Reaction-restriction Fragment Length Polymorphism Research Articles

Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits

Abstract. Setyorini YW, Kurnianto E, Sutopo, Sutiyono. 2023. Identification polymorphism of LHR and FSHR genes in Indonesian Holstein dairy cattle associated with productive and reproductive traits. Biodiversitas 24: 2898-2905. Luteinizing and follicle-stimulating hormones and their receptors play an important role in the reproductive system’s hormonal activity and physiological function. This study aimed to identify the genotype variation of the Luteinizing Hormone Receptor (LHR) gene exon 11 and the Follicle-stimulating Hormone Receptor (FSHR) gene exon 10 and to elucidate the associating between polymorphism with the milk and reproductive traits in dairy cattle. A total of 100 samples of deoxyribonucleic acid (DNA) from Indonesian Holstein dairy cattle were used in this study. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) was performed for genotyping of the Single Nucleotide Polymorphism (SNP) of the LHR and FSHR genes with the HhaI and AluI restriction enzymes, respectively. The collected phenotype data were milk production, quality, and reproductive trait. In addition, the general linear model procedure in the SAS program was carried out to investigate the association of genotypes with productive and reproductive traits. The results showed that the LHR gene in the population of this study was monomorphic, while the FSHR gene was polymorphic with three genotypes, namely CC, CG, and GG. The sequences of the FSHR gene indicated the presence of a mutation at nucleotide number 2037 that substitutes cytosine for guanine. Cows with the CC genotype showed better parameter service per conception (P<0.05) than the other genotypes but did not correlate with other reproductive parameters and milk traits. Therefore, it was concluded that SNP g.2037C>G polymorphism at FSHR gene exon 10 was identified in the Indonesian Holstein Dairy cattle population and associated with service per conception parameters but not with milk trait.

Genetic heterogeneity of thalassemia major patients in Rembang Regency, Central Java, Indonesia

Link of Video Abstract: https://www.youtube.com/watch?v=RG95aJkUndg Background: Thalassemia and hemoglobinopathies are caused by mutations in the globin genes that reduce haemoglobin synthesis. In many Asian countries, including Indonesia, thalassemia is a common inherited disorder. Thalassemia has specific characteristics due to the wide variability of the mutations from various populations. Therefore, screening protocol should be designed on specific populations’ regional gene mutation patterns. This study aims to investigate the mutational diversity of the globin genes in thalassemia major patients in Rembang Regency, Central Java, Indonesia. Methods: Genetic mutations were carried out in all major thalassemia patients who were recorded and underwent routine blood transfusions at the Rembang District Hospital from 2018 – 2020. Mutation identification was carried out using a PCR- RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) based method, the ACRS (Amplified Created Restriction Site) method, and genome sequencing. Results: The most common genotypes are double heterozygous HbE and Cd 35 mutation (-C) (42.87%), HbE and IVS1-nt.5 mutations (G> C) (28.57%), HbE and IVS1- nt.2 mutations (T> C), HbE and codon 8/9 (insertion + G), codon 8/9 (insertion + G) and Hb Malay, as well as Hb Adana and Hb Constant Spring (1.74%). Conclusion: The β-thalassemia and α-thalassemia mutations most commonly found in Rembang Regency are Cd 35 (-C) and non-deletional mutations.

A Pilot Study on the Prevalence of Transcription Factor 7-Like 2 Gene (TCFL2), Rs290487 in Ethnic Groups with Type 2 Diabetes Mellitus in Bayelsa State of Nigeria

