Molecular basis of alpha-thalassemia in Iran

Alpha-thalassemia [alpha-thal] is probably the most prevalent monogenic condition in the world. Deletions are the most common types of mutations in alpha-thal, followed by point mutations and small insertion/deletion. In the context of national screening program for prevention of thalassemia and hemoglobinopathies in Iran, alpha-thal carriers have come to more attention. Therefore, the frequency and distribution of alpha-globin mutations in various regions of the country have been studied in recent years. A comprehensive search was performed in PubMed, Scopus, and national databases for finding reports on mutation detection in alpha-thal carriers and HbH disease with Iranian origin. The mutation data of 10849 alpha-thal carriers showed that -alpha[3.7] and alpha[-5NT] were the most common deletional and nondeletional mutations, respectively. In HbH disease cases, the -alpha[3.7]/-[-MED] was the most prevalent genotype. Overall, 42 different mutations have been identified in alpha-globin cluster reflecting the high heterogeneity of the mutations in Iranian populations

High-resolution HLA-A typing in normal Iranian population

Background: Human leukocyte antigen [HLA] gene is a highly polymorphic region. HLA typing is required to match patients and donors for transplantation; therefore, development of HLA registries is necessary for finding HLA match donors. HLA system is highly informative, and numerous studies have been conducted on HLA allele distribution in different populations

Methods: In this study, 100 unrelated Iranian individuals were typed for HLA-A locus using sequence-based typing method. Samples were subjected to the PCR, followed by Sanger sequencing and software analysis

Results: A*02:01 [13%] and A*24:02 [12%] were the two most frequent alleles, while A*01:14, A*02:05, A*02:11, A*02:34, A*02:50, A*11:04, A*23:02, A*24:34, A*25:01, A*26:09, A*26:43, A*29:67, A*30:54, A*31:02, A*31:66, A*32:03, A*32:04, A*33:03, and A*66:15 alleles had the least frequencies [1%]

Conclusion: This is the first report of HLA-A allele level typing in a randomized population of Iran and can be useful for development of national registries of HLA-typed volunteer marrow donors and local cord blood banks

[Expression levels of key components of the hedgehog pathway and an investigation of the correlation of this pathway with expression levels of claudin-1 in invasive breast carcinoma]

Objective: Breast cancer is considered a heterogeneous disease, characterized by different biological and phenotypic features which make its diagnosis and treatment challenging. We have sought to investigate the expression levels of key components of the Hedgehog signaling pathway, correlation between the signal transducer Smo, and clinicopathologic features [lymph node metastasis and metastasis stage] in invasive breast carcinoma. Also, we examined the inverse correlation between expression levels of Smo and Claudin-1 [an important gene involved in cell tight junctions]

Methods: In this case-control study, we assessed 36 pairs of tumor and adjacent normal tissue specimens obtained from patients with invasive ductal breast carcinoma. The expression levels of key components of Hedgehog signaling [Smo, Gli1 and Ptch], Claudin-1, E-cadherin, and MMP2 were measured by qRT-PCR. The correlations between Smo expression with some clinicopathologic parameters were also analyzed

Results: We found up-regulation of Hedgehog signaling in invasive breast carcinoma samples compared to normal adjacent tissues. Upregulation of the signal transducer Smo correlated with tumor stages and lymph node metastasis of the breast tumors. Interestingly, this correlation was affected by the expression of Her2. A significant correlation existed between expression levels of the signal transducer Smo and Claudin-1, E-cadherin as an epithelial cell marker, and MMP2 as a metastasis-related gene in advanced metastatic tumor samples

Conclusion: Taken together, our study revealed a new layer of molecular complexity which should be considered in the management of patients with invasive breast carcinoma. The results suggested a key role for Hedgehog signaling in invasive breast carcinoma. In terms of the inverse correlation between expression levels of Claudin-1 and Hedgehog signaling, Claudin-1 could serve as a candidate gene in diagnostic studies. Thus, its clinical significance should be further clarified

Expression enhancement in trastuzumab therapeutic monoclonal antibody production using genomic amplification with methotrexate

Trastuzumab [Herceptin] is a humanized monoclonal antibody [mAb] which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2[+] tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase [DHFR]. It is usually accepted that DHFR gene can be amplified in DHFR CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR

[ study of intron 22 inversion type I and II of coagulation factor 8 gene in patients with severe Hemophilia A using IS-PCR technique]

Hemophilia A [HA] is an X-linked recessive bleeding disorder. The disease is caused by mutations in the F8 gene. Inversion of intron 22 is the most common causative mutation of severe hemophilia A, which is detectable by southern blot and polymerase chain reaction [PCR] methods. The aim of this study was to determine intron 22 inversion type I and type II inversion in severe hemophilia A patients by inverse shifting-PCR method. This study was performed on 30 patients with severe hemophilia A with less than 1% of normal level activity of Factor 8. After obtaining consent from the patients, genomic DNA was extracted from peripheral blood leukocytes. The extracted DNAs were used as template for IS-PCR amplification after circularization [digestion by BcII and then ligation of the produced fragments by ligase enzyme]. In 30 severe hemophilia A patients, 40% of the patients had intron 22 inversion type I, and 6.6% of the patients had intron 22 inversion type II. The results of this study revealed that recombination between intron 22h-1 within the F8 gene and its copies [22h-2 and 22h-3], which lie in opposite direction to intron 22h-1, respectively cause the inversions of intron 22 type II and type I. Inversion of intron 22 type I is more frequent than type II. Also, by application of IS-PCR as a cost effective method, we could save time and improve the molecular detection of inversion. This method and can be used for detection of carriers and patients and for prenatal diagnosis of hemophilia A disease

[Production of camel polyclonal antibody against vascular endothelial growth factor receptor-2 and performance evaluation in ELISA test]

In molecular approach, serum of camel contains a unique type of antibodies devoid of light chains since the light chain is missing, the heavy-chain antibodies should bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain. Vascular endothelial growth factor receptor-2 [VEGFR-2] is one of the important proteins in angiogenesis which over-expressed in many types of tumors. Two male dromedaries [Camelus dromedarius] were immunized against VEGFR-2. ELISA was used to evaluate the immunization process. Camel immune sera were fractionated with protein A and G affinity chromatography. Heavy chain antibodies tested for its specific reactivity in cell-based ELISA in recognition of VEGFR-2 on the cell surface of three cell lines; 293/KDR, HUVECs and A431. In ELISA test, the camel sera in 1/12800 dilution had the acceptable results. Furthermore, cell-based ELISA demonstrated that the polyclonal heavy chain antibodies bind to VEGFR-2 in 293/KDR and HUVECs cell lines. Single chain polyclonal antibodies against VEGFR-2 can be used for detection of soluble form of this protein by ELISA, and also flowcytometry, western blotting and immunohistochemistry

[ occurrence and contribution of germline BRCA1/2 sequence alterations in Iranian patients with breast cancer]

Breast cancer is the most common form of hereditary cancer worldwide and is an important cause of morbidity and mortality. Approximately 5-10% of breast and ovarian cancers are due to the highly penetrating germline mutations in cancer predisposing genes. Two genes, BRCA1 and BRCA2, account for at least half of these cases. The demand for BRCA1 and BRCA2 mutation screening is rapidly increasing as their identification will affect the medical management of people at increased risk for the disease. Therefore, the aim of this study was to investigate BRCA1/2 mutations in 100 high risk Iranian families. One hundred families who met the minimal risk factors for breast/ovarian cancer were screened among the families referred to Kawsar Human Genetics Research Center for the diseases in 2009-2011. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened for by direct sequencing and MLPA in both patients and the controls. In the present study, we could detect the following novel mutations: p.Gly1140Ser, p.Ile26Val, p.Leu1418X, p.Glu23Gln, p.Leu3X, p.Asn1403His, p.Asn1403Asp, p.Lys581X, p.Pro938Arg, p.Thr77Arg, p.Leu6Val, p.Arg7Cys, p.Leu15Ile, p.Ser177Thr, IVS7+83[-TT], IVS8-70[-CATT], IVS2+9[G>C], IVS1-20[G>A], IVS1-8[A>G], p.Met1Ile, IVS2+24[A>G], IVS5-8 [A>G], IVS2[35-39]TTcctatGAT, IVS13+9 G>C in BRCA1 and p.Glu1391Gly, p. Val1852Ile, IVS6-70[T>G], 1994-1995 [InsA] in BRCA2. Ten mutations seemed to be pathogenic and the disease-causing mutations were seen in 16% of the families. In addition, from the total number of substitutions and reassortments [42], 80% related to BRCA1 and 20% to mutations in BRCA2 genes

