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]

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

[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

[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

[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>

[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

[ syndrome resemble to CODAS: the first reported Iranian girl]

CODAS is a syndrome consisting of cerebral, ocular, dental, auricular, and skeletal abnormalities. Patients with this syndrome have psychomotor delay, mental retardation, generalized hypotonia, cataract, ptosis, abnormally shaped teeth, malformed ears, deafness, grooved nasal tip, radiological findings of spondylo-epiphyseal dysplasia, with delayed skeletal maturation and coronal vertebral clefts and short stature. We report on an Iranian girl with features resembling CODAS syndrome. She presented with facial dysmorphism, cataract, abnormally shaped teeth, malformed ear, radiological findings of metaphysio-epiphyseal dysplasia and growth and developmental delay. The underlying defect responsible for CODAS syndrome remains unknown. Many of the features suggest a possible underlying collagen gene defect. The fact that this child is the product of consanguineous marriage suggests the possibility of autosomal recessive inheritance

[CTG expansion and haplotype analysis of DM1 gene in healthy Iranian population]

Myotonic dystrophy type 1 [DM1] is due to an unstable expansion of CTG repeat in the DMPK gene [19q13.3]. The CTG repeat is highly polymorphic [5 to 37] in healthy individuals. According to the hypothesis that expanded [CTG]n alleles originated from larger normal alleles, there may exist a correlation between the prevalence of DM1 and the frequency of large size normal alleles. Strong linkage disequilibrium between different length alleles and the three biallelic markers, Alu, Hinf1 and Taq1, has been reported. Therefore, we decided to determine the distribution of normal alleles, the frequency of larger normal alleles and analysis of the three biallelic markers, in healthy Iranian controls. Polymerase chain reaction was conducted on two hundred unrelated healthy individuals from different ethnic groups living in Iran to determine the size of the alleles. Markers were analyzed by PCR/RFLP on 174 chromosomes from other control healthy individuals. Our data reveals that 23.7% of alleles had 5 CTG repeats and 7.2% of alleles had >18 CTG repeats. The analysis of haplotypes revealed that 75% of CTG5 and 80% of CTG>18 had the [+++] haplotype. In this study, we find that the frequency of alleles with CTG>18 is similar to that of Western Europe and Japan

[Survival motor neuron gene deletions among Iranian patients with spinal muscular atrophy]

Spinal muscular atrophy [SMA] is a common neuromuscular disorder with progressive paralysis caused by the loss of alpha-motor neuron in the spinal cord. SMN is encoded by two genes, SMN1 and SMN2, which essentially differ by a single nucleotide in exon 7. The most frequent mutation is biallelic deletion of exon 7 of the SMN1 gene. A small percentage of SMA patients present compound heterozygosity with a point mutation on one allele and deletion on the other. In the remaining cases, the disease is unlikely to be related to SMN1 defects. In spinal muscular atrophy [SMA], SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. The aim of our study was to estimate the frequency of the common exon 7 SMN1 deletion in the families who referred to our center for carrier detection and prenatal diagnosis. Between March 1999 and March 2006, one hundred sixty seven families with history of at least one affected member were referred to us. We performed detection of deletion exon 7 SMN1 for the patients and carrier detection for their parents, prenatal diagnosis in subsequent pregnancies to couples who previously had an affected child became possible [63 prenatal diagnosis]. From 167 families, 139 categorized in type I of the disease, 21 in type II, and 7 in type III. Carrier detection for the parents indicated that in 96 families with history of affected member with type I SMA both parents carried the deletion in exon 7 and in 20 families, one of the parents was carrier. These rates were 16 to 1 for SMA type II, and 3 to 2 for type III SMA. Sixty-four children affected with SMA were studied, 58 of them were found to be homozygous for the loss of exon 7 of the SMN1 gene, except two patients who were heterozygote for exon 7 deletion [frequency of homozygocity: 90.7%]. Eleven of sixty-three [17.5%] fetal samples were found to be affected and these pregnancies were terminated. The molecular analysis of the biallelic exon 7 of the SMN1 deletion is a standard and reliable test in cases of SMA

[Introduction to diagnosis of neuromuscular disorders]

Neuromuscular disorders are the most common progressive group of heterogenous disorders. There are considerable genetic heterogeneity in these group of disorders which show involvement of over hundreds genes in neuromuscular disorders. The major symptoms of neuromuscular disorders are generalized muscle weakness, extra ocular ophthalmoplagia, muscle wasting, respiratory and cardiac involvement and exercise intolerance. Since 1950 molecular diagnosis of neuromuscular disorders have been considerably developed and so with clinical evaluation of neuromuscular patients we can set up molecular and immunohisotchemical techniques. Classification of these disorders are as follow: 1- Myopathies muscular dystrophies, 2- Neuromuscular junction congenital myasthenia syndromes, 3- Neuropathies like Charcot- Marie -Tooth, Spinal Muscular Atrophy and 4- Motor neuron Disorders

