Homozygous Type Research Articles

Association analysis of Glutathione S-transferase omega-1 and omega-2 genetic polymorphisms and ischemic stroke risk in a Turkish population

ABSTRACTObjectives: Oxidative stress is a known risk factor for the pathogenesis of atherosclerosis, the main cause of ischemic stroke. Glutathione S-transferase (GST) omega-1 and omega-2, members of phase II enzymes, play a role in the antioxidant system. The single nucleotide polymorphisms (SNPs), C419A and A424G in GST omega genes can cause a decrease in enzyme activity. The aim of this study was to investigate the possible association between these polymorphisms and ischemic stroke risk in a Turkish population.Methods: The genotypes and allele frequencies for 239 patients and 130 controls were determined by the PCR/RFLP method. No significant differences were found between patients and controls in terms of genotype and allele frequencies.Results: The frequency of the polymorphic ‘A’ allele was 0.358 in patients and 0.342 in controls for the C419A polymorphism in the GSTO1 gene. The frequency of the polymorphic ‘G’ allele for GSTO2 A424G SNP was 0.370 in patients and 0.404 in controls. The combined homozygous wild type genotype ‘CCAG’ was significantly higher in control group than in the patients.Conclusion: No significant difference was observed between the stroke patients and controls in terms of genotypes and allele distributions. Double combine haplotype CCAA was found to be protective against ischemic stroke when compare to other haplotypes. However, different genotypes of GSTO1 and GSTO2 were observed to have effects on stroke risk in subgroups of diabetics and smokers. In conclusion, the current study is the first to report this finding.

Validation of the POLLED Celtic variant in South African Bonsmara and Drakensberger beef cattle breeds

An increased awareness of animal welfare necessitates the breeding of genetically polled animals, especially since more than 70% of South African beef cattle are rounded off in commercial feedlots. The Bonsmara and the Drakensberger, two locally developed breeds, play a major role in beef production in South Africa. The causative mutation for polledness in these breeds have not been confirmed, therefore, this study aimed to validate the POLLED Celtic variant as the causative mutation of polledness in the South African Bonsmara and Drakensberger beef cattle breeds. A total of 386 animals, consisting of Bonsmara, Drakensberger and Herefords (included as a Bos taurus control), were tested for the Celtic mutation by PCR-based screening. Phenotypically polled and scurred animals were found to carry at least one copy of the Celtic allele (PC) whereas horned animals were homozygous wild type. The highest frequency of homozygous polled animals (PC/PC = 0.337) was observed in the Bos taurus control (Hereford breed) while the majority of the Bonsmara animals were heterozygous polled (PC/p = 0.591). For the Drakensberger, a heterozygous (PC/p) genotypic frequency of 0.346 was observed, with the majority of animals being horned (p/p = 0.639). In the Bonsmara and Hereford breeds, a high proportion of heterozygous polled animals were phenotypically scurred, emphasizing the importance of correct phenotyping at farm level. This research validates the Celtic mutation as causative mutation for polledness in indigenous South African beef cattle breeds. It also demonstrates the current challenges with regards to both phenotypic and genetic verification of the scurs phenotype and requires further investigation in South African beef cattle breeds.

Association between KISS1 rs5780218 promoter polymorphism and onset of growth hormone secreting pituitary adenoma

ObjectivesThis study analyzed the KISS1 c.-145delA (rs5780218) promoter polymorphism in a cohort of patients with growth hormone secreting pituitary adenoma (somatotropinoma) and controls, to investigate its role in the incidence of acromegaly and to assess patient/tumor characteristics.Material and methods rs5780218 allelic and genotypic distributions were compared between 49 somatotropinoma patients and 167 healthy controls. rs5780218 was also assessed in relation to patient characteristics and tumor aggressiveness, as characterized by tumor invasion and resistance to conventional therapy. The relationship between KISS1 mRNA expression and the rs5780218 genotype was also assessed in available pituitary tumor samples. ResultsThe homozygous -/- variant genotype was associated with high rates of somatotropinoma (P<0.01), but not with tumor invasiveness, patient characteristics or hormonal remission. KISS1 mRNA expression was much lower in somatotropinomas carrying the deleted allele than in homozygous wild type AA. ConclusionsIn this pilot study, the rs5780218 promoter polymorphism was evaluated in pituitary adenoma, and showed a possible association with the incidence of somatotropinoma but not with tumor progression.

Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients.

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world's population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori.

OR31. Characterization of the CCR5 delta 32 mutation by race in unrelated donors listed on the national marrow donor program’s be the match registry

Aim Donors homozygous for the CCR5 delta 32 deletion are of interest in hematopoietic stem cell transplantation (HCT) for patients infected by both HIV-1 and a hematologic malignancy. The mutation has been published to confer natural HIV resistance to individuals carrying two copies, while heterozygous individuals show increased resistance and lower viral loads compared to wild type. The aim of this study was to characterize the CCR5 delta 32 deletion frequency in the diverse unrelated donor populations listed on the National Marrow Donor Program’s Be The Match Registry. Methods Next Generation Sequencing by the Illumina MiSeq was used to target the 32 base pair deletion in the CCR5 gene for 495,530 URDs between 2016-2018. The frequency of donors identified as homozygous, heterozygous and wild type for the mutation was calculated for each of broad race/ethnic groups; Black, Asian/Pacific Islander, White, Hawaiian, Hispanic and Native American Indian. Results Overall, .73% URDs were identified as CCR5 delta 32 homozygous in our cohort. The frequency of homozygotes found in unrelated registry donors self-identified as race group of White was similar (.95%) to previously published data on European individuals (1%). The frequency of homozygotes in the remaining populations was low with .04% observed in Black, .01% in Asian/Pacific Islander, .21% in Hispanic and .24% in Native American. Within donors carrying the homozygous genotype, the population was highly homogeneous, with 98.9% self-identifying as White, given predominate donor recruitment numbers and higher frequency. Conclusions The frequency of self-identified White U.S. registry donors homozygous for the CCR5 delta 32 deletion is consistent with previous studies on individuals of European descent. Given the low frequency of homozygous CCR5 delta32 genotypes in the unrelated donor population, additional treatment methods using multiply mismatched donors can extend the feasibility of this use case for concurrent HCT and infection therapy in HIV patients. Download : Download high-res image (283KB) Download : Download full-size image

The Involvement of ERCC2/XPD and ERCC6/CSB Wild Type Alleles in Protection Against Aging and Cancer.

DNA helicases maintain genome stability, and their deficiency is associated with disorders resembling premature aging as well as contributes to carcinogenesis. Their functions are determined by the respective genes encoding nucleotide excision repair initiating proteins, e.g. XPD and CSB. The present study aimed to investigate the influence of genetic variations in ERCC2/XPD (rs1799793, rs13181) and ERCC6/CSB (rs2228526, rs2228528) loci on lifespan and developing age-related bladder cancer focusing on homozygous wild type alleles. The allelic variants were identified in 354 clinically healthy controls and 418 bladder cancer patients using the PCR-RFLP method. The age-depended increase in frequencies of homozygous carriers of wild-type XPD 312Asp and XPD 751Lys alleles was observed among controls, especially among subjects over 80 years (r = 0.67, p = 0.012). The statistically significant correlation was also found between the frequency of homozygous wild type alleles at all tested loci and age in healthy population over 60 years (r = 0.35, p = 0.046) suggesting the relationship between lifespan and longevity, on one hand, and normal functioning of these genes and their products, on the other hand. Homozygous carriers of wild type alleles were less susceptible to bladder cancer, tumor invasion, increase in grade of malignancy and recurrence, but their effects were specific with respect to clinicopathological and lifestyle characteristics. Homozygous wild type alleles encoding XPD and CSB proteins with optimal properties were shown to affect human lifespan, risk of developing bladder cancer, its progression and recurrence under certain conditions.

