Mohammad Hamid

A novel 355–357delGAG mutation and frequency of connexin-26 (GJB2) mutations in Iranian patients

The common form of autosomal recessive non-syndromic deafness is caused by the mutation in gap junction beta 2 (GJB2) gene (GenBank M86849, OMIM# 121011) which is located at the DFNB1 locus at 13q11. GJB2 is a small gene about 5500-bp length with two exons, of which only one contains the coding region (Kelley et al. 2000). The sequence of the coding region consists of 681 bp, encoding a gap-junction protein with 226 amino acids (Schrijver 2004). The genetics of hearing loss is highly heterogeneous and more than 100 mutations in connexin 26 (GJB2) genes are reported to be responsible for 30%–40% of hereditary hearing loss in deaf subjects (Ballana et al. 2001; Schrijver 2004). The most frequent mutation 35delG has been detected in different populations; especially in European countries where it is established to be due to founder effect (Van Laer et al. 2001; Rothrock et al. 2003). In this study, we performed mutation screening in 33 families who met clinical criteria of non-syndromic hereditary hearing loss (NSHHL) to evaluate the type and frequency of GJB2 mutations in Iranian population.

Molecular characterization of β-thalassemia intermedia: a report from Iran

Thalassemia intermedia is a clinical definition applied to patients whose clinical phenotype is milder than thalassemia major. To characterize different common mechanisms involving in pathogenesis of moderate to severe β-thalassemia intermedia, we have studied four factors in 38 Iranian patients with thalassemia intermedia: β-globin gene mutation, deletion in α-globin genes, presence of XmnI polymprphism and RFLP haplotype at β-globin gene cluster. The results showed that 84.4% of patients were associated with severe mutations in β-globin gene, mainly IVSII-1(G to A) (56.4%). The positive XmnI polymorphism was seen in 76.9% of the studied alleles which showed strong linkage to β° mutations and high level of fetal hemoglobin. Co-existence of α-globin gene deletions, β+ mutation and the most frequent of RFLP haplotype (−/−, +/+, −/+, +/+, +/+, +/+, −/−) were seen in 7.7, 12.8 and 17.9%, respectively. In this group of our study it seems the main ameliorating factor in the patients was co-inheritance of a positive XmnI polymorphism with β° mutation especially IVSII-1, which were associated with increased production of fetal hemoglobin. However, the other probable genetic factors should be investigated to describe genotype-phenotype correlation in thalassemia intermedia patients.

Molecular Analysis of γ-Globin Promoters, HS-111 and 3′HS1, in β-thalassemia Intermedia Patients Associated with High Levels of Hb F

The nucleotide (nt) variations in the promoter region of the γ-globin genes, HS-111 and 3′HS1 regions, were studied in Iranian patients with β-thalassemia intermedia (β-TI), β-thalassemia major (β-TM) and healthy individuals. Of the five nt variations at the 5′ end of the Aγ-globin gene, −369 (C>G), −611 (−T) and −603/604 (GA>AG) were found in all samples, whereas −588 (A>G) and −AAGC at −222 to −225 were found at different frequencies in the studied groups. Therefore, the −369, −611 and −603/604 variations were considered common mutations in this population, and the difference with respect to the −AAGC deletion was not significant. However, the A allele of the −588 variation and [+] allele of the XmnI polymorphism were more frequent in β-TI patients, especially those who had the IVS-II-1(G>A)/IVS-II-1(G>A) genotype. The + allele of XmnI also had complete correlation with the A allele of −588 variation. The HS-111 (−21 A) variation also showed association with β-TI patients who had high levels of Hb F. Bearing in mind that the −588 variation lies within the postulated adult-specific silencer region and that the majority of β-TI patients had allele A, then it can be envisaged that this allele could have a role in altering the repressor function at this region. Therefore, the A allele of −588, [+] allele of XmnI and HS-111 (−21 A) variation are useful genetic markers to differentiate between β-TM and β-TI patients. However, these nt changes alone may not be the only elements raising the level of Hb F, other regulatory and modifying factors also play a role in Hb F production.

