Multiplex Snapshot minisequencing for the detection of common PAH gene mutations in Iranian patients with Phenylketonuria

Background: Phenylketonuria is a common inborn defect of amino acid metabolism in the world. This failure is caused by an autosomal recessive insufficiency of the hepatic enzyme hyperphenylalaninemia (PAH), which catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. More than 1,040 different disease-causing mutations have already been identified in the PAH gene. The most prominent complication of Phenylketonuria, if not diagnosed and treated, is severe mental retardation. Hence, early diagnosis and initiation of nutritional therapy are the most significant measures in preventing this mental disorder. Given these data, we developed a simple and rapid molecular test to detect the most frequent PAH mutations. Methods: Multiplex assay was developed based on the SNaPshot minisequencing approach to simultaneously perform genotyping of the 10 mutations at the PAH gene. We optimized detection of these mutations in one multiplex PCR, followed by 10 single-nucleotide extension reactions. DNA sequencing assay was also used to verify genotyping results obtained by SNaPshot minisequencing. Result: All 10 genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. Sequencing results of these frequent mutations showed that by using this method, a 100% detection rate could be achieved in the Iranian population. Conclusion: SNaPshot minisequencing can be useful as a secondary test in neonatal screening for HPA in neonates with a positive screening test, and it is also suitable for carrier screening. The assay can be easily applied for accurate and time- and cost-efficient genotyping of the selected SNPs in various population.

SNHG1 Long Noncoding RNA is Potentially Up-Regulated in Colorectal Adenocarcinoma.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. However, the molecular mechanisms involved in CRC initiation and progression is remained to be unknown. It seems that lncRNAs, as the main and lengthy functional transcripts of the genome, have important roles in different cancers such as CRC. CRC-related lncRNAs are reported to be involved in diverse molecular processes such as metastasis, invasion, cell proliferation, and apoptosis. This study was aimed to analyse the expression level of lncRNA SNHG1 in colorectal adenocarcinoma and normal tissues. We performed an in silico analysis on a large cohort and confirmed the results by experimental analysis of clinical samples through real-time PCR. Our findings demonstrated that that SNHG1 is potentially overexpressed in tumor tissues compared with adjacent normal tissues. The expression level of SNHG1 was shown to be potentially associated with clinicopathological features of tumors. The current study suggests the potential role of SNHG1 in colon cancer progression.

Use of embryonic fibroblasts associated with graphene quantum dots for burn wound healing in Wistar rats.

Burn is one of the common wounds in the world and using modern methods such as cell therapy can be considered as an effective strategy in the treatment of these wounds. The aim of this study is investigating the effects of using graphene quantum dots (GQDs) associated fibroblasts on treating third-degree burns in Wistar rats. In this experiment, cells were obtained by isolating fibroblasts from 13-day embryos of Wistar rats. MTT assay was performed to determine the dose of nanoparticle and cell tracker. For this study, 40 Wistar rats were burned and randomly divided into two groups of control and treatment. The treatment group was divided into three groups of daily injection of GQD nanoparticle with a concentration of 100 µg/ml, cell therapy, and cell therapy + GQDs. On days 20 and 40, skin tissue sections were prepared and stained with hematoxylin-eosin (H&E) and trichrome Masson for microscopic examination. Macroscopic and microscopic observations showed that in the treatment groups, the recovery was higher than the control. Also, cell therapy and GQD injection and simultaneous injection of cell therapy + GQDs accelerated the wound healing process and the cell therapy + GQDs were significantly more effective than nanoparticles and cell injection alone after 20 and 40 days. Histological studies indicated a significant increase in angiogenesis, number of cells, collagen synthesis, thickness of skin layers, and ultimately acceleration wound healing in treatment samples compared to controls. Based on these results, it can be concluded that simultaneous cell therapy and GQDs accelerate the repair of skin lesions in the animal models more significantly.

Genetic Variations of DAOA (rs947267 and rs3918342) and COMT Genes (rs165599 and rs4680) in Schizophrenia and Bipolar I Disorder.