Haplo-Insufficiency or Knockout of the Serotonin Transporter Does not Affect Heroin Self-Administration but Decreases BDNF in the Frontal Cortex

(1) Background: Drug addiction places a heavy burden on those affected by it but only a small percentage of individuals (~20%), regardless of their drug of abuse, go on to develop the compulsive behaviours that define drug addiction. Clinical studies have shown that genetic variations that reduce serotonin transporter (SERT) activity increase the vulnerability to developing a variety of psychiatric disorders such as depression, anxiety and drug addiction. (2) Methods: To investigate the influence of reduced SERT function in the laboratory, we studied the effects of heroin self-administration in a SERT knockout rat model. Groups of homozygous knockout (HOM), heterozygous (HET) or wild type (WT) animals completed a series of heroin self-administration experiments. In addition, quantitative reverse transcription polymerase chain reaction was used to measure relative abundance of brain derived neurotrophic factor (BDNF) transcripts of in the frontal cortex and striatum in animals that had previously self-administered heroin. (3) Results: There were no differences between the genotypes for acquisition, maintenance and progressive ratio responding for heroin self-administration. However, relative expression of total BDNF and the BDNF III and BDNF VI isoforms were significantly decreased in the frontal cortex of animals that had self-administered heroin for all genotypes, however, no differences in BDNF expression were found in RNA isolated from the striatum relative to untreated control animals. (4) Conclusions: These data suggest that reduced SERT function does not augment the addictive properties of heroin and support the hypothesis that different classes of addictive substances act through different neurobiological pathways.

P-220 - MTHFR, TSER and DPYD gene mutation is associated with toxicity and response in pre-operative chemo-radiotherapy for local advanced rectal cancer

Introduction: Radiotherapy and 5 FU based chemotherapy is the most common pre-operative regimen used for cT3-T4, N1 rectal cancer (RC). Evaluation of predictive markers of response and toxicity to radio-chemotherapy is a challenging approach for patients (pts) and drug selection. In the present experience we have analyzed the predictive role of the genetic polymorphisms (MTHFR, TSER and DPYD) on toxicity and response to pre-operative radio-chemotherapy. Methods: We have enrolled eighteen patients with locally advanced RC treated with pre-operative radiotherapy and fluoropyrimidines based chemotherapy. Genetic polymorphisms of MTHFR C677T, MTHFRA 1298C, DPYD IVS 14 + 1G>A, DPYDA 2846T, DPYD T 1679 G, TSER 28 bp VNTR were analyzed by PCR and pyrosequencing of genomic DNA extracted from peripheral blood samples. Genetic markers were correlated with toxicity to treatment (chemotherapy and radio-chemotherapy) and clinical response. Results: Patients characteristics were: male 15 pts, female 3 pts, median age 66 years, ECOG PS 0-1 all pts. We found DPYD IVS 14 + 1 G>A G/G homozygous wild type, DPYD A2846T, T/T homozygous wild type and DPYD T1679 g, T/T homozygous wild type in 100% of pts, homozygous wild type MTHFR C677T in 10% of pts, MTHFR C677T homozygous mutated in 50% of pts, heterozygous MTHFR A1298C in 60% of pts and homozygous wild type MTHFR A 1298C in 40% of pts. Adverse events G-3 (diarrhea, neutropenia, asthenia, mucositis) were observed in 60% of pts with heterozygous MTHFR A 1298C and in 10% of pts with homozygous mutated MTHFR C 677t treated with chemo radiotherapy combination. DPYD homozygous wild type was not associated with severe toxicity. Rectal surgery with TME/TEM will be performed 8 weeks after the end of pre-operative chemo-radiotherapy. We obtained 9 pathological complete response and 9 partial pathological response. Adjuvant chemotherapy was well tolerated without G3-G4 adverse events. Five pts with pathological complete response were treated with Transanal Endoscopic Microsurgery (TEM) and they are alive without recurrence to twelve months after surgery. Conclusion: Concomitant assessment of genetic polymorphisms of MTHFR and DPYD is promising to predict severe toxicity during preoperative chemo-radiotherapy approach for pts with locally advanced rectal cancer. This result does not exclude the need to consider other non-genetic factors that might influence the individual enzyme activities.

