Novel CFTR mutations in two Iranian families with severe cystic fibrosis

Background: Cystic fibrosis [CF] is a common autosomal recessive disorder that affects many body systems and is produced by mutations in the cystic fibrosis transmembrane conductance regulator [CFTR] gene. CF is also the most frequently inherited disorder in the West. The aim of this study was to detect the mutations in the CFTR gene in two Iranian families with CF

Methods: After DNA extraction using the salting out method, a mutation panel consisting of 35 common mutations was tested by PCR, followed by reverse hybridization Strip Assay. To confirm the mutations, we have also performed Sanger sequencing for all 27 exons, intronic flanking regions, and 5' and 3' UTRs of the CFTR gene

Results: Carrier testing in a spouse revealed a novel nonsense mutation in the CFTR gene [c.2777 T>A [p.L926X]] in exon 17 for husband and a previously described heterozygous splice site pathogenic mutation [c.1393-1G>A] in his wife. The other novel compound heterozygous missense mutation [c.3119 T>A [p.L1040H]], which was previously reported as nonsense c.3484C>T [p.R1162X] mutation, was found in exon 19 in patient screening

Conclusion: Two novel CFTR mutations in exons 17 and 19 are responsible for CF with severe phenotypes in two Iranian families. These two mutations supplement the mutation spectrum of CFTR and may contribute to a better understanding of CFTR protein function

Association study of the TREM2 gene and identification of a novel variant in Exon 2 in Iranian patients with Late-Onset Alzheimer's disease

Objective: To analyze the association between TREM2 exon 2 variants and late-onset [sporadic] Alzheimer's disease [AD] in an elderly Iranian population

Materials and Methods: Exon 2 of TREM2 in a total of 131 AD patients and 157 controls was genotyped using polymerase chain reaction and Sanger sequencing. Fisher's exact test was used to compare the allele and genotype frequency between the 2 study groups

Results: One homozygous and 2 heterozygous carriers of rs75932628-T in the AD patients and 1 heterozygous carrier in the control group were identified. One novel damaging variant, G55R, was also detected in the AD patient group. The frequency of rs75932628-T as well as the amount of rare variants were higher in the AD patients than in the controls, but this did not reach a statistically significant association with AD [odds ratio: 4.8; 95% confidence interval: 0.54 to 43.6; p = 0.270]

Conclusion: The rs75932628-T allele frequency in the elderly Iranian population [0.86%] was high

Investigating Seven Recently Identified Genes in 100 Iranian Families with Autosomal Recessive Non-syndromic Hearing Loss

Objectives: Hearing loss [HL] is the most common sensory disorder, and affects 1 in 1000 newborns. About 50% of HL is due to genetics and 70% of them are non-syndromic with a recessive pattern of inheritance. Up to now, more than 50 genes have been detected which are responsible for autosomal recessive non-syndromic hearing loss, [ARNSHL]. In Iran, HL is one of the most common disabilities due to consanguineous marriages. The aim was to investigate the prevalence of three new ARHL genes [GJB4, GJC3, and SLITRK6] reported in neighboring countries among Iranian families with ARNSHL.

Methods: One hundred unrelated families with at least two affected siblings in consanguineous marriage, who were negative for GJB2 gene mutations, were selected. By using three STR markers for each gene, homozygosity mapping was performed.

Results: Two families showed linkage to GJB4, six families were linked to GJC3 and only one family linked to SLITRK6. The samples of these families who showed linkage were sent for Sanger sequencing to detect the causative mutations. However, after analyzing the sequencing results, no mutation could be detected in either of the families. Molecular analysis for these nine families is underway in order to determine the pathogenic mutations using whole exome sequencing.

Discussion: These data demonstrate a very low prevalence of mutation in these three genes [GJB4, GJC3, and SLITRK6] in the Iranian population, since no mutation was detected in our study group of 100 families.