The negative impact of type 2 diabetes mellitus (T2DM) on individuals, families, health system and the economic development as a whole, is an important justification for research in this field. The present study is a hospital-based case-control type to ascertain the genetic susceptibility of T2DM among a sample population of various ethnic groups resident in Bayelsa State, Nigeria. Also evaluated was the relationship between transcription factor 7-like 2 (TCFL2) rs12255372 and rs290487 genetic polymorphisms with development of T2DM. Genotyping of TCFL2 rs12255372 and rs290487 were carried out using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Among the indigenous groups, the allelic frequencies determined for the abnormal (CC) and the normal (TT) genotypes were 94.4 and 5.6 (%), respectively; whereas, the values 91.7 and 8.3 (%), in that other, were determined for the non-indigenous groups. A convincing significant difference was found between subjects with T2DMne and their counterpart controls within indigenous people of Bayelsa State with respect to confounders, including age (t=8.046, p<0.00001), BMI (t =2.628, p<0.0190), waist circumference (t=2.800, p=0.0091) and fasting blood sugar (t=3.212, p< 0.0006). This study verified the association of TCFL2 rs290487 with the development of T2DM in indigenous and non-indigenous people in Bayelsa State. The risk conferred by the homozygous CC genotype was higher than that of the heterozygous TC state – a fact indicative of an additive model of inheritance.

The association between PTPN22 C1858T gene polymorphism and type 1 diabetes mellitus: an Indonesian study

Background Type 1 diabetes mellitus (T1DM) is disease caused by the destruction of β pancreatic cells. The activation of T-lymphocyte and proliferation inhibitor are induced by protein tyrosine phosphatase non-receptor type 22 (PTPN22). However, the link between PTPN22 C1858T gene polymorphism and T1DM is still controversy. This study aimed to analyse the C1858T gene polymorphism in Indonesian children with T1DM. Materials and methods This case-control study was conducted from March 2021 to May 2022 in the Endocrinology Outpatient Clinic at Dr. Soetomo Hospital and Tropical Disease Center Universitas Airlangga. Patients with controlled T1DM during the study period were included. The PTPN22 analysis used polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Results Sixty-two children voluntarily participated in this study, and were equally divided into the T1DM and control groups. Most of the patients (94%, 58/62) are Javanese. This study revealed a more frequent CC genotype (9.4%) and allele-C (54.6%) polymorphism in the T1DM group, while more frequent CT genotype (100%) and allele-T (50%) polymorphism were in the control group. The C- and T-allele frequency was 54.6% and 45.4% in the T1DM group, respectively. The T1DM and control groups did not significantly differ (p= .2381). Conclusions PTPN22 homozygous genotype-CC and allele-C polymorphisms are more frequent in patients with T1DM. However, the PTPN22 C1858T gene polymorphism did not significantly correlate to T1DM children in this study. Key Messages: The PTPN22 C1858T gene polymorphism does not significantly affect the susceptibility of T1DM in Indonesian children. PTPN22 homozygous genotype-CC polymorphism was more observed in the T1DM group; thus, this genotype may play as a risk factor for T1DM children in the Indonesian population.

Molecular Detection of Phytoplasmas of the 16SrⅠ and 16SrXXXⅡ Groups in Elaeocarpus sylvestris Trees with Decline Disease in Jeju Island, South Korea

Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrⅠ and 16SrXXXⅡ groups) in Jeju Island (Republic of Korea).

Correlation analysis and prognostic value of miR-29a-3p expression and CYP2C19 genotypes in exfoliated cells from tongue coating of patients with gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is a highly prevalent and troublesome disease. Several differentially expressed microRNAs (miRNAs) have been found in GERD. This study was to analyze the correlation of miR-29a-3p expression and CYP2C19 genotypes in exfoliated cells from tongue coating of GERD patients and its prognostic value. Tongue coating specimens were collected from 130 GERD patients and 70 healthy volunteers and their clinical baseline information was recorded. miR-29a-3p expression in exfoliated cells from tongue coating was determined via RT-qPCR, and its diagnostic efficiency on GERD was evaluated via receiver operating characteristic curve. CYP2C19 genotypes and their correlation with miR-29a-3p were analyzed via polymerase chain reaction restriction fragment length polymorphism technique. The adverse events of patients were documented via 12-month follow-up. The impact of miR-29a-3p expression on the healing rate of patients was analyzed via Kaplan-Meier method. Qualification of miR-29a-3p as an independent prognostic factor of GERD patients was analyzed via multivariate Cox regression analysis. miR-29a-3p was highly-expressed in exfoliated cells from tongue coating of GERD patients. miR-29a-3p expression had high specificity and sensitivity in diagnosing GERD. CYP2C19 genotypes in GERD patients comprised rapid metabolizers, intermedia metabolizers, and poor metabolizers. miR-29a-3p expression showed a correlation with CYP2C19 genotypes. Higher miR-29a-3p expression predicted higher cumulative incidences of adverse outcomes. Highly-expressed miR-29a-3p was an independent prognostic factor for adverse outcomes of GERD patients. High expression of miR-29a-3p aided the diagnosis and predicted poor prognosis of GERD patients.