Relative frequency of GJB2 gene mutations in autosomal recessive non-syndromic hearing loss [ARNSHL] patients in lurastan population

Congenital hearing loss with many genetic and environmental causes affecting 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 study was to study the frequency of GJB2 mutations in Lurastan non-syndromic deaf population using ARMS/PCR, DHPLC and direct sequencing. For this purpose, 106 chromosomes from 53 patients were studied. Eighteen chromosomes [17%] carry GJB2 mutations including 35delG, 314dell4, 512insAACG, -3170G>A, W24X, V95M and 510insCGAA. The last mutation is a novel GJB2 mutation, 35delG mutation was diagnosed in 10 chromosomes [9/4%]; 4 patients were homozygote and 2 patients were heterozygote. Also polymorphism VI531 were found in 3 families. This frequency is significantly higher compared to that of the whole population of Iran

[ novel mutation of Ach receptor in an Iranian family with Congenital Myasthenic Syndrome]

Congenital Myasthenic Syndromes [CMS] are characterized by fatigable weakness involving ocular, bulbar, and limb muscle with onset at or shortly after birth. CMS is a group disorders which arise from inherited structural or functional abnormalities of the neuromuscular junction. Diagnosis of CMS is based on clinical and electrophysiological findings. Several genes encoding different proteins express at the neuromuscular junction are currently known to be associated with CMS, these include the gene encoding different subunits of the acetylcholine receptor [CHRNE, EAchR-subunit; CHRNAl: aAchR-subunit; CHRNB1: 13 AchR-subunit; CHRND: oAchR-subunit] the gene encoding the collagenic tail subunit of the acethylcholinesterase [COLQ]; the gene of the choline acetyltransferase [CHAT]; and the gene encoding rapsyn [RAPSN]. A 17-Year-old presented with fluctuating ocular and generalized muscle weakness, ptosis and dysphagia which has begun a few month after birth. At the beginning she had feeding difficulty and recurrent apnea. On examination she has ptosis, external ophthalmoplagia and limb muscle weakness. Her parents are first cousin. Laboratory investigation showed normal creatine phosphokinase [CPK]. EMG revealed nonspecific neuropathic changes. Molecular investigation performing by a collaborative center in Paris has shown a EAchR subunit mutation in the AchR gene. It is the first report of mutation in the Ach receptor gene in an Iranian family. Although rapsyn mutation is a common mutation in Iranian-Jewish population

[Screening of GJB2 Mutations in autosomal recessive non-syndromic kashan deaf patients]

Hearing loss is the most common sensory neural defect in humans, affecting 1 of 1000/2000 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, encoding Connexin26, are major cause of inherited deafness in the most populations. We studied 34 probands from 34 families with autosomal recessive nonsyndromic hearing loss [ARNSHL] in Kashan populasions. Mutations Screening of GJB2 was performed by Amplification Refractory Mutation System [ARMS]-PCR for detection of 35delG and then we analyzed all samples excluding 35delG homozygote by DHPLC and Direct Sequencing.We identified 2 mutations [35delG 2.9%, 312de114 2.9%] and 2 polymorphisms V1531 in this study. Regards to our data,GJB2 mutations are very low in comparison with other part of world and finally, further studies need to find other genes that have a causal role in NSHL in this population

[Hb Dhonburi [Neapolis] [beta126[H4] Val-Gly] identified in a family from northern Iran]

Thalassemias are the most common hereditary disease in Iran, resulting from defects in synthesis of one or more hemoglobin [Hb] subunits. The majority of patients suffer from beta-thalassemia [thal], but cases with microcytic hypochromic anemia and normal electrophoretic patterns are suspected to have alpha or silent beta-thalassemia. A family from the northern part of Iran, an area where thalassemias, are highly prevalent was referred to us for prenatal diagnosis. The hematological data of the father indicated a pattern of beta-thal minor. Reverse hybridization analysis for the most common beta-globin mutations identified IVS-II-1 [G-A] in the heterozygous state. The maternal laboratory data indicated a case more compatible with alpha-thal. Iron deficient anemia was ruled out, and common alpha-thal point mutations and deletions were investigated. As no mutation was detected, globin chain synthesis was performed and showed an alpha/beta chain ratio of 2.1, that was in the range of beta-thal minor. DNA sequencing of the entire beta-globin gene identified a heterozygous GTG-GGG [Val-Gly] mutation at codon 126, also known as Hb Dhonburi [Neapolis]. Prenatal diagnosis of the fetal DNA showed the absence of the IVS-II-1 and codon 126 anomalies. This result demonstrates the importance of screening of individuals with mild microcytic hypochromic anemia for both alpha- and silent beta-thal mutations