Association of polymorphisms at LDLR locus with coronary artery disease independently from lipid profile

Coronary artery disease [CAD] is the leading cause of mortality in many parts of the world. Genome-wide association studies [GWAS] have identified several genetic variants associated with CAD in Low-density lipoprotein receptor [LDLR] locus. This study was evaluated the possible association of genetic markers at LDLR locus with CAD irrespective to lipid profile and as well as the association of these SNPs with severity of CAD in Iranian population. Sequencing of 2 exons in LDLR gene [Exon 2, 12] and part of intron 30 of SMARCA4 gene include rs1122608, was performed in 170 Iranian patients angiographically confirmed CAD and 104 healthy controls by direct sequencing. Sullivan's scoring system was used for determining the severity of CAD in cases. Our results showed that homozygote genotypes of rs1122608 [P<0.0001], rs4300767 [P<0.005] and rs10417578 [p<0.007] SNPs have strong protective effects on the CAD. In addition, we found that rs1122608 [GT or TT] was at higher risk of three vessel involvement compared to single vessels affecting [P=0.01]

Optimizing a lipocomplex-based gene transfer method into heLa cell line

One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mammalian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV: Beta -Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were carried out in different volumes of FuGENE-HD, Lipofectamine[TM] 2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4 Micro l along with 10[5] HeLa cells, transfection efficiency was higher [43.66 +/- 1.52%] in comparison with the cationic lipids lipofectamine[TM] 2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency

Mutational screening of ARX gene in Iranian families with X-linked intellectual disability

Mutations in the human aristaless-related homeobox [ARX] gene are amongst the major causes of developmental and neurological disorders. They are responsible for a wide spectrum of phenotypes, including nonsyndromic X-linked intellectual disability [NS-XLID], and syndromic [XLIDS] forms such as X-linked lissencephaly with abnormal genitalia [XLAG], Partington syndrome [PRTS], and X-linked infantile spasm syndrome [ISSX]. The recurrent 24 bp duplication mutation, c.428_451dup[24 bp], is the most frequent ARX mutation, which accounts for 40% of all cases reported to date. We have screened the entire coding sequences of the ARX gene in 65 Iranian families with intellectual disabilities in order to obtain the relative prevalence of ARX mutations. At first these families were screened for the most recurrent mutation, the c.428_451dup[24 bp]. For samples with negative results, single strand conformation polymorphism [SSCP] analysis was performed. We identified one family with the c.428_451dup[24 bp] duplication. Three shifts [one shift in exon 5 and two shifts in exon 4] were also identified among the total families. According to the results of the sequencing analysis, two shifts were not associated with any mutation and the other one was a c.1347C>T [p.G449G] substitution in exon 4. Hence, we suggest that molecular analysis of ARX mutations as a second cause of XLID should be considered as routine diagnostic procedure in any male who presents with either NS-XLID or XLIDS

[Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia]

Background and Aim: Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED [Direct repeat erythroid-definitive]. DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene

Materials and Methods: In this study, lipofectamin[Trade Mark] 2000 was used to transfect siRNA molecules and pSV-beta-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels

Results: Our findings showed that K562 cells were transfected by 40% using Lipofectamine[Trade Mark] 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times

Conclusion: Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation; first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression

[Study of factor V Leiden polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the Factor V Leiden polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a teststrip presenting a parallel array of allele-specific oligonucleotide probes. The prevalences of mutant Factor V Leiden [1.2%] in our cohort was below previously published figures on the population of Tehran [2.7%]. Here we describe the distribution of mutant allele FV Leiden in different ethnicities of Iranian population and compare the results to previously reported data. Our data represent the most comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

[Study of ACE Ins/Del polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study, in order to assess the distribution of the ACE Ins/Del polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a test strip presenting a parallel array of allele-specific oligonucleotide probes. The allele frequencies of mutant ACE D/I polymorphism [0.62] was remarkably high. Mutant ACE D/I polymorphism in Iran occurred as high as East Mediterranean populations, and exceeded the lower frequencies known from Europe, India and most of Asia. Here we describe the distribution of mutant allele ACE D/I polymorphism in different ethnicities of Iranian population. Our data represent the only comprehensive study to date with respect to ACE D/I gene polymorphism in Iran

[Study of beta-fibrinogen-455 G/A polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the beta-Fibrinogen-455 G/A polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a teststrip presenting a parallel array of allele-specific oligonucleotide probes. The allele frequency of mutant FGB-455 G/A [0.22] is comparable to that of Europeans, but exceeded the much lower frequencies known from the most of Asia. Here we describe the distribution of mutant allele FGB-455 G/A in different ethnicities of Iranian population. Our data represent the only comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

[Study of MTHFR A1298C polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the MTHFR A1298C polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a teststrip presenting a parallel array of allele-specific oligonucleotide probes for each mutation. The prevalence of mutant MTHFR A1298C in our study population [0.42], was remarkably high. Mutant MTHFR A1298C in Iran occurred less frequently than among Europeans, but exceeded the much lower frequencies known from India and most of Asia. Here we describe the distribution of mutant allele MTHFR A1298C in different ethnicities of Iranian population and compare the results to previously reported data. Our data represent the only comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