The influence of endothelial cell protein C receptor gene 6936A/G, 1651C/G, 4678G/C polymorphisms and soluble endothelial protein C receptor levels on in vitro fertilization outcomes

Hypercoagulability could be intrinsic or caused by the hormone treatment preceding the IVF procedure. Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C by the thrombin–thrombomodulin complex. Soluble EPCR plays a role in the maintenance of pregnancy evidenced by the findings that EPCR expression is critical for embryo development. Specific gene variants linked with altered soluble EPCR levels were associated with poor pregnancy outcome. The aim of this study was to assess the predictive value of EPCR gene polymorphisms (6936A/G, 1651C/G, and 4678C/G) and sEPCR level on the IVF outcome in Egyptian women with repeated IVF failure. They were compared to healthy control patients eligible for IVF. The present study was conducted on 45 women with repeated IVF failure, three or more previous IVF-embryo transfer cycles, and 45 healthy age-matched women eligible for IVF. PCR-RFLP for the EPCR polymorphisms (6936A/G, 1651C/G, 4678G/C) was done for cases and control groups. Plasma-soluble EPCR levels were measured with ELISA. As regards the mutant, EPCR (6936A/G) genotypes (AG, GG) were higher than the wild type (AA) (P < 0.001, OR 4.125, 95% CI 2.198–7.740). The homozygous mutant genotype (GG) was higher in comparison to the wild type (AA). The mutant allele (G) was higher than the wild allele (A) (P < 0.001, or 2.549, 95% CI 1.601–4.061). Higher frequencies of the (1651C/G) genotype and lower soluble EPCR levels were noted both in (C/C) (P = 0.004; Z = −0.2867) and (C/G) (P = 0.006; Z = −0.2767) genotype carriers. Regarding, EPCR polymorphism (4678G/C), the homozygous mutant genotype (CC) was significantly lower than the homozygous wild type (GG), (P = 0.014, OR 0.289, 95% CI 0.108–0.776). Our data suggest that the 6936A/G and 1651C/G EPCR gene variants coupled with procoagulant diminished levels of sEPCR may be associated with a higher tendency for repeated implantation failure.

The Pharmacologic Role and Clinical Utility of PCSK9 Inhibitors for the Treatment of Hypercholesterolemia

In addition to monoclonal antibodies against proprotein convertase subtilisin-kexin type 9 (PCSK9), vaccines against PCSK9 and smaller molecule inhibitors as well as RNA inhibitors of PCSK9 production have been created. The monoclonal antibodies against PCSK9 and the PCSK9 RNA inhibitors can reduce low-density lipoproteins (LDLs) by over 50%, non-high-density lipoprotein (HDL) cholesterol and triglycerides, and increasing HDL. Although effective in several homozygous familial hypercholesterolemia patient types, PCSK9 inhibitors does not work in all patient types. Outcome trials show no effects on mortality but do show reductions in atherosclerotic events such as myocardial infarctions, strokes, and need for coronary revascularization. PCSK9 inhibitors have a very attractive safety profile with no significant elevations in measures of muscle or liver damage. The current and more advanced experimental agents all require subcutaneous dosing, and injection site reactions are among the most common adverse events. Therapy for the Food and Drug Administration(FDA) approved agents is markedly expensive, and this is the primary barrier to utilization. However, it is possible to identify patients with a number needed to treat to prevent an atherosclerotic event low enough to render it cost-effective and one such factor is whether or not you require a 50% reduction in LDL in order to achieve your LDL goal.

Effect of double knockout of cartilage-specific mitogen-inducible gene 6 and transforming growth factor-alpha on joint homeostasis