Macrophage Migration Inhibitory Factor and Gene Polymorphism (rs755622G>C) in Unstable Vitiligo Patients

Vitiligo pathogenesis is related to the macrophage migration inhibitory factor (MIF) protein. This study aimed to assess the lesional MIF levels and gene polymorphisms (rs755622G>C) in patients with vitiligo. To assess the consequences of combining narrow‐band ultraviolet B with oral minipulse prednisolone as opposed to a combination with oral methotrexate on MIF levels in vitiligo patients, 50 unstable vitiligo patients and 50 controls were randomly chosen for comparison. MIF levels in skin homogenates and MIF (rs755622G>C) single nucleotide polymorphisms were assessed using the ELISA and the polymerase chain reaction restriction fragment length polymorphism (RFLP‐PCR) techniques. We found significantly higher lesional MIF levels, a higher frequency of both (GC) and (CC) genotypes, and a significantly more frequent mutant allele (C) in patients than in controls. In addition, there was a significantly lower frequency of the allele (C) among patients who exhibited moderate to marked therapeutic improvement than among those who showed minimal to mild improvement. In conclusion, tissue MIF and gene polymorphisms were associated with vitiligo. In addition, oral corticosteroids, narrow‐band ultraviolet B, methotrexate‐targeted tissue MIF, and gene polymorphisms can improve unstable vitiligo.

CTLA-4 +49A/G And -318 C/T Polymorphisms and Cytokines Level on Inhibitors Development of Haemophilia A among Different Ethnicity in Malaysia

Introduction: Haemophilia A (HA) is an inherited X-chromosome recessive disorder characterized by the deficiency of factor VIII (FVIII). About 25-30% of HA patients which received FVIII concentrate developed inhibitors towards FVIII. Ethnicity has been associated as a predisposing factor for the formation of FVIII inhibitors. This study characterizes the polymorphism of +49A/G and -318C/T of Cytotoxic T-Lymphocytes Antigen 4 (CTLA-4) which relates to the development of inhibitors among Malaysian HA patients of Malay, Chinese and Indian ethnicity. Cytokines level of tumour necrosis factor alpha (TNFα), interleukin 4 (IL-4) and interleukin 10 (IL-10) were also measured to assess the link to inhibitors development. Method: Severe HA patients with and without inhibitor who were being treated at the National Blood Centre, Kuala Lumpur were recruited and consented where their collected blood was genotyped for both polymorphisms using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) and their cytokines levels were measured using the Enzyme-linked Immunosorbent Assay (ELISA). Results: Analysis of the total 64 respondents who fulfilled the study criteria found polymorphisms of CTLA-4 +49A/G and CTLA-4 -318C/T were not significantly different among patients with and without inhibitors. However, HA patients without inhibitors revealed that the Chinese population exhibited a higher +49G allele which is protective towards inhibitors development. Indian patients expressed a higher level of inhibitors titre. Patients without inhibitors showed statistical differences between ethnicity for both genotypes and allele frequencies of CTLA-4 +49A/G (P < 0.05). Conclusion: The mixed pattern of polymorphisms and cytokine profiles were observed in multi-ethnicity. This finding requires further verification with a larger sample size involving multicenter for further confirmation.