INTRODUCTION: Genetic and environmental factors are involved in the incidence of schizophrenia and bipolar disorder. Many reports confirm that several common genes are connected with these two psychotic disorders. Several neurotransmitters may be involved in the molecular mechanisms of schizophrenia and bipolar disorder. We aimed to estimate the role of two talent genes: DAOA in neurotransmission of glutamate and COMT in neurotransmission of dopamine to guide the treatment of schizophrenia and bipolar disorder. METHODS: Blood samples (n=100 for schizophrenia, n=100 for bipolar I disorder and n=127 for case control) were collected from individuals unrelated in the southwest of Iran. The SNPs (rs947267 and rs3918342 for DAOA gene/rs165599 and rs4680 for COMT gene) were genotyped using the PCR-RFLP method. Our finding was studied by logistic regression and Mantel-Haenszel Chi-square tests. RESULTS: We observed an association in rs3918342, rs165599 and rs4680 single nucleotide polymorphisms and schizophrenia and bipolar I disorder. In addition, our data demonstrated that the rs947267 was related to bipolar I disorder but there was no association between this SNP and schizophrenia. CONCLUSION: In conclusion, this result supports the hypothesis that variations in DAOA and COMT genes may play a role in schizophrenia and bipolar disorder.

Association of PTPN22 rs2476601 Polymorphism with Rheumatoid Arthritis and Celiac Disease in Khuzestan Province, Southwestern Iran.

BACKGROUND: Single-nucleotide polymorphism (SNP) rs2476601 within protein tyrosine phosphatase non-receptor type 22 gene (PTPN22) has been shown to be a risk factor for different autoimmune diseases. This study explored the association of 1858 C/T SNP with rheumatoid arthritis (RA) and celiac disease (CD) in a region covering south-west of Iran. METHODS: Totally, 52 patients with CD, 120 patients with RA, and 120 healthy subjects were selected. The samples were genotyped for the rs2476601 in PTPN22 gene using the tetra-amplification refractory mutation system polymerase chain reaction. RESULTS: The frequency of +1858T risk allele was significantly increased in both RA (P=0.021, OR=2.56, 95%CI=1.19-5.47) and CD (P=0.002, OR=3.87, 95%CI=1.68-8.95) patients, as compared to the control group. However, no association was found between the +1858C/T PTPN22 gene SNP and the anti-cyclic citrullinated peptide and rheumatoid factor positivity in RA patients. CONCLUSIONS: PTPN22 gene could play a crucial role in people's susceptibility to certain autoimmune diseases.

Additive effect of MTHFR and GRIN1 genetic polymorphisms on the risk of schizophrenia.

Schizophrenia is a complex disorder with polygenic inheritance. The MTHFR gene (OMIM: 607093) plays an important role in the folate metabolism. It has been suggested that C677T (rs1801133) and A1298C (rs1801131) genetic polymorphisms in the MTHFR gene lead to the decreased activity of the methylenetetrahydrofolate reductase enzyme which may have significant effect on developing schizophrenia. We used a case-control study to establish the possible association between the C677T and the A1298C polymorphisms and susceptibility to schizophrenia in an Iranian population. The genotypes of the polymorphisms were determined using PCR-RFLP. The data were analyzed by logistic regression model. Data analysis revealed that the combination genotypes of 677CT/1298AA, 677CC/1298CC, 677TT/1298AA, 677CT/1298AC and 677CT/1298CC increase the risk of schizophrenia. In order to evaluate the effect of combined genotypes of the three mentioned polymorphic loci, the frequencies of the compound genotypes were compared between control and patient groups (Table 4). Base on the results, the existence of >4 risk factors showed about 32-fold increased risk for schizophrenia (OR=32.3, 95% CI: 5.52-188, P=<0.001).

Association study of single nucleotide polymorphism rs165599 of COMT gene, with schizophrenia and bipolar mood disorder in the south-west of Iran.