Purpose: Osteoarthritis (OA) is a prevalent joint disease that involves the degeneration of smooth articular cartilage found at interfacing bone surfaces in synovial joints. The mRNA for transforming growth factor-alpha (Tgfa, a potential ligand of epidermal growth factor receptor (EGFR)), has been seen upregulated in a subset of OA cases, both in rat models and human patients (Appleton et al., 2007). Mitogen-inducible gene 6 (Mig-6) is induced by EGFR signaling and acts as a negative feedback regulator by sterically inhibiting additional downstream signaling and targeting EGFR for internalization. In previous studies, mice with a cartilage-specific knockout (KO) of Mig-6 have exhibited anabolic increases in articular cartilage thickness, suggesting that decreased EGFR signaling can mitigate OA effects (Pest et al, 2014). However, development of chondro-osseous nodules (CONs) within the joint capsule in this phenotype restricts movement of the joint, which can be debilitating. We aim to attenuate EGFR signaling by inhibiting Tgfa production in Mig-6-KO mice to further understand the role of this pathway in joint homeostasis. Methods: Male mice displaying cartilage-specific Mig-6 deletion with both homozygous Tgfa wild type (Mig-6fl/flCol2a1-Cre+ Tgfa+/+) and Tgfa-null (Mig-6fl/flCol2a1-Cre+ Tgfa-/-) genotypes, along with male mice displaying control genotypes (Mig-6fl/flCol2a1-Cre- Tgfa+/+ and Mig-6fl/flCol2a1-Cre- Tgfa-/-), were generated and raised to 12 weeks of age. Mice from different litters were age-matched in order to achieve N ≥ 5. Joint structure was then assessed using toluidine blue histological staining. Average articular cartilage thickness and cell density were obtained from sagittal sections of the elbow and knee joint, as well as from frontal sections of the knee. Immunohistochemical analysis was also performed for SOX9, a transcription factor and key marker for both chondrocyte development and phenotype maintenance. Results: Regardless of the absence or presence of TGFa, a significant increase in articular cartilage thickness was found at the surfaces of the humerus, ulna, femur, and tibia in Mig-6-KO models compared to controls. Similarly, cartilage-specific Mig-6-KO, but not whole-body TGFa KO, significantly increased cell density in mice 12 weeks of age. Formation of CONs were found in the knee joints of Mig-6-KO mice regardless of TGFa presence. Unexpectedly, Tgfa-null Mig-6-KO mice appeared to develop more severe CONs that were larger in area with increased proteoglycan staining compared to Mig-6-KO mice with TGFα. Immunochemical staining for SOX9 also appeared more intense in Mig-6-KO mice without TGFα compared to controls. Conclusions: This study indicates that MIG-6 plays an integral role in regulating articular cartilage growth in synovial joints and deletion of Mig-6 results in an anabolic phenotype. However, at the time point examined, TGFα did not have an effect on articular cartilage thickness or cell density. This suggests that the anabolic phenotype in Mig6-deficient mice arises from the role of MIG-6 in other non-EGFR specific mechanisms, or through alternate ligand activation of EGFR.

Linked homozygous BMPR1B and PDHA2 variants in a consanguineous family with complex digit malformation and male infertility.

In affected members of a consanguineous family, a syndrome, which is concurrence of set of medical signs, is often observed and commonly assumed to have arisen from pleiotropy, i.e., the phenomenon of a single gene variant affecting multiple traits. We detected six sibs afflicted with a unique combination of digit malformation that includes brachydactyly, symphalangism and zygodactyly plus infertility in males owing to azoospermia, sperm immotility or necrospermia, which we hypothesised to have arisen from a defect in a single gene. We mapped the disease locus and by exome sequencing identified in patients homozygous missense variants bone morphogenetic protein receptor type IB (BMPR1B) c.640C>T (p.(Arg214Cys)) and alpha-2 pyruvate dehydrogenase (PDHA2) c.679A>G (p.(Met227Val)). Structural protein modelling, protein sequence conservation and in silico analysis indicate that both variants affect protein function. BMPR1B is known to be responsible for autosomal dominant brachydactyly and autosomal recessive acromesomelic chondrodysplasia. Our findings show that also recessive complex digit malformation can be caused by BMPR1B variant and not all biallelic BMPR1B variants cause acromesomelic dysplasia. PDHA2 is a novel candidate gene for male infertility; the protein product is a mitochondrial enzyme with highest expression in ejaculated sperm. Our findings are a unique example of two linked variants, ~ 711 Kb apart, in different genes that together manifest as a novel syndrome. They demonstrate that exome sequencing and not candidate gene approach should be employed in disease gene hunt, defining new diseases and genetic testing, to rule out the coincidental presence of two variants contributing together to the phenotype, which may be discerned as a novel disease.

Whole Genome Resequencing of Capsicum baccatum and Capsicum annuum to Discover Single Nucleotide Polymorphism Related to Powdery Mildew Resistance