First report on the Co-inheritance of beta-globin IVS-I-5 [G-C] thalassemia with delta globin CD12 [Asn-Lys [AAT-AAA]] hba2- NYU in Iran

Background: co-inheritance of beta- and delta-globin mutations in Iran is not uncommon. This situation may interfere with correct diagnosis and genetic counseling of alpha- and beta-thalassemia in screening programs. Here we report the co-inheritance of beta- and delta-globin gene mutations in an individual with microcytosis, hypochromia and a normal hemoglobin A2 [HbA2] level

Methods: genomic DNA extraction, amplication refractory mutation system [ARMS] polymerase chain reaction and direct DNA sequencing of delta- and beta-globin genes were exploited for detection of the mutations in these two genes in an individual with low hematological indices and normal HbA2

Results: ARMS-PCR technique revealed the beta+ IVSI-5 [G to C] mutation and direct DNA sequencing of the delta-globin gene detected a previously reported delta codon 12 [AATàAAA] HbA2-NYU. This study reports HbA2-NYU in association with the beta IVSI-5 [G to C] mutation in Iran

Discussion: this report emphasizes that normal HbA2 expression in a beta-goblin carrier is due to mutation in the delta-globin gene and may cause misdiagnosis of thalassemia

Co-inheritance of hemoglobin D and beta-thalassemia traits in three Iranian families: clinical relevance

Here we report the result of three cases referred to our lab that had a combination of beta-thalassemia and hemoglobin D [Hb D] traits. These individuals had no symptoms of profound anemia and hematological indices were similar to that of a beta-thalassemia heterozygote. In all three cases, the Hb D level was elevated and no HbA was detected electrophoretically. The electrophoresis pattern suggested that all cases were homozygotes for Hb D. PCR followed by digestion with EcoRI and sequencing of the beta-globin gene confirmed the presence of Cd 121 GAA>CAA in the heterozygous form with another beta-globin mutation. In all cases, the mutations in the beta-globin gene were detected by ARMS PCR technique and they were either IVSII-I or IVSI-5. Hematological studies of the family members showed that thalassemia which caused the mutations and Hb D were in the Trans position

Transient expression assay of [A]gamma-588 [A/G] mutations in the k562 cell line

In the previous study, we have shown that the presence of A allele at position -588 in [A]gamma -globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele [A allele at -588] could result in an increase in [A]gamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients. Three constructs containing ji locus control region, [A]gamma -globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of, [A]gamma -globin gene [A and G alleles at -588]. A construct with T to C base substitution at -175 of, [A]gamma -globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of, [A]gamma -globin gene was determined by quantitative real-time reverse transcription-PCR. There was not a significant increase in the expression of, [A]gamma -globin gene in the construct containing A allele comparing the one with G allele at -588. -588 [A>G] mutation does not play a major role in regulation of, [A]gamma -globin gene, suggesting that other factors may be involved

[Investigation of connexin 26 mutations and three large deletions spanning connexin 30 in 63 Iranian families with autosomal recessive non-syndromic hearing loss]

Hearing loss is the most frequent neurosensory defect in human. Mutations in GJB2 and GJB6 are responsible for 50% of autosomal recessive non-syndromic hearing loss [ARNSHL] cases. Here we report on the frequencies of GJB2 and GJB6 mutations and three large deletions spanning the GJB6 gene including Del [GJB6-D13S1830], Del [GJB6-D13S1854] and a>920 kb deletion in patients affected by ARNSHL referred to Kawsar's Human Genetics Research Center. In this study, 94 patients from 63 families with ARNSHL were investigated. Patient's homozygote for 35delG were screened and left out of the study and the remaining samples were analyzed by sequencing of GJB2 and GJB6 genes. Also the three large deletions spanning the GJB6 gene were analyzed by Real Time PCR In this study we found GJB2 mutations in 13 families [20.6%] out of 63.The 35delG mutation was the most common mutation in the studied population [61.5%]. Other GJB2 mutations were delE120, R127H, W24X, and V37I. The heterozygous or negative cases for the GJB2 mutations were screened for mutation in the GJB6 gene by sequencing and no mutation was observed. Also, we checked the three large deletions in GJB6, we found no mutations. Low frequency of mutations in the GJB2 gene implies that other genes may be involved in causing non-syndromic hearing loss in our country