[Study of Prothrombin G20210A polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the Prothrombin G20210A polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a teststrip presenting a parallel array of allele-specific oligonucleotide probes for each mutation. The allele frequencies of mutant Prothrombin G20210A [0.005] in our cohort were below previously published figures on the population of Tehran [1.5]. Here we describe the distribution of mutant allele Prothrombin G20210A in different ethnicities of Iranian population and compare the results to previously reported data. Our data represent the most comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

[Study of PAI-1 4G/5G polymorphism as a genetic risk factor for cardiovascular disease in the Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the PAI-1 4G/5G polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a test strip presenting a parallel array of allele-specific oligonucleotide probes. The allele frequency of mutant PAI-1 4G/5G [0.45] is comparable to that of Europeans, but exceeded the much lower frequencies known from the most of Asia. Here we describe the distribution of mutant allele PAI-1 4G/5G in different ethnicities of Iranian population. Our data represent the only comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

[Determination of gamma-globin induction levels by different concentrations of hydroxyurea in K 562 cell lines for beta thalassemia treatment

Hydroxyurea [HU] is currently used for beta thalassemia treatment through induction of fetal gamma-globin, In this study, effects of different concentrations of hydroxyurea on induction of gamma-globin gene in K562 cells, and inhibitory effect of siRNA against candidate gene which may be involved in HU mediated gamma-globin induction were assessed. In this eperimantal study, K562 cells were treated by different concentrations of HU [0, 50, 100 and 200 microM]. siRNA against candidate gene in HU mediated gamma-globin gene induction was transfected to K562 cells using lipofectamine 2000. The level of gammal-globin gene induction and inhibition were determined by quantitavie real-time PCR. There were 1.75-, 2.45- and 2.55- fold inductions in gamma-globin gene using 50, 100 and 200 microM HU, respectively. There has been 79.5% down regulation on candidate gene in siRNA transfected K562 cells. This study showed that gamma-globin induction in 100 microM HU is similar to 200 micro M HU treated K562 cells. There was also efficient inhibitory effect on candidate gene which may be involved in HU mediated gamma-globin induction

[Large deletion in VLGR1 causes Usher syndrome type IIC in male and female patients of an Iranian family]

Usher syndrome [USH] is a clinically and genetically heterogeneous disease. The three recognized clinical phenotypes, USH1, USH2 and USH3, are caused by mutations in nine different genes. USH2C is characterized by moderate-to-severe hearing loss, retinitis pigmentosa and normal vestibular function. Earlier reports describe mutations in VLGR1 in five families segregating this phenotype. We ascertained an Iranian family, in which nine family members were found to have hearing loss. Five patients were diagnosed to have Usher syndrome, while two individuals have nonsyndromic hearing loss. Consistent with these clinical findings, the five subjects with USH carried a haplotype linked to the USH2C locus, while the two subjects with nonsyndromic hearing loss did not, indicating that their hearing loss is due to another genetic cause. We identified a new mutation in VLGR1 segregating with the USH2C in this family. The mutation is a large deletion g.371657-507673del containing exons 84 and 85 and is presumed to lead to a frameshift. The deletion probably arose by replication slippage caused by the presence of a short 7-bp repeat at the deletion breakpoints. No genotype-phenotype correlation could be found for USH2C. In the family described, the first male subjects with USH2C and a VLGR1 mutation have been identified

[Nucleophosmin gene mutations in acute myeloid leukemia]

Heterozygous mutation of the nucleophosmin gene [NPM1] has recently been described as one of the most frequent genetic lesions in acute myeloid leukemia [AML]. NPM1 gene mutations tend to occur more frequently in women, and also tend to be associated with a higher white blood cell count. There is no significant age difference. NPM1-mutated AML is preferentially associated with AML monocytic differentiation [in particular FAB M5b], lack of CD34, normal cytogenetics, FLT3 gene mutations, and a trend toward favorable clinical outcome, specially in patients without FLT3 gene mutation. NPM1 gene mutations cause a framshift in the C-terminus of exon 12, disrupting the NPM nucleolar-localization signal or generating leucin-rich nuclear export motif, resulting in abnormal cytoplasmic accumulation of NPM. Detection of NPM1 gene mutation may allow dissection of the heterogeneous group of AML with normal karyotype into prognostically different subgroups. Exploring the mechanisms may lead to a better understanding of how mutant NPM protein becomes leukemogenic, thereby providing insights for the development of new chemotherapeutic agents