Associative role of polymorphism of the gene of <i>MMP-9</i> (rs11697325) in development of arterial hypertension in patients with the rheumatoid arthritis

Aim. To study a contribution of polymorphism A-8202G (rs11697325) of a gene of matrix metalloproteinase-9 (MMP-9) in development of arterial hypertension (AH) in patients with the rheumatoid arthritis (RA).
 Materail and methods. 143 patients with RA were examined, among which a group of patients with RA without AH (n=50) and a group of patients with RA in association with AH (n=93) were identified. Healthy volunteers (n=151) were also divided into 2 groups comparable in age, sex and number with the main groups (control group 1 n=54, control group 2 n=97). The work used a range of clinical, laboratory, instrumental methods. A molecular genetic study was also performed. Blood samples were taken from all study participants. DNA isolation was carried out by the standard phenol-chloroform method. Genotyping for the MMP-9 gene was performed by PCR-RFLP analysis (polymerase chain reaction restriction fragment length polymorphism). PCR was carried out with a set of primers to the corresponding regions of the genome. PCR products were analyzed by electrophoresis in 4% polyacrylamide gel followed by staining with ethidium bromide.
 Results. In the course of a molecular genetic study, a statistically significant predominance of the homozygous AA genotype and the A allele of the MMP-9 (rs11697325) gene polymorphism was observed in the group of patients with RA, both separately and in association with AH, in comparison with the data of control groups. The risk of developing RA estimated by the odds ratio (OR) in carriers of the AA genotype of the MMP-9 gene is 1.8 times higher (95% confidence interval CI 1.1362.950; p=0.02) than in carriers of the GG and GA genotypes; in carriers of the A allele, the risk of developing RA is 1.6 times higher compared to the G allele (95% CI 1.1192.578; p=0.01). The OR risk of developing RA in association with hypertension in carriers of the AA genotype of the MMP-9 gene is 2.8 times higher (95% CI 1.2836.54; p=0.04) compared to GA and GG genotypes, 2.1 times higher in carriers of the A allele (95% CI 1.1134.127; p=0.02) compared to the G allele.
 Conclusion. Thus, the results of the study indicate that the homozygous AA genotype and the A allele of the MMP-9 (rs11697325) gene polymorphism are the predictors of the development of RA both as a separate nosological unit and in association with AH.

Genetic Variations of ferroportin-1(FPN1-8CG), TMPRSS6 (rs855791) and Hemojuvelin (I222N and G320V) Among a Cohort of Egyptian β-Thalassemia Major Patients

Iron overload remains a major cause of morbidity and mortality among β-thalassemia major (β-TM) patients. Iron regulatory proteins and their genetic variants together with changes in hepcidin levels in thalassemic patients could affect the disease manifestations. This work aimed to study genetic variations of ferroportin-1 (FPN1-8CG), Transmembrane Serine Protease 6 (TMPRSS6 rs855791) and hemojuvelin (HJV I222N and G320V) genes within a cohort of 97 β-TM Egyptian patients by Polymerase chain reaction Restriction Fragment Length Polymorphism (PCR-RFLP) in comparison to fifty normal control subjects. Among β-TM patients; the CG variant of FPN1 was significantly higher, while the TT and TC variants of TMPRSS6 were significantly lower in comparison to controls. Liver Iron Concentration (LIC) was significantly higher among β-TM patients harboring the FPN1 (GG) genotype and we found that FPN1gene mutation acts as independent predictor of MRI LIC (p = 0.011), Pulmonary artery pressure (PAP) was significantly higher in patients harboring the mutant FPN1 (GG and CG) genotypes (p value 0.04). β-TM patients having the HJV I222N (AA) genotype were having significantly higher cardiac iron overload (p value = 0.026). The studied genetic variants of iron regulatory proteins could alter the manifestations of iron overload thus resulting in different clinical phenotypes of thalassemic patients, these findings need to be confirmed by larger cohorts of patients with longer follow-up periods.

Identification of immune-related mechanisms of cetuximab induced skin toxicity in colorectal cancer patients.