[Transfection of human hematopoietic stem cells by a gene targeting construct containing the beta-globin gene]

Replacment of CD133+ cell's beta globin gene by using a gene targeting construct containing the beta globin gene and essential elements for homologous recombination. pFBGGT was amplified, then digested using the NheI and XhoI restriction enzymes, and finally, a 13.3 kb band [naked DNA] was extracted from the agarose gel. Biological activity of positive and negative selection markers were checked by transfection of COS-7 cells with linear plasmid. Hematopoietic stem cells [HSCs] were separated and transfected with linear plasmids using lipofection followed by positive and negative selection. Polymerase chain reaction [PCR] were done on DNA from the selected cells and the products were sequenced. The results of biological activity assays showed that selection markers were active. PCRs for hygromycin, neomycin and joining segments were positive but PCRs for TK1 and TK2 genes were negative. Sequencing PCR product joining segment confirmed the formation of homologous recombination. In this novel strategy gene replacement was achieved and biological activities of its components were observed

Antibacterial effect of the Persian Gulf sea cucumber Holoturia. SP extracts on three strain of Escherichia coli

Sea cucumber is a traditional food and medical item and has been reported to exhibit Antioxidant, antifungal, anti tumoral and antibacterial bioactivity. The objective of this study is to describe the antibacterial activity of 4 extracts of Holoturia. Sp [sea cucumber], collected from Hengam Island of Persian Gulf. Methanol, hexane, aqueous and chloroform extracts from body wall tissue of the sea cucumber were screened for antibacterial activity against three strains of Escherichia coli Top 10 F', TG1 and K12 using disc diffusion and broth microdilution tests methods. The growth of all these strains were inhibited using concentration from 0.78 to 100 mg/ml of methanol, hexan and chloroform extracts. Among the extracts just methanol and chloroform with 100 mg/ml had bactericidal effect on TG1 and K12 strains. On the other hand, Aqueous extract had induced growth in of the all strains. The results suggest the possibility of applying sea cucumber as source of potential anti bacterial agents, whose compounds can be good candidates to make antibiotic products

[Detection of unknown deletions in alpha globin genes in alpha thalassemia carriers using real-time PCR]

Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more alpha-globin genes. Common alpha-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Real-time PCR was performed using intercalating dye SYBR Green I and alpha1, alpha2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method [delta delta CT] for determination of Gene dosage of alpha1-globin and alpha2-globin genes. The results showed the ratio of 0.90 +/- 0.16 for normal individuals and the ratio of 0.32 +/- 0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers

Cross-resistance to vincristin and etoposide in a sub line of the human breast cancer T47D cells selected for adriamycin-resistance

Breast cancer is one of the most common malignancies among women. Although chemotherapy remains a major therapeutic approach to treat cancers, drug therapy often fails for several reasons, particularly the drug resistance. Resistance to multiple chemotherapeutic agents is one of the most important problems in the treatment of different types of cancers. Therefore, in this study a resistant sub line of the human breast cancer T47D cells was isolated in vitro by stepwise exposure to increasing concentrations of Adriamycin [ADR] to compare the characteristics of parent and resistant cells. We also evaluated the phenomenon of cross-resistance to some other chemotherapeutic drugs. A significant increase in doubling time of resistant cells, named T47D/ADR, [94 h] was observed when compared to the parental T47D cells [50 h] that indicates a relatively slow growth rate pattern of these cells. T47D/ADR cells were 4 fold resistant to adriamycin and also showed cross-resistance to vincristin [VCR, 3.5 fold] and to etoposide [VP-16, 5.5 fold] when compared to parent cells. Therefore, our results indicate that T47D/ADR cells are also cross-resistant to structurally and functionally different chemotherapeutic agents and can be used as a model for studying molecular changes of drug resistance