The present study deals with genome wide identification of single-nucleotide polymorphism (SNP) markers related to powdery mildew (PM) resistance in two pepper varieties. Capsicum baccatum (PRH1- a PM resistant line) and Capsicum annuum (Saengryeg- a PM susceptible line), were resequenced to develop SNP markers. A total of 6,213,009 and 6,840,889 SNPs for PRH1 and Saengryeg respectively have been discovered. Among the SNPs, majority were classified as homozygous type SNPs, particularly in the resistant line. Moreover, the SNPs were differentially distributed among the chromosomes in both the resistant and susceptible lines. In total, 4,887,031 polymorphic SNP loci were identified between the two lines and 306,871 high-resolution melting (HRM) marker primer sets were designed. In order to understand the SNPs associated with the vital genes involved in diseases resistance and stress associated processes, chromosome-wise gene ontology analysis was performed. The results revealed the occurrence that SNPs related to diseases resistance genes were predominantly distributed in chromosome 4. In addition, 6281 SNPs associated with 46 resistance genes were identified. Among the lines, PRH1 consisted of maximum number of polymorphic SNPs related to NBS-LRR genes. The SNP markers were validated using HRM assay in 45 F4 populations and correlated with the phenotypic disease index.

Independent validation of missense polymorphism in HSD3B1 in Japanese men treated with primary androgen-deprivation therapy for metastatic prostate cancer.

179 Background: Recently, genetic polymorphism in HSD3B1 has been shown to be associated with oncological outcome when treated with androgen-deprivation therapy (ADT) for prostate cancer. However, its impact on prognosis among different ethnicities remains unclear. Then, we aimed to investigate the significance of missense polymorphism in HSD3B1 gene encoding 3β-hydroxysteroid dehydrogenase among men treated with primary ADT for metastatic prostate cancer. Methods: This study included 104 Japanese patients with metastatic prostate cancer treated with primary ADT. The association of HSD3B1 (rs1047303, 1245C) with clinicopathological parameters and prognosis, including progression-free survival and overall survival, was examined. Results: Clinicopathological parameters were comparable between men with homozygous wild-type and men with heterozygous and homozygous variant types in HSD3B1 gene. Men carrying heterozygous and homozygous variant types in HSD3B1 gene showed higher progression risk [hazard ratio (95% confidence interval), 2.34 (1.08–4.49), P = 0.033], but not any-caused death risk [hazard ratio (95% confidence interval), 1.36 (0.52–2.92), P = 0.50], compared with men carrying homozygous wild-type. Conclusions: This study confirmed the finding that HSD3B1 genetic variation is associated with high risk of progression in Japanese men treated with primary ADT for metastatic prostate cancer, suggesting universal significance among different ethnicities.

Angiotensin-converting enzyme activity in Cavalier King Charles Spaniels with an ACE gene polymorphism and myxomatous mitral valve disease.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is particularly common in the Cavalier King Charles Spaniel (CKCS) breed and affected dogs are frequently managed with angiotensin-converting enzyme inhibitors (ACE-I). We have previously identified a canine ACE gene polymorphism associated with a decrease in angiotensin-converting enzyme (ACE) activity. The aim of this study was to evaluate for the prevalence of the ACE polymorphism in CKCS with mitral valve disease and to determine whether the presence of the polymorphism is associated with alterations in ACE activity at different stages of cardiac disease. Seventy-three dogs with a diagnosis of mitral valve disease were evaluated and a blood sample was drawn for ACE polymorphism genotyping and ACE activity measurement. Forty-three dogs were homozygous for the ACE polymorphism; five were heterozygous and 25 were homozygous wild type. The mean age and the median severity of disease were not different for dogs with the polymorphism and dogs with the wild-type sequence. The median baseline ACE activity was significantly lower for the ACE polymorphism (27.0 U/l) than the wild-type sequence dogs (31.0 U/l) (P=0.02). Dogs with more severe disease and the ACE polymorphism had significantly lower levels of ACE activity than dogs with the wild-type sequence (P=0.03). The CKCS appears to have a high prevalence of the ACE variant. Dogs with the ACE variant had lower levels of ACE activity even in more advanced mitral valve disease than dogs without the variant. The clinical significance of this finding and its impact on the need for ACE-I in dogs with the polymorphism and heart disease deserves further study.

Polymorphism in cancer-associated fibroblasts (CAFs) related genes and clinical outcome in metastatic colorectal cancer (mCRC) patients (pts) enrolled in two independent randomized phase III trials: TRIBE and FIRE-3.

