Investigation of genetic causes of intellectual disability in Kerman province, south east of Iran

Intellectual disability [ID] has a worldwide prevalence of 1-3% and results from extraordinary heterogeneous. To shed more light on the causes of ID in Kerman Province, in Southeast Iran, we set out in 2008 to perform systematic clinical studies and homozygosity mapping in large Iranian families with ID. Fifty seven families with a minimum of two mentally retarded children from Kerman Province were initially tested for metabolic disorders, by Tandem mass spectrometry. Fragile X testing and standard karyotyping were performed for all probands of families. Cases with autosomal recessive [AR] pattern of inheritance and microcephaly were subjected to homozygosity mapping by using several microsatellite markers for known MCPH loci. Three out of seven families with X-linked pattern of inheritance were positive for fragile X syndrome. Chromosome abnormality was not observed in any of dysmorphic patients and all families were negative for metabolic tests. Among the remaining 50 families of AR ID, six were found to be microcephalic, of which 2 linked to two MCPH loci [33.3%]. The rest 4 families were not linked to any of the known loci. The results of this study showed that ID with microcephaly comprised 12% of ID cases in Kerman Province. In two families with apparent linkage to the MCPH5 and MCPH6 locus, mutation screening was not successful, which might indicate that either the mutation is located in the regulatory sequences of the gene or that there might be another genes present in these regions, which is mutated in such cases

Clinical and molecular aspects of Sjogren-Larsson syndrome reported in an iranian consanguineous family with triplet affected individuals

Sjogren Larsson Syndrome [SLS; OMIM: 270200] is an autosomal recessive neurocutaneous disorder characterized by mental retardation, congenital ichthyosis and spastic paraplegia. SLS is caused by mutations in aldehyde dehydrogenase 3A2 isoform 2 [ALDH3A2], which encodes fatty aldehyde dehydrogenase [FALDH]. This enzyme metabolizes the NAD-dependent oxidation of long chain aldehyde derived from lipid metabolism. Up to now, more than 72 mutations have been reported in SLS patients. DNA was extracted from peripheral blood of all the five patients, one healthy sibling and their parents using standard procedures. SNP genotyping was performed using the GeneChip [registered sign]. Multipoint linkage analyses and non-parametric linkage analysis was performed too. Results: Here, we report an interesting family with five affected individuals with a novel splice site mutation [c.1107+1delGTA] in ALDH3A2. In absence of capability to measure FALDH activity in Iran, DNA sequencing of the ALDH3A2 gene could lead to the identification of causative mutation and confirm the diagnosis

[Screening of DFNB4 locus in Iranian families with hereditary hearing impairment]

Mutations in the SLC26A4 gene in the DFNB4 locus is responsible for syndromic [Pendred syndrome] and non-syndromic hereditary hearing loss [HHL]. In many populations, mutations in this gene have been reported as a second cause of HHL. The objective of this study was to investigate the prevalence of SLC26A4 mutations in our HHL consanguineous families. After completing clinical evaluation and obtaining signed consent forms from each family, we included 80 families with two or more affected individuals, referred to the Genetics Research Center [GRC]. All families that previously tested negative for the DFNB1 locus were candidates for homozygosity mapping using STRs for DFNB4 locus. Families localized to this region were subjected to complete DMA sequencing. Twelve out of 80 families were mapped to DFNB4. Sequence analysis of 12 linked families revealed 10 mutations in 8 families. [T420I, 1197delT, G334V, R409H, T721M, R79X, S448L, L597S, 965insA, and L445W]. The T420I, G334V, L597S and R79X were novel mutations; we did not find any mutation in the four linked families, nor did we detect any nonsyndromic families with mutation in the SLC26A4 gene. We have been able to identify mutation in the SLC26A4 gene in only 8 of 80 families. In 12 families, we detected some degree of diffuse or nodular goiter; three out of 12 families showed thyroid function impairment and in five of 12 families there were positive prechlorate discharge tests. Eight families that showed mutation had normal temporal bone scan. This investigation, demonstrated that the SLC26A4 gene mutation is the most prevalent syndromic hereditary hearing loss in Iran, a finding in accordance with reports from other countries

Evaluating the effect of DMSO concentrations on y-globin expression in Hull cells