[Prevelancey MTHFR C677T polymorphism as a genetic risk factor for cardiovascular disease in the normal Iranian population]

Two hundred and eight asymptomatic individuals from different origins of Iran were included in this study in order to assess the distribution of the MTHFR C677T polymorphism responding for cardiovascular disease [CVD] in the general Iranian population using a novel technique reverse hybridization Strip Assay for the rapid and simultaneous detection. The test is based on multiplex PCR and hybridization to a tests trip presenting a parallel array of allele-specific oligonucleotide probes for each mutation. The allele frequencies of mutant MTHFR C677T [0.24] in our cohort were quite comparable with those reported by Golbahar et al. [2005] in Iran. Mutant MTHFR C677T in Iran occurred less frequently than among Europeans, but exceeded the much lower frequencies known from India and most of Asia. Here we describe the distribution of mutant allele MTHFR C677T in different ethnicities of Iranian population and compare the results to previously reported data. Our data represent the most comprehensive study to date with respect to thrombophilic gene polymorphism in Iran

Deletions in the survival motor neuron gene in Iranian patients with spinal muscular atrophy

<p><b>INTRODUCTION</b>Spinal muscular atrophy (SMA) is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The survival motor neuron (SMN) protein is encoded by 2 genes, SMN1 and SMN2. The most frequent mutation is the biallelic deletion of exon 7 of the SMN1 gene. In SMA, SMN2 cannot compensate for the loss of SMN1, due to the exclusion of exon 7. The aim of our study was to estimate the frequency of the common SMN1 exon 7 deletion in patients referred to our centre for carrier detection and prenatal diagnosis.</p><p><b>MATERIALS AND METHODS</b>We performed the detection of exon 7 deletion of the SMN1 gene for the affected patients and fetuses suspected to have SMA.</p><p><b>RESULTS</b>Of 243 families, 195 were classified as SMA type I, 30 as type II, and 18 as type III according to their family histories. The analysis of exon 7 deletion among living affected children showed that 94% of the patients with SMA type I, 95% with type II families and 100% with type III had homozygous deletions. Of the prenatal diagnoses, 21 (22.8%) of the 92 fetuses were found to be affected and these pregnancies were terminated.</p><p><b>CONCLUSIONS</b>The homozygosity frequency for the deletion of SMN1 exon 7 for all 3 types was (94%), similar to those of Western Europe, China, Japan and Kuwait.</p>

[Chromosomal abnormality and genetics factors in recurrent pregnancy loss]

Pregnancy loss is a common problem in reproductive-aged women. Although most cases of pregnancy loss are sporadic, some couples experience recurrent pregnancy loss. A variety of possible etiologies have been described for recurrent pregnancy loss. In most cases, the cause is not apparent and often requires intensive and expensive clinical and laboratory investigations, despite which there is still a limited understanding of it. This review focuses on the genetic abnormalities that may contribute to this clinical problem and delineates strategies for genetic evaluation and clinical management in subsequent pregnancies

[Genotype-phenotype correlations in an iraninan myotonic dystrophy cohort]

Myotonic Dystrophy type I [DM1] the most common form of muscular dystrophy in adults affecting 1/800 individuals is a dominantly inherited disorder with a multi-systemic pattern affecting skeletal muscle, heart, eye, endocrine and central nervous system. DM1 is associated with the expansion and instability of a trinucleotide [CTG] repeat in the 3 untranslated region of the myotonic dystrophy protein kinase [DMPK] gene located on choromosome 19q13.3. The normal copy number is 5-37 CTG repeat whereas it is expanded in DM1 patients and the expansion size broadly correlates with the severity of the symptoms. The aim of this study was to determine the clinical and genetic characteristic of DM1 in Iranian patients for genotype-phenotype correlation methods. Clinical assessment was based on the muscular disability rating scale [MDRS] and a sum of symptoms score [SSS]. Molecular analysis [PCR and Southern blot] was used to clarify uncertain clinical diagnoses and confirm the clinical findings. Forty six patients from twenty five DM1 families were reviewed. In all the DM1 patients, the wide clinical symptoms confirmed the reported phenotypic vaiability of disorder. The range of CTG expansion of the mutated allele was 97-833 CTG repeats and an inverse correlation between age of onest and repeat length was observed. A clear relation between the size of the CTG repeat and the clinical disease score [MDRS] was found but not with SSS. No correlation was seen between the endocrine dysfunction and the expansion size in DM1 patients