645 Background: Tumor microenvironment plays a critical role for CRC development and progression. CAFs are the most dominant cellular components which orchestrate tumor cell proliferation, survival and invasion and have been shown to be associated with prognosis in pts with CRC. We aimed to evaluate whether single-nucleotide polymorphisms (SNPs) in CAFs related genes predict outcome in mCRC pts treated with first-line FOLFIRI/bevacizumab (bev) in TRIBE and FIRE-3 trials. Methods: The OncoArray, a custom array by Illumina including 530K SNP markers, was used to genotype genomic DNA from blood samples and served as our database, from which 15 functional SNPs within 9 genes (OPN, CD44, TCN, CD63, TIMP1, SLIT2, ROBO1, LOXL2, INHBA) related to CAF pathway were analyzed. Results: A total of 451 pts were included. Those treated with FOLFIRI/bev in TRIBE (N = 215, median PFS/OS: 9.7/26.3 months (mts)) and FIRE-3 (N = 107, median PFS/OS: 11.6/31.5 mts) served as discovery and validation cohorts respectively, while FIRE-3 FOLFIRI/cetuximab arm (N = 129, median PFS/OS: 12.8/49.8 mts) was used as the control. In overall pts in discovery set, OPN (osteopontin) rs11728697 any C variants showed longer PFS (median 10.8 vs 9.0 mts; HR = 0.68, 95%CI = 0.49-0.95; p = 0.025) and OS (median 30.6 vs 23.8 mts; HR = 0.69, 95%CI = 0.50-0.95; p = 0.025) than the homozygous wild type genotype (T/T) in multivariable analysis. These data were validated in the FIRE-3 cohort in OS both in univariate (median 49.3 vs 23.7 mts; HR = 0.40, 95%CI = 0.21-0.77; p &lt; 0.001) and in multivariable analyses (HR = 0.31, 95%CI = 0.16-0.60; p &lt; 0.001). However, the favorable impact on outcome was not shown among C allele carriers treated with FOLFIRI/cetuximab in the control arm. Conclusions: Our findings confirm that CAFs play a crucial role in mCRC and suggest that OPN polymorphisms could serve as predictive biomarkers for mCRC pts treated with first-line FOLFIRI/bev.

Peculiar laboratory phenotype/ genotype relationship due to compound inherited protein C defects in a child with severe venous thromboembolism

A 7-years-old child who developed unprovoked deep vein thrombosis (DVT) and pulmonary embolism (PE) was tested for inherited thrombophilia. Protein C (PC) antigen level (87 %) and PC coagulometric and amidolytic activities (12 % and 11 %, respectively) were consistent with a homozygous PC type IIA phenotype.The patient was carrier of two heterozygous missense mutations causing p.Arg32Cys substitution associated with a type I PC defect ("null allele", from the paternal side) and p.Gly433Ser substitution responsible for a type IIA PC defect (from the maternal side). Thus, the apparently normal PC antigen level in the proband was misleading in the interpretation of phenotype/genotype relationship of this compound PC defect. The child was also carrier of heterozygous prothrombin G20210A variant.Severe venous thromboembolism can occur in otherwise healthy children with complex inherited thrombophilia. Careful laboratory characterization of the phenotype/genotype relationship can be crucial to correctly classify PC defects and for their management with anticoagulants or replacement therapy.

Differentially expressed Secretoglobin 1C1 gene in nasal polyposis.

Nasal polyps (NP) are the most common pathological change that occurs in the nasal mucosa and is characterized by mucosal inflammation. Although its etiology and pathogenesis have not been clearly explained, its pathophysiology is arranged by the balance between pro-inflammatory and anti-inflammatory cytokines. The Secretoglobin 1C1 gene synthesizes odor molecule binding proteins (OBPs) in the nasal mucosa and regulates some cytokines. The Secretoglobin 1C1 gene expression could be disrupted by polymorphisms that may act as a possible cause of a disruption in the regulation of the promotor of the gene. Therefore, the main aim of this study was to determine the effects of Secretoglobin 1C1 gene promotor polymorphisms on the gene expression in NP. In this study, to determine the relationship between the Secretoglobin 1C1 gene promotor polymorphisms and the gene expression, the levels of 48 subjects were sequenced (24 patients with NP and 22 controls without sinonasal disease). The levels' expression of Secretoglobin 1C1 in the subjects' nasal mucosa was also detected using RT-PCR. In this study, the level of Secretoglobin 1C1's expression increased in NP (P= 0.003). Three polymorphisms were detected in the Secretoglobin 1C1 gene's promotor. The rs113795008 and rs2280540 variations were significantly high in NP (P= 0.005, P= 0.045). The the rs113795008 homozygous mutant type genotype (G/G) was associated with a high mRNA expression level of Secretoglobin 1C1 in NP (P= 0.009). A correlation was found between a high level of Secretoglobin 1C1 expression and its promotor polymorphism, which thus might increase and/or contribute to the susceptibility of developing NP in the Turkish population. These findings suggested that promotor variations in the function of the Secretoglobin 1C1 gene can alter the gene expression biology in NP.