Evaluating the effect of DMSO concentrations on g-globin expression in Hu11 cells Asgharian AM1, Banan M2, Deilami Z1, Gharesouran J3, Ghasemi S4, Behjati F2, Javadi GR5, Kahrizi K6, Najmabadi H7 1 PhD Student of cell and molecular biology, Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran. 2 Assistant professor, Genetics Research Center, University of Social Welfare and Rehabilitation Sciences. Tehran, Iran. 3 Student of MSc of Genetics, Genetics Research Center, University of Social Welfare and Rehabilitation Sciences. Tehran, Iran. 4 MSc of Genetics,Genetics Research Center, University of Social Welfare and Rehabilitation Sciences. Tehran, Iran. 5 Associate Professor, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 6 Associate Professor, Genetics Research Center, University of Social Welfare and Rehabilitation Sciences. Tehran, Iran. 7 Professor, Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran Abstract Background: Understanding of the mechanisms involved in gamma- to beta-globin switching may be important for development of treatment options for b-thalassemia. Such studies require the availability of relevant cellular model systems. One such cell type is Hu11, a mouse erythroleukemia [MEL] cell line containing the human b-locus. MEL and Hu11 cells differentiate in the presence of the chemical dimethyl sulfoxide [DMSO]. Nevertheless, levels of gamma-globin induction in Hu11 cells after DMSO treatment have not been determined. In the present study, we determined gamma-globin levels in Hu11 cells after treatment with various DMSO concentrations. Materials and methods: Hu11 cells were cultivated in various DMSO concentrations and the levels of gamma-globin were determined by real-time PCR. Results: Our study showed that hemoglobin in Hu11 cells treated with 1% and 2% DMSO was increased by approximately 5 and 10 folds. Moreover real-time PCR results showed that g-globin levels using the indicated DMSO levels were increased by 66 and 298 folds, respectively. Hu11 cells differentiate in the presence of DMSO, and in doing so, their gamma-globin levels are increased. Therefore these cells can be used to study the mechanisms of gamma-globin induction. Such studies may be beneficial for the treatment of beta-thalassemia

Use of immunoglobulin heavy chain and kappa light chain gene rearrangements by PCR for molecular diagnosis of minimal residual disease in Iranian children suffering from beta-precursor acute lymphoblastic leukemia

Diversity of IgH and IgK molecules is generated during B and T Lymphocyte differentiation through the rearrangement of variable, diversity, junction and constant gene segments. Additionally, random insertion and deletions of nucleotides between gene segments make unique sequences which are cell or clone specific. Similar IgH and IgK genes rearranged in normal cells of lymphoid leukemia cases can be used as a marker of clonality and for evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of IgH chain and IgK gene rearrangements using polymerase chain reaction [PCR] in beta-precursor acute lymphoblastic leukemias [ALL] to follow the MRD at day 14, day 28 [end of remission induction], week 10, 3-6 months and 6-12. month after the initiation of treatment. In our prospective study bone marrow aspirates of 183 children at the mean age of 63.6 months with diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 cases with diagnosis of beta-precursor ALLs were selected for study. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, IgH and IgK [V[K] I-IV / Kde] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned with the sequences homologous for IgH and IgK published by Gene Bank. The follow up specimens were collected at day 14, day 28 [end of remission induction], day 45-month 3, and 3-6 months and 6-12 months after initiation of treatment. After routine cytomorphologic analysis, similar PCR was done on follow up extracted DNAs in parallel with diagnosis DNA. MRD was considered to be approved positive if bands similar to those at the time of diagnosis were present. Statistical analysis using SPSS software [version 11.5] was performed. 90.5% of patients had clonal IgH gene rearrangements. Monoclonal, biclonal and oligoclonal patterns were observed in 57.8%, 34.9% and 5.5% of patients with IgH [CDR III] rearrangement, respectively. Clonal patterns of IgK-Kde were detected in 59 [67%; n: 88] of BP-ALLs. According to cytomorphology about 92% of patients were in complete remission. MRD positivity decreased from more than 90% to 20% using different gene rearrangements in defined time points. Four patients who relapsed during follow up were MRD positive using 1-3 rearrangements and all except one were in clinical remission. Clonal rearrangement of IgH had a pattern similar to other populations. IgK was slightly more frequent than previously reported and the VKI [25%] was the most common type. These differences can be explained by different techniques, DNAs and clonality markers. According to the results, these clonal markers can be used in diagnosis and follow up of MRD

pattern of cross lineage T-cell receptor delta/gamma gene rearrangements in B-precursor acute lympho-blastic leukemia of Iranian children using polymerase chain reaction