Skin rash is a well-known predictive marker of the response to cetuximab (Cmab) in metastatic colorectal cancer (mCRC). However, the mechanism of skin rash development is not well understood. Following exposure to EGFR-targeted therapies, changes in IL-8 levels have been reported. The aim of this study was to evaluate the association between skin rash and inflammatory cytokine levels, including IL-8. Between 2014 and 2017, we prospectively enrolled 38 mCRC patients who underwent chemotherapy with either Cmab or bevacizumab (Bmab) at two hospitals. We performed multiplex cytokine ELISA with 20 inflammatory cytokines including E-selectin, GM-CSF, IFN-alpha, IFN-γ, IL-1 alpha, IL-1 beta, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IP-10, MCP-1, MIP-1 alpha, MIP-1 beta, P-selectin, sICAM-1, and TNF-alpha at baseline before cycle 1, 24 h after cycle 1, before cycle 2 (= 14 d), and before cycle 3 (= 28 d). Cytokine levels were compared using ANOVA after log-transformation. IL-8 genotypes in 30 patients treated with Cmab were determined using the polymerase chain reaction restriction fragment length polymorphism technique. Depending on the RAS mutational status, 30 and eight patients were treated with Cmab and Bmab-based chemotherapy, respectively. Skin rash developed in 23 (76.6%) of the 30 patients treated with Cmab plus FOLFIRI, after cycle 1. Only the mean log-transformed serum IL-8 level in patients with skin toxicity was statistically lower (2.83 ± 0.15) than in patients who did not experience skin toxicity (3.65 ± 0.27) and received Bmab (3.10 ± 0.26) (ANOVA test, p value = 0.0341). In addition, IL-8 polymorphism did not affect IL-8 levels, skin toxicity, or tumor response in Cmab treated patients. This study suggests that the inflammatory cytokine levels might be affected by Cmab exposure and are associated with the development of skin rash in mCRC patients. Further studies are warranted to evaluate this interaction in Cmab treated patients.

Evaluation of Tumor Necrosis Factor-Alpha Gene (−308 G/A, −238 G/A and −857 C/T) Polymorphisms and the Risk of Gastric Cancer in Eastern Indian Population

Introduction: Gastric cancer (GC) is one of the leading causes of cancer-related decimations worldwide. The gastric infection at both the stomach and duodenum with Helicobacter pylori causes inflammation by the tumor necrosis factor-alpha (TNF-α). The aim of the study was to associate and evaluate the three TNF-α gene polymorphisms at positions −308 G/A, −238 G/A, and −857 C/T with the risk of GC. Methods: A total of 156 individuals (consecutively diagnosed 95 GC patients and 61 controls) above the age of 18 years were enrolled in the study. Healthy individuals with normal upper gastrointestinal endoscopy (UGIE) irrespective of their family history of GC or peptic ulcer were included as controls. The cited three TNF-α gene polymorphisms were evaluated using polymerase chain reaction-restriction fragment length polymorphism (RFLP). Results: There was no significant difference in the distribution of gene polymorphisms as genetic factors, TNF-α−308 GA/AA (22.1% vs. 14.8%, p = 0.2), TNF-α−238 GA/AA (21% vs. 19.6%, p = 0.8), and TNF-α−857 CT/TT (8.4% vs. 11.5%, p = 0.5), between GC cases and healthy controls. A subgroup analysis of H. pylori-positive patients showed that there was no significant difference in the distribution of GA/AA polymorphisms in TNF-α−308 (15(45.5%) vs. 3(23%); p = 0.17) and −238 (12(36.3%) vs. 2(15.4%); p = 0.17), and the distribution of TT/CT −857 CT/TT (13(39.4%) vs. 2(15.4%); p = 0.13), among the GC cases and controls. Conclusion: The statistical comparisons of GA/AA vs. GG genotypes at −308 (with OR = 1.6, 95% CI: 0.6–3.8), −238 (OR = 1.09, 95% CI: 0.4–2.4) and TT/CT vs. CC genotypes at −857 (OR = 0.7, 95% CI: 0.2–2.1) did not suggest any association of TNF-α with GC in the population herein. Hence, the TNF-α (−308 G/A, −238 G/A and −857 C/T) may not be the associating factor for GC incidence determined by the PCR–RFLP method.