The polymorphisms of MyoD1 gene in Manych Merino sheep and its influence on body conformation traits

Candidate genes associated with meat productivity often affect the performance of myostatin and muscle development in general. The MyoD1 gene is a member of the myogenic regulatory factor (MRF) family and plays a key role in the differentiation of skeletal muscle cells in vertebrates. We investigated the structure of the MyoD1 gene. The effect of polymorphisms on meat production was studied in the Russian sheep breed Manych Merino. To detect alleles, we used NimbleGen sequencing technology (Roche, USA). In the Manych Merino breed, we found 14 single nucleotide polymorphisms (SNPs) associated with substitutions, all in the second exon, namely, c.244C&gt;T, c.246G&gt;T, c.253G&gt;T, c.259G&gt;C, c.261C&gt;T, c.269C&gt;G, c.274C&gt;A, c.276C&gt;G, c.277C&gt;A, c.279C&gt;T, c.281C&gt;A, c.287C&gt;A, c.325T&gt;C, and c.483C&gt;T. All of these SNPs, except from c.325T&gt;C, were detected here for the first time. The Manych Merino breed had 13 substitutions that were present as homozygous type. Only SNP c.325T&gt;C occurred as the wild type of homozygotes and heterozygotes in the ratio 2:1. We found that SNP c.325T&gt;C was associated with some vital body parameters, including parameters of height and croup measurement. Thus, the determination of allelic variants of the MyoD1 gene may be used in marker assisted genetic selection schemes.

Administration of Clopidogrel in patients after percutaneous coronary intervention

Objective To provide supportive evidence for using Clopidogrel in patients with coronary heart disease(CHD)after percutaneous coronary intervention(PCI). Methods From June 2015 to May 2016, 105 cases of CHD were admitted to the Department of Cardiology, the First Affiliated Hospital of Zhengzhou University.Genetic testing for CYP2C19 polymorphisms combined with thromboela-stogram was used to detect the rate of Clopidogrel resistance, and adverse cardiovascular events were recorded for 12 months after PCI.Moreover, multivariate Logistic regression was used to analyze risk factors of Clopidogrel resistance. Results A total of 98 cases completed genetic testing for CYP2C19 polymorphisms.Cases with the wild type allele, heterozygous mutations and homozygous mutations accounted for 35.7%, 57.1% and 7.1%, respectively.Compared that for wild type cases(49.6±18.5)%, the platelet inhibition rate was significantly reduced for hybrid mutation cases(38.4±15.2)% and homozygous mutation cases(24.8±12.9)%(t=3.142, 3.370; P=0.002, 0.001, respectively). Meanwhile, based on pharmacokinetic characteristics, the rates of Clopidogrel resistance for patients with the intermediate metabolic type(the heterozygous mutant type)and the slow metabolic type(the homozygous mutant type)were significantly higher than that for patients with the normal metabolic type(the wild type)(χ2=5.687, 6.363; P<0.05, respectively). Follow-up results showed that the incidences of adverse cardiovascular events for wild-type and heterozygous mutation patients were 2.9% and 3.6%, respectively, which were significantly lower than that for homozygous mutant patients(28.6%)(χ2=5.815, 6.540; P<0.05, respectively). Multivariate Logistic regression analysis showed that risk factors for clopidogrel resistance were glycosylated hemoglobin Alc level, total cholesterol and white blood cell count. Conclusions Genetic testing for CYP2C19 polymorphisms combined with thromboela-stogram can be used as an effective way to evaluate whether Clopidogrel should be used.It is important to measure the platelet aggregation inhibition rate for early detection of Clopidogrel resistance to insure appropriate drug use. Key words: Angioplasty, transluminal, Percutaneous Coronary; Clopidogrel