Production and Evaluation of the Properties of HIV-1-Nef-MPER-V3 Fusion Protein Harboring IMT-P8 Cell Penetrating Peptide.

BACKGROUND: Finding a safe and effective vaccine for HIV-1 infection is still a major concern. OBJECTIVE: This study aimed to design and produce a recombinant Nef-MPER V3 protein fused with IMT-P8 using E. coli expression system to provide a potential HIV vaccine with high cellular penetrance. METHODS: After synthesizing the DNA sequence of the fusion protein, the construct was inserted into the pET-28 expression vector. The recombinant protein expression was induced using 1 mM IPTG and the product was purified through affinity chromatography. Characterization of cellular delivery, toxicity and immunogenicity of the protein was carried out. RESULTS: The recombinant protein was expressed and confirmed by the anti-Nef antibody through western blotting. Data analyses showed that the protein possessed no considerable toxicity effect and has improved the IMT-P8 penetration rate in comparison to a control sample. Moreover, the antigen immunogenicity of the protein induced specific humoral response in mice. CONCLUSION: It was concluded that IMT-P8-Nef-MPER-V3 fusion protein has a high penetrance rate in mammalian cell line and low toxicity, thus it can be potentially considered as a vaccine against HIV-1.

The rs4846049 polymorphism in the 3'UTR region of the MTHFR gene increases the migraine susceptibility in an Iranian population.

INTRODUCTION: Migraine is a painful complex neurovascular disease characterized by recurrent moderate-to-severe headaches. Increased level of homocysteine is related to dilation of cerebral vessels and endothelial injury that could trigger migraine attacks. Functional polymorphisms in the MTHFR gene affect homocysteine metabolism and, therefore, play an important role in the etiology of the disease. OBJECTIVES: We aimed to investigate the possible association between MTHFR gene rs4846049, C677T, and A1298C polymorphisms and the risk of migraine in Iranian population. METHODS: In this genetic association study, 498 individuals were enrolled, including 223 migraine patients and 275 healthy controls. Genotyping was performed using tetra-primer ARMS-PCR for rs4846049 and PCR-restriction fragment length polymorphism for C677T and A1298C polymorphisms. RESULTS: The association between rs4846049 and C677T polymorphisms and migraine was observed. For the rs4846049 polymorphism, the association was detected under a dominant model (P=0.007; odds ratio [OR] =0.60; 95% confidence interval [CI], 0.41-0.87), and for the C677T polymorphism, the TT genotype frequency was significantly different in the studied groups (P=0.009; OR =2.48; 95% CI, 1.25-4.92). No significant differences in the genotype or allele frequencies were found for the A1298C polymorphism between the migraineurs and controls. CONCLUSION: Present data provide evidence for the association of rs4846049 and C677T polymorphisms in the MTHFR gene and migraine. Further studies are required to validate the significance of the studied genetic variations in diverse ethnic populations.

Homozygosity mapping in albinism patients using a novel panel of 13 STR markers inside the nonsyndromic OCA genes: introducing 5 novel mutations.

Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented hair, skin and eyes. It is associated with decreased visual acuity, nystagmus, strabismus and photophobia. Six genes are known to be involved in nonsyndromic oculocutaneous albinism (OCA). In this study, we aimed to find the disease causing mutations in albinism patients using homozygosity mapping. Twenty three unrelated patients with nonsyndromic OCA or autosomal recessive ocular albinism were recruited in this study. All of the patients' parents had consanguineous marriage and all were screened for TYR mutations previously. At first, we performed homozygosity mapping using fluorescently labeled primers to amplify a novel panel of 13 STR markers inside the OCA genes and then the screened loci in each family were studied using PCR and cycle sequencing methods. We found five mutations including three mutations in OCA2, one mutation in SLC45A2 and one mutation in C10ORF11 genes, all of which were novel. In cases where the disease causing mutations are identical by descent due to a common ancestor, these STR markers can enable us to screen for the responsible genes.

Evaluation of IGK and IGL molecular gene rearrangements according to the BIOMED-2 protocols for clinical diagnosis of Hodgkin lymphoma.