CC Genotype of GNAS c.393C>T (rs7121) Polymorphism Has a Protective Effect against Development of BK Viremia and BKV-Associated Nephropathy after Renal Transplant

The GNAS gene encodes the alpha-subunit of the stimulatory G-protein (Gαs) in humans and mice. The single-nucleotide polymorphism of GNAS, c.393C>T, is associated with an elevated production of Gαs and an increased formation of cyclic adenosine monophosphate (cAMP). In the present study, we analyzed the effect of this GNAS polymorphism on a renal allograft outcome. We screened a cohort of 436 renal allograft recipients, who were retrospectively followed up for up to 5 years after transplant. GNAS genotypes were determined with polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays. The 393T allele was detected in 319 (73%) recipients (113 recipients with TT and 206 with CT genotype) and the CC genotype in 117 (27%). The CC genotype was associated with a significantly lower frequency of BK viremia (CC, 17 recipients (15%); T 84 (26%)); p = 0.01; TT, 27 vs. CC, 17, p = 0.07; TT, 27 vs. CT, 57, p = 0. 46; CT, 57 vs. CC, 17, p = 0.01) and BKV-associated nephropathy (CC, 3 recipients (3%); T, 27 (8%); p = 0.03; TT,10 vs. CC, 3, p = 0.04; TT, 10 vs. CT,17, p = 0.85; CT, 17 vs. CC,3, p = 0.04) after transplant. BKV-associated nephropathy-free survival was significantly better among CC genotype carriers than among T allele carriers (p = 0.043; TT vs. CC, p = 0.03; CT vs. CC, p = 0.04; TT vs. CT, p = 0.83). Multivariate analysis indicated an independent protective effect of the CC genotype against the development of both BK viremia (relative risk. 0.54; p = 0.04) and BKV-associated nephropathy after renal transplant (relative risk. 0.27; p = 0.036). The GNAS 393 CC genotype seems to protect renal allograft recipients against the development of BK viremia and BKV-associated nephropathy.

Association of CD40 gene polymorphisms and immune thrombocytopenic purpura in the adult Egyptian population.

BackgroundThe pathophysiology underlying primary adult immune thrombocytopenic purpura (ITP) has not yet been identified. However, many mechanisms affect the immune system, causing defective tolerance to self-platelets and megakaryocytes. Cluster of differentiation 40 (CD40) contributes to both humoral and cell-mediated immune responses.MethodsThis case‒control study was conducted to detect rs4810485G>T and rs1883832C>T polymorphisms of CD40 in Egyptian patients with persistent/chronic ITP to clarify their possible association with chronic disease evolution. This study included 50 patients with persistent/chronic ITP and 50 healthy controls. Genotyping was performed using the polymerase chain reaction‒restriction fragment length polymorphism technique.ResultsGenotyping of rs1883832 and rs4810485 revealed no statistically significant differences between the two groups. However, combined gene polymorphism genotyping showed a statistically significant difference between the two groups (P<0.01).ConclusionOur results indicate a strong association between the combined polymorphism of both genes and susceptibility to developing ITP among adult Egyptian patients. Targeting this pathway using novel therapeutic approaches is promising.

Risk Prediction of Type 2 Diabetes Mellitus (T2DM) in Indian Families Using Antioxidant Gene Variants