Diversity in heavy chain immunoglobulin [IgH] and T-cell receptor [TCR] molecules occures during B- and T-lymphocyte differentiation through the rearrangement of variable [V], diversity [D], junction [J] and constant [C] gene segments. Lymphoid leukemia cells are similar to normal precursors and have rearranged IgH, IgK and TCR [cross-lineage rearrangement] genes which can be used as a marker of clonality and evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of TCR- delta/gamma gene rearrangements using Polymerase Chain Reaction [PCR] in B-precursor acute lymphoblastic leukemia [ALL] in Iranian children. In our prospective study, bone marrow aspirates of 183 children with early diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 subjects with diagnosis of B-precursor ALLs were selected for study. Sixteen were excluded from our study due to various reasons including cellular degeneration. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, hyper-variable regions TCR-delta [V delta2-D delta3 and D delta2-D delta3] and TCR-gamma [V gamma; V gamma I and V gamma II] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were then compared and aligned to the homologous sequences of Gene Bank for confirmation. T-test, Mann whitney, Fisher exact test and Chi-square were used for data analysis. Clonal rearrangement of TCR-gamma [V gamma] and V gamma l/Il were present in 79.3% and 64.9% of patients respectively and only 5% of cases showed biclonal pattern. The V gamma ll rearrangement was the most common [46.8%] type in TCR-gamma. 47 [45.2%] and 11 [16.6%] of patients had V delta2- D delta3 and D delta2-D delta3 partial gene rearrangements, respectively. Biclonal/oligoclonal patterns were present respectively in 27.7% and 4.3% of cases with Vdelta2-D delta3 rearrangement. Only one patient had biclonal D delta2 D delta3 rearrangement. Clonal rearrangement of TCR-delta [Vdelta2-Ddelta3 and D delta2-D delta3] genes had a pattern similar to other populations. Frequency of TCR- gamma [V gamma I and V gamma II] rearrangements was slightly higher than previous reports, and in contrary to others except for Brazilian report the V gamma II rearrangement was the most common type. We found no significant correlation between presence of different types of rearrangements and quantitative variables. The only significant point was the reduction of Vdelta2Ddelta3 with increase in age. According to preliminary results, these clonal markers can be used in diagnosis and follow up of MRD

[Molecular diagnosis of clonality in B-precursor acute lymphoblastic leukemia of children using immunoglobulin heavy and kappa light chain gene rearrangements]

Enormous diversities of heavy chain immunoglobulin [IgH] and IgK molecules are generated during B- and T-lymphocyte differentiation through the rearrangement of gene segments. Additionally, random insertion and deletion of nucleotides between gene segments make unique sequences which are cell or clone specific. IgH and IgK gene rearrangements are the most and relatively common reported rearrangements in B-precursor acute lymphoblastic leukemia, respectively. The purpose of this study is to evaluate the pattern of IgH and IgK gene rearrangements using polymerase chain reaction [PCR] in BP-ALL in Iranian children. For this prospective study, bone marrow aspirates of 183 patients with the diagnosis of acute leukemia were collected at admission before any chemotherapy. Having reviewed cytomorphology [L[1]:44%, L[2]:41%] and immunophenotyping, only 140 cases with the diagnosis of B-precursor ALL were selected. Mononuclear cells including leukemic blasts were isolated by density gradient. Having DNA extracted, hyper-variable regions of IgH and IgK were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned to the sequences homologous for IgH and IgK published by Gene Bank. IgH gene rearrangements were found in 114 [90.4%] of patients using consensus primers for CDR-III and CDR-I regions [monoclonal 57.8% biclonal 34.9% oligoclonal 5.5%]. Four of nine patients with T-ALL had clonal rearrangements of IgH. Clonal pattern of Ig?-Kde were present in 59 [67%] of cases [biclonal 10%]. VKI [25%] and VK? [22.7%] were the most common type of rearrangements. Clonal rearrangement pattern of IgH gene was similar to other populations. Using FRI and FRIII primers in multiplex PCR increased the rate of detection and reducing turnaround time. IgK was slightly more frequent than previous reports while VKI [25%] was the most common type

[Spectrum of GJB2 gene mutations in nonsyndromic autosomal recessive deaf patients in Yazd]

Hearing loss is the most common sensory neural defect in humans, affecting 1 in 1000 neonates, with over half of these cases predicted to be hereditary in nature. Most hereditary hearing loss is inherited in a recessive fashion, accounting for approximately 80% of non-syndromic hearing loss [NSHL]. Mutations in GJB2 gene are major cause of inherited deafness in the European and American populations. To date, more than 90 mutations have been reported in this gene. Although most of these mutations are rare but 35delG mutation is the most common deafness causing allelic variant of GJB2 in most parts of t he world. In this project, 120 probands from 120 families with ARNSHL in Yazd Province were studied. Mutations Screening of GJB2 was performed by Amplification Refractory Mutation System [ARMS]-PCR for detection of 35delG and then all samples excluding 35delG homozygote were analyzed by DHPLC and Direct Sequencing. GJB2-related deafness was present in 7.5% of this population. We identified 4 mutations [35delG, 312del14, 314del14 and 167delT] and 4 polymorphisms [V153I, V27I, E114G and R127H] in this study. Prevalence of GJB2 mutations in this population was lower than American and European populations, and also other provinces of Iran. Interestingly, 312del14 rather than 35delG was the most common mutation found in the population under study. 56.25% of GJB2 mutant alleles carried 312del14 mutation. To date, this frequency has not been reported in any of the world populations