BACKGROUND: Although the analysis of molecular clonality rearrangements of the immunoglobulin light chains (IGK and IGL) is an alternative approach for diagnosis of B cell non-Hodgkin lymphomas (NHLs) using BIOMED-2 protocols, NHLs have not been extensively confirmed for Hodgkin lymphoma (HL) cases. We evaluated BIOMED-2 protocols in HL cases, which have been suggested previously as gold standard method for molecular clonality analysis on formalin fixed, paraffin-embedded (FFPE) tissue in NHL patients. METHODS: We recruited 50 consecutive FFPE tissues of HL samples to evaluate IGK and IGL clonality gene rearrangements using BIOMED-2 and Heteroduplex methods. RESULTS: Our findings revealed a total of 94% (47/50) positive clonality, which consisted of 70% (35/50) for IGK and 44% (22/50) for IGL. In three cases, clonality was not detected in any of the immunoglobulin gene segments. CONCLUSIONS: Analysis of clonality gene rearrangements in IGK and IGL genes using BIOMED-2 protocols could be implemented as a valuable method for improving clonality detection rate in HL cases and sensitivity (94%) and accuracy of HL diagnosis similar to that of the NHL samples will be increased.

Molecular Analysis of IGH and Incomplete IGH D-J Clonality Gene Rearrangements in Hodgkin Lymphoma Malignancies.

BACKGROUND: We evaluated molecular clonality in immunoglobulin heavy chain (IGH) and incomplete IGH D-J genes for improvement of clinical diagnosis of Hodgkin's lymphoma (HL). We applied BIOMED-2 protocols in HL cases, which were previously approved by clonality detection in non-Hodgkin lymphoma (NHL) cases. METHODS: We investigated 50 consecutive FFPE samples of classical HL (cHL) patients to assess IGH and IGH D-J clonal gene rearrangements by multiplex PCR protocols, which were provided by the European Biomedicine and Health (BIOMED-2) Concerted Action Project BMH4-CT98-3936. RESULTS: In the present study, there was a monoclonality of 86% (43/50) including a clonality of 74% (37/50) for IGH and a clonality of 42% (21/50) in IGHD-J. In addition, a lack of clonality was detected in 14% (7/50) of cases. Frequent gene rearrangements were detected in framework (FR) III (54%) and FRII (20%), whereas no clonality was seen in FRI. Furthermore, a monoclonality of 28% and 14% was detected in the DH(1-6)-JH and DH(see symbol)-JH gene rearrangements, respectively. CONCLUSIONS: The present study suggests that the complete IGH and incomplete IGH D-J clonality gene rearrangement assays using BIOMED-2 protocols could be considered a valuable method for detection of clonal gene rearrangements, especially in HL cases.

Correlation between the EGF gene intronic polymorphism, rs2298979, and colorectal cancer.

Colorectal cancer (CRC) is an important disorder that results from genetic and epigenetic alterations in one colonic epithelial cell. Epidermal growth factor (EGF) is critical in the development of tumors in epithelial tissues. Variations in the DNA sequence of the EGF gene may be particularly significant with regard to susceptibility to CRC. The present study aimed to investigate the effect of the EGF gene single nucleotide polymorphism (SNP), rs2298979, on CRC. In this prospective study, 220 samples were collected from patients with CRC and compared with 220 matched healthy controls. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, and the result was validated by direct sequencing. A significant correlation was observed between the rs2298979 variant in the EGF gene and CRC. The frequency of the A/G genotype in the control group was higher than in the patients with sporadic CRC [odds ratio (OR), 0.488; 95% confidence interval (CI), 0.307-0.774; P=0.002]. In this study there were no individuals with a G/G genotype. Although the frequency of the G and A alleles was similar in the healthy control and CRC patient groups, individuals with the A/G genotype were less susceptible to CRC compared with those with the A/A genotype.

The effect of 5'untranslated region polymorphism in EGF gene, rs4444903, on colorectal cancer.

AIM: The purpose of this study was to determine the relationship of rs4444903 (EGF+61A/G) SNP genotype with colorectal cancer and tumor stage in an Iranian population. BACKGROUND: Epidermal growth factor (EGF) is one of the important proteins that determine survival of cells. EGF binds to its receptor on the cell surface and then activates some of the cell signaling pathway networks within cells that lead to activation or deactivation of factors which are responsible for growth and apoptosis of cells. In this study we assessed the association in EGF polymorphism rs4444903 with colorectal cancer (CRC) in Iranian population. PATIENTS AND METHODS: We conducted case-control study to investigate the association of polymorphism rs4444903 in EGF, with colorectal cancer risk in Iranian population. Analyzed Polymorphism of EGF rs4444903 with restriction fragment length polymorphisms (RFLP) among two groups of subjects consisting of including 220 cases with colorectal cancer and 220 healthy individuals as controls. Mutations were confirmed in 10% of the samples by direct sequencing. RESULTS: The frequencies of AA, AG and GG genotypes among cases with colorectal cancer were 28.2, 46.8, and 25.0 % respectively and in controls genotype frequencies were 23.2, 56.4, and 20.5 %, respectively. Frequency of A allele among case group was 51.6% and for control group was 51.4%. The frequency of G allele in case and control was, respectively 48.4% and 48.6% (OR= 1.009, 95% CI= 0.775-1.315; P= 0.946). The percentage of Stage 0, I, II, III, IV were 5%, 9.35%, 38.84%, 30.21% and 16.54%, respectively, among the cases. However, no significant association between this polymorphism and CRC stage was observed (p=0.626). CONCLUSION: Our data suggest a SNP rs4444903 may not represent a risk factor in the development and progression of CRC among Iranian population.