Background: Type 2 diabetes mellitus (T2DM) is a complex metabolic disease that is caused by insulin dysfunction. It is an output of oxidative stress that results from defective redox reactions and increased reactive metabolites (RMs) and is neutralized by antioxidant enzymes. It has been reported that decreased levels of antioxidant enzymes are due to genetic alterations in the respective genes. Therefore, the present study has undertaken a genetic analysis of antioxidant genes and their interaction in the family to assess T2DM risk. Objective: This study aimed to investigate the individual susceptibility/risk to T2DM using antioxidant gene variants and their interactions in family members with a diabetic history. Methods: Genotypic analysis of antioxidant genes was done by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), haplotype analysis, and gene-gene interactions using statistical tools. Pedigrees were constructed by face-to-face interviews with members of nine families. Results: Genotypes AT (CAT-21A&gt;T), IV (GSTP1+313(105I&gt;V), and CT (GPx1 +599C&gt;T) were found to be frequent in diabetic individuals. For instance, in one family, if only the mother had diabetes, all siblings were found to have the risk genotypes AT (CAT- 21A&gt;T) and CT (GPx1 +599C&gt;T) with 2.12- and 2.11-folds risk of developing T2DM. The risk haplotypes, NNV (GSTM1 N&gt;P(Null&gt;Present), GSTT1 N&gt;P(Null&gt;Present), GSTP1105I&gt;V and TCC (CAT-21A&gt;T, SOD2+47C&gt;T, GPx1+599C&gt;T) were observed in most of the diabetic individuals and non-diabetics possessing the risk haplotypes manifested altered BMI. Conclusion: The present study suggests that the GSTP1105I&gt;V, CAT-21A&gt;T, SOD2+47C&gt;T and GPx1+599C&gt;T gene variants can be prognostic biomarkers for the assessment of T2DM risk in healthy individuals.

Effect of IL-6 -174G/C and -572G/C variants on susceptibility to osteoarthritis in Turkish population

Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage in which inflammatory mechanisms play a major role in the pathogenesis. Interleukin-6 (IL6), a multifunctional cytokine, can trigger osteoclast differentiation and bone resorption. Our purpose in this study was to evaluate the association of IL-6 −174 G/C (rs1800795) and −572 G/C (rs1800796) variants with the susceptibility to OA. One hundred fifty OA patients and 150 healthy individuals were enrolled in the study. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was used for genotyping the IL-6 gene variants. The results of analyses were evaluated for statistical significance. The pain intensity was assessed using the Visual Analogue Scale (VAS). There was a statistically significant difference in the genotype and allele frequencies of the IL-6 −174 G/C variant between patients with OA and control groups (p = 0.001, p = 0.002, respectively). IL-6 −174 G/C GG genotype and G allele were more prevalent in patients with OA. We found that the IL-6 –572 G/C variant was not different between patients and controls in either genotype distribution and allele frequency. IL-6 174 G/C and −572 G/C loci GG-GG combined genotype was significantly higher in OA patients (p = 0.00). Our study suggests that there was a strong association between the IL-6 −174 G/C variant and OA in the Turkish population. Further studies on populations of different ethnic background are necessary to prove the association of IL-6 variants with OA.

Effects of gene polymorphisms in tumor necrosis factor-α on the susceptibility of type I diabetes mellitus in an Iraqi population

Background: Type 1 diabetes mellitus (T1DM) is a chronic, immune-mediated disease characterized by the destruction of insulin-producing β cells in the pancreas. This study investigated the influence of single nucleotide polymorphisms (SNP) in tumor necrosis factor (TNF-α) –308 G/A (rs1800629,) on T1DM patients. Method: The study was included 75 T1DM patients and 25 healthy subjects. The SNP–308 G/A TNF-α gene was detected by using polymerase chain reaction restriction fragment length polymorphism (RFLP-PCR) technique in T1DM patients and controls. Results: The genotype distribution results of the -308G/A SNP of Tumor Necrosis Factor - α (TNF- α)-308 G/A gene showed significant difference (p&lt;0.05) between controls (GG: n= 24, 96%; GA: n=1, 4%; AA: n=0, 0%) and T1DM patients (GG: n= 44, 58.67%; GA: n=21, 28%; AA: n=10, 13.33%).These results showed an increased in GA, AA genotype and G allele of the TNF- α(-308) G/A in T1DM patients than controls, and they were significantly more likely than controls to have the mutant allele (OR=18.431, 95%CI= 2.464-137.855, p=0.00001). These results indicate a possible role for the G genotype in T1DM disease.