novel mutation of SLC26A4 gene in an Iranian family with pendred syndrome

In the diagnosis of Pendred syndrome, assessment of individuals by molecular analysis of the SLC26A4 gene is recommended. Here we report a novel mutation in the SLC26A4 gene as revealed by denaturing high performance liquid chromatography [DHPLC] and DNA sequencing of the entire coding region of the SLC26A4 gene in five members of an Iranian family affected with Pendred syndrome. This is the first report of the molecular investigation of Pendred syndrome in Iran and the first report of the R79X mutation

Expression of recombinant human serum albumin in the methylotrophic yeast; Hansenula polymorpha

Human serum albumin [HAS] is the major protein component of human plasma. It plays a very important role in transporting of macro molecules and maintaining the normal osmolarity. It is used as a therapeutical protein in patients with hypoalbuminemia and acute bleeding and burning. Albumin consumption in the world is about 500 ton/year. The aim of this research is to study the production of rHSA in shake flask culture by Hansenula polymorpha. H. polymorpha was used for the production of recombinant human serum albumin [rHSA] in several of shake flask culturing; expression of rHSA was investigated relating several parameters affecting the expression of HSA. To optimize the secretory expression of rHSA under the control of FMD promoter in H polymorpha RB-11 incubation time, culture media temperature and protease inhibitors were analyzed. This study not only established production of rHSA in yeast but also analyzed the correlation between affecting parameters and the level of HSA expression. Comparison of the HSA levels in the culture supernatants showed that the highest HSA yield was 17.6mg/l. The research shows that among three different temperatures [25°C,30°C and 37°C] 37°C was the best temperature and amongst three different incubation times [24h,48h and 72h] 48h was the optimum time and YNB 1% glycerol with buffer was the best derepression medium in comparison with others. Using these optimized conditions, stable production of rHSA of around 17.6mg/l was achieved. Our results suggest that affecting experssion factors improved in this study are suitable for production of recombinant albumin

[ investigation of Duchenne muscular dystrophy deletions in Iranian families and prenatal diagnosis]

Duchenne muscular dystrophy [DMD] is one of the most common X-linked genetic disorders, commonly seen in children. It is caused by mutations in Dystrophin gene and clinically manifests with severe muscle weakness and eventually leads to death in the second or third decades of life. In the absence of an appropriate cure, prenatal diagnosis [PND] appears to be the best approach to reduce the burden of the disease on the individual family and ultimately on the society. During the last five years, prenatal diagnosis was offered on request to 85 families, having one or more affected male children. Initially, the deletions in the DMD gene were identified by Multiplex PCR, screening for 20 exons. Then, three intragenic RFLPs [PERT 87-15lBamH1, PERT87-8ITaq1, PERT 87- 151XmnI] and two main CA repeats [5'- DYS and 3' DYS microsatellite analysis] that have-been shown to be highly heterozygous in the previous studies, were used to perform carrier detection and linkage analysis. Deletions were found in 43 affected boys [50.6%]. Most of the deletions were found in exons 49 and 50. However, there were not any mutations identified in the promoter region. In 42 families these three intragenic RFLPs were utilized and in 26 of them one or more RFLPs were informative [61.9%]. In 30 families two microsatellite repeat analysis were done to identify the mutant alleles and in 12 families, 5'-Dys was informative. Prenatal diagnosis was performed in 25 families [16 CVS cases and 9 amnion cases]. The cases were 14 male fetuses [5 cases affected, 9 cases not affected] and 11 female fetuses [4 cases were carriers and the remaining cases were normal]. Therefore, it is concluded that multiplex PCR technology and linkage analysis, can be used effectively for PND and carrier detection in Iran

[Relative frequency of 35delG mutation in GJB2 gene in autosomal recessive non-syndromic hearing loss [ARNSHL] patients in Kerman population]

Congenital hearing loss with many genetic and environmental causes affects 1 in 1000 newborns. Mutations in the GJB2 [Gap Junction Beta-2] gene encoding the gap junction protein connexin 26 have been established as the main cause of autosomal recessive non-syndromic hearing loss. The aim of this stand was to study the frequency of one mutation [35delG] of GJB2 gene in Kerman non-syndromic deaf population. For this purpose, 130 chromosomes from 65 patients were studied and 35delG mutation was diagnosed in 3 [2.3%] chromosomes [one patient was homozygote and the other one was heterozygote]. This rate of frequency is significantly higher comparing to that in the whole population of Iran