene effects on litter size and other relevant traits can be estimated more accurately.MethodsOur study included 5775 ewes genotyped since 2010 and 1025 sires genotyped since 2002. Performances and pedigrees were extracted from the French national database for genetic evaluation and research. Analysis of the effect of the gene on different traits was based on linear or threshold genetic animal models using the ASReml software.ResultsThe female population was composed of 71% homozygous wild type ewes (++), 27% heterozygous ewes for the FecL mutation (L+) and 2% homozygous mutant (LL) ewes. On average, L + ewes produced 0.5 more lambs per lambing than ++ ewes. The FecL gene not only affected the mean litter size but also its variability, which was lower for ++ than for L + ewes. Fertility after insemination was higher for L + ewes than for ++ ewes. Lambs from ++ dams were heavier (+300 g) than the lambs of L + dams and the mortality of twin lambs born from ++ dams was lower than those from L + dams. In addition, bias in estimated breeding values for prolificacy when ignoring the existence of this major gene was quantified.ConclusionsThe effect of the FecL gene on prolificacy was estimated more accurately and we show that this gene affects both the mean and the variability of litter size and other traits. This paper also shows that ignoring the existence of this major gene in genetic evaluation of prolificacy can lead to a large overestimation of polygenic breeding values.