Polymorphism of growth hormone gene and its association with body measurement traits in Boer goat does

The study examined single nucleotide polymorphisms (SNPs) in the growth hormone gene (GH1) in Boer goat does and their relationship with body measurement traits, namely bodyweight, body length, heart girth, rump height, rump width, ear length, cannon circumference, and head width. Seventy-six Boer goat does between the ages of 2 and 4 years were used as experimental animals. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and deoxyribonucleic acid (DNA) sequencing techniques were used to detect SNPs. Chi-square test was used to measure the genetic equilibrium and a general linear model was used for the marker-trait association analysis. The PCR-RFLP and DNA sequencing results discovered one SNP (G505C) in the exon 5 of the candidate gene with two genotypes observed (AA and AB). The association analysis indicated that bodyweight was associated with the genotypes (P &lt;0.01), but not with any of the morphometric traits. A chi-square analysis indicated that the genotypic frequencies were in Hardy-Weinberg equilibrium. The polymorphism discovered in this study is a putative marker that might assist farmers in improving their does’ bodyweight through marker-assisted selection.

Frequency of the DI*A, DI*B and Band 3 Memphis polymorphism among distinct groups in Brazil

The Band 3 is a red blood cell protein that carries the Dia and Dib antigens from the Diego blood system. The SLC4A1 gene encodes Band 3; Band 3 Memphis is a polymorphism of normal Band 3 and has two variants, but only the variant II carries the Dia antigen. Describe the frequencies of the DI*A and DI*B alleles and the Band 3 Memphis among blood donors, sickle cell disease (SCD) patients and Amazonian Indians. A total of 427 blood samples were collected and separated into three groups: 206 unrelated blood donors, 90 patients with SCD and 131 Amazonian Indians. We performed DI*A/B, normal Band 3 and Band 3 Memphis genotyping, using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP). The frequency of the DI*A/DI*A genotype was 0.5% in blood donors and it was not found in other groups. The frequency of the DI*A/DI*B was higher in Amazonian Indians (33.6%) and the frequency of the DI*B/DI*B was highest in blood donors (92.2%). All 105 individuals tested were positive for the presence of normal Band 3 and of these individuals, only 5/105 (4.8%) presented the Band 3 Memphis mutation. We observed a higher frequency of the DI*B allele in blood donors and a low frequency of the DI*A/DI*A genotype in all groups studied. The Band 3 Memphis was found in a higher frequency in the blood donor group. Our findings highlight the importance of analyzing different population groups to gain a better understanding of the genetic association of blood group antigens.

15 ROLE OF NEUROPEPTIDE Y GEN RS 16147 POLIMORFISM AS A RISK FACTOR FOR HYPERTENSION

Background: According to World Health Organization (WHO), hypertension has been a medical problem that associated with 7.5 millions (12.8 percents) death in wide world. Neuropeptide Y (NPY) is the most often found neurotransmitter in nervous system, comes from expression of NPY gen rs16147, has effect in symphatetic tonus enhancement result in increasing blood pressure (BP). Objective: To determine role of NPY Gen RS 16147 Polimorfism as a risk factor of hypertension. Method: Analytic study with case control design to determine relationship between NPY Gen RS 16147 Polimorfism: C-399T and BP with Chi Square test. Population consist of 100 sample (50 male,50 female) with consecutive sampling. Sample taken from venous blood, analyzed using Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. p value &lt;0,05 means statistically significant. Result: Study enrolled subject with median age 51,5 (22—83) years (26 normotension, 74 hypertension), subject with alel “C” NPY RS 16147 genotype has abnormal BP, while subject with non alel “C” NPY RS 16147 (TT genotype) has both normal and abnormal BP, (p: 0.017). Average systolic blood pressure (SBP) in non alel “C” NPY RS 16147 genotype is 139,05±21,42 mmHg, while average SBP in alel “C” genotipe NPY RS 16147 genotype is 151,32±22,26 mmHg. Conclusion: Neuropeptide Y Gen RS 16147 polimorfism is a risk factor for hypertension. The presence of alel “C” in NPY RS16147 related to high BP significantly.