"Familial" versus "Sporadic" intellectual disability: contribution of common microdeletion and microduplication syndromes.

BACKGROUND: Interstitial Microdeletion and Microduplication syndromes have been proposed as a significant cause of sporadic intellectual disability (ID) but the role of such aberrations in familial ID has not yet been investigated. As the balanced chromosomal abnormalities commonly lead to the recurrent ID or multiple congenital anomalies, this study was designed to evaluate whether it was justified to investigate such aberrations in familial ID patients. Three hundred and twenty eight patients from 101 unrelated Iranian families with more than two ID patients in the first-degree relatives, have been investigated. Assessment of a panel of 21 common Microdeletion and Microduplication syndromes (CMMS) was carried out using Multiplex Ligation-Dependent Probe Amplification (MLPA) technique. RESULTS: Among the families studied, 27.7% had 4-12, 35.6% had 3 and 36.6% had 2 affected individuals in the first-degree relatives. An autosomal dominant inheritance of Williams-Beuren syndrome (WBS) was detected in a family with no clinical suspicion of WBS. The prevalence of CMMS was therefore,0.99%. CONCLUSION: This is the first investigation of a panel of CMMS in a large sample set of "familial ID patients". The findings of this study showed the low prevalence of CMMSs in "familial ID" patients in spite of the significant contribution of such aberrations in "sporadic ID" which has a very useful practical impact by avoiding unnecessary diagnostic tests in "familial ID" patients.

BRCA1 and BRCA2 germline mutations in 85 Iranian breast cancer patients.

Breast cancer is the most common cancer in Iranian women (Mousavi et al in Asian Pac J Cancer Prev 9(2):275-278, 2008). Genetic predisposition accounts for 15% of all breast cancers and germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2 are responsible for a substantial proportion of high-risk breast and breast/ovarian cancer families (Collaborative Group on Hormonal Factors in Breast Cancer in Lancet 350:1047-1059, 1997; Lee et al in Int Nurs Rev 55:355-359, 2008; Hulka and Stark in Lancet 346:883-887, 1995; Kelsey in Epidemiol Rev 15:256-263, 1993; Tischer et al in J Biol Chem 266:11947-11954, 1991; Newman et al in: Proc Natl Acad Sci USA 85:3044-3048, 1988). Therefore, the aim of this study was to investigate mutations of BRCA1/2 in high risk Iranian families. We screened 85 patients who met our minimal criteria. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened by direct sequencing. In the present study, we could detect the novel following mutations: p.Glu1735 p.Gly1140Ser, p.Ile26Val, p.Leu1418X, p.Glu23Gln, p.Leu3X, p.Asn1403His, p.Lys581X, p.Pro938Arg, p.Thr77Arg, p.Arg7Cys, p.Ser177Thr, IVS7+83(TT), IVS8-70(-CATT), IVS2+9(-GC), IVS1-20(-GA), IVS1-8(-AG), IVS2+24(AG), IVS5-8 (A-G), IVS2(35-39)TTcctatGAT in BRCA1 and p.Glu1391Gly, 1994_1995 (Ins A), IVS6-70-T>G in BRCA2. In agreement with findings in other populations, we found that family history is a good predictor of being a mutation carrier. Five pathogenic BRCA1 mutations and one pathogenic BRCA2 mutation were detected in 85 index cases.

Frequent MSI mononucleotide markers for diagnosis of hereditary nonpolyposis colorectal cancer.

BACKGROUND: Failure in the DNA mismatch repair system is commonly accompanied by microsatellite instability and leads to colorectal cancer. The aim of this study was to find the most frequent of five mononucleotide markers in order to devise the simplest diagnostic strategy for identification of patients with hereditary nonpolyposis colorectal cancer (HNPCC) who were defined by defects in mismatch repair system. MATERIALS AND METHODS: 78 patients with colorectal cancer were recruited for this investigation. Five mononucleotide markers, NR-27, NR-21, NR-24, BAT-25 and BAT-26, were used as a pentaplex panel to determine MSI status. RESULTS: Two out of five mononucleotide markers, NR-21 (25.6%) and BAT-25 (23.1%) showed more instability than the others. CONCLUSION: In defining individuals with colorectal cancer, BAT25 and NR-21 may provide diagnostic assistance.