Linkage studies and epidemiological findings indicate that some possible genes in schizophrenia (SCZ) and bipolar mood disorder (BPD) are common. Numerous evidences for linkage of two diseases on chromosome 22 have been found. These findings suggest that one or more genes in the 22q11.21 region may be involved in the development of both disorders. In the present case-control study, association between the mentioned disorders and a genetic polymorphism (rs165599) of catechol O- methyltransferase (COMT, OMIM: 116790) was studied. Here 100 BPD patients, 100SCZ patients, and 100 healthy controls were included in the study. The samples were matched in terms of gender and ethnicity. Statistical analysis showed that there was a significant association this polymorphism and risk of SCZ. The AG (OR=7.41, 95% CI: 3.21-17.1, P<0.001) and GG genotypes (OR=13.9, 95% CI: 5.61-34.4, P<0.001) increased the risk of SCZ compared with the GG genotypes. The AG (OR=14.3, 95% CI: 4.16-49.4, P<0.001) and AA genotypes (OR=54.2, 95% CI: 15.3-191, P<0.001) significantly associated with the risk of BPD. The risk of SCZ (x2=37.4, P<0.001) and BPD (x2=66.2, P<0.001) significantly increased as a function of numbers of the A allele. The present study revealed that this polymorphism associated with risks of SCZ, and BPD.

Study of the association between glutathione S-transferase (GSTM1, GSTT1, GSTP1) polymorphisms with type II diabetes mellitus in southern of Iran.

Diabetes Mellitus is characterized by chronic hyperglycemia and associated with an increased production of reactive oxygen species (ROS). Oxidative stress is the result of accumulation of free radicals in tissues which specially affects beta cells in pancreas. Glutathione S-transferases (GSTs) are a family of antioxidant enzymes that include several classes of GSTs. These enzymes have important roles in decreasing of ROS species and act as a kind of antioxidant defense. To investigate the association between GSTs polymorphism with type 2 diabetes mellitus (T2DM), we investigated the frequency of GSTM1, T1 and P1 genotypes in patients with T2DM and controls. The genotypes of GSTT1, M1 and P1 were determined in 171 clinically documented T2DM patients and 169 normal cases (as controls) by multiplex polymerase chain reaction and PCR-RFLP. In diabetic patients, the frequency of GSTM1-null genotype was significantly (OR = 1.74; 95 % CI = 1.13-2.69, P = 0.016) higher than that in control. However, the frequency of GSTT1 (OR = 1.29; 95 % CI = 0.07-2.14, P = 0.367) and GSTP1 (OR = 0.83; 95 % CI = 0.53-1.30, P = 0.389) genotypes were not significantly different comparing both groups. Also, the frequency of both GSTT1-null and GSTM1-null genotypes in patients (19.88 %) was significantly higher compared to controls with the same genotypes (11.83 %, P = 0.022). Our results indicated that GSTM1 and GSTT1 genotypes might be involved in the pathogenesis of T2DM in south Iranian population.

Molecular Characterization of Cosenza Mutation among Patients with Glucose-6-Phosphate Dehydrogenase Deficiency in huzestan Province, Southwest Iran.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common hereditary enzymatic disorders in human, increases the vulnerability of erythrocytes to oxidative stress. It is also characterized by remarkable molecular and biochemical heterogeneity. According to previous investigations, G6PD Cosenza (G1376C) is a common G6PD mutation in some parts of . Therefore in the present study we have characterized mutation among G6PD deficient individuals in Khuzestan province. In order to identify G6PD Cosenza, we analyzed the G6PD gene in 64 samples out of 231 deficient individuals who had not G6PD Mediterranean mutation, using PCR- restriction fragment length polymorphism (RFLP) method. G6PD Cosenza mutation was found in 6 males of 231 samples, resulting in the relative rate of 2.6% and allele frequency of 0.023 among Khuzestanian G6PD deficient subjects. A comparison of these results with previous findings in some parts of suggests that G6PD Cosenza is a common mutation in Khuzestanian G6PD deficient individuals.