Multigene Next-Generation Sequencing Panel Identifies Pathogenic Variants in Patients with Unknown Subtype of Epidermolysis Bullosa: Subclassification with Prognostic Implications

Please cite this article as: Vahidnezhad H, Youssefian L, Saeidian AH, Touati A, Sotoudeh S, Abiri M, Barzgar M, Aghazadeh N, Mahmoudi H, Norouz-zadeh S, Hamid M, Zahabiyon M, Bagherian H, Zeinali S, Fortina P, Uitto J, Multigene Next Generation Sequencing Panel Identifies Pathogenic Variants in Patients with Unknown Subtype of Epidermolysis Bullosa: Subclassification with Prognostic Implications, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.07.830.

A 13-bp deletion in the 3' untranslated region of the β-globin gene causes β-thalassemia major in compound heterozygosity with IVSII-1 mutation.

Objective: To describe hematological and molecular features of a 13-bp deletion in the 3′ untranslated region(3′ UTR) of the β-globin gene in carrier individuals and a compound heterozygous patient. Subjects and Methods: Five members of an Iranian family of Persian ethnic origin were studied. Red blood cell indices and hemoglobin analysis were carried out according to standard methods. Genomic DNA was obtained from peripheral blood cells by salting-out procedures. β-Globin gene amplification and DNA sequencing were performed. Results: One patient had a 13-bp deletion in the 3′ UTR of the β-globin gene that causes the β-thalassemia phenotype in combination with the IVSII-1 (G→A) mutation. The patient had inherited the IVSII-1 (G→A) mutation from his mother, while the second β-globin gene (inherited paternally) had a 13-bp deletion at nucleotide 90 downstream of the termination codon (CD +90 del 13 bp).The patient’s father and paternal grandmother, who are carriers of this deletion, had no hematological abnormalities. Conclusion: This case showed a patient with a 13-bp deletion in the 3′ UTR of β-globin gene that could cause a slight decrease in the stability of the mRNA, but did not have a hematological effect in the heterozygotes. The 13-bp deletion could be clinically important only in situations where β-chain synthesis in trans is compromised.

Genome-wide single nucleotide polymorphism-based autozygosity mapping facilitates identification of mutations in consanguineous families with epidermolysis bullosa

Autozygosity mapping (AM) is a technique utilised for mapping homozygous autosomal recessive (AR) traits and facilitation of genetic diagnosis. We investigated the utility of AM for the molecular diagnosis of heterogeneous AR disorders, using epidermolysis bullosa (EB) as a paradigm. We applied this technique to a cohort of 46 distinct EB families using both short tandem repeat (STR) and genome‐wide single nucleotide polymorphism (SNP) array‐based AM to guide targeted Sanger sequencing of EB candidate genes. Initially, 39 of the 46 cases were diagnosed with homozygous mutations using this method. Independently, 26 cases, including the seven initially unresolved cases, were analysed with an EB‐targeted next‐generation sequencing (NGS) panel. NGS identified mutations in five additional cases, initially undiagnosed due to the presence of compound heterozygosity, deep intronic mutations or runs of homozygosity below the set threshold of 2 Mb, for a total yield of 44 of 46 cases (95.7%) diagnosed genetically.

Exon sequencing of PKD1 gene in an Iranian patient with autosomal-dominant polycystic kidney disease.

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders with the incidence of 1 in 1,000 births. ADPKD is genetically heterogeneous with two genes identified: PKD1 (16p13.3, 46 exons) and PKD2 (4q21, 15 exons). Eighty five percent of the patients with ADPKD have at least one mutation in the PKD1 gene. Genetic studies have demonstrated an important allelic variability among patients, but very few data are known about the genetic variation among Iranian populations. Methods: In this study, exon direct sequencing of PKD1 was performed in a seven-year old boy with ADPKD and in his parents. The patient’s father was ADPKD who was affected without any kidney dysfunction, and the patient’s mother was congenitally missing one kidney. Results: Molecular genetic testing found a mutation in all three members of this family. It was a missense mutation GTG>ATG at position 3057 in exon 25 of PKD1. On the other hand, two novel missense mutations were reported just in the 7-year-old boy: ACA>GCA found in exon 15 at codon 2241 and CAC>AAC found in exon 38 at codon 3710. For checking the pathogenicity of these mutations, exons 15, 25, and 38 of 50 unrelated normal cases were sequenced. Conclusion: our findings suggested that GTG>ATG is a polymorphism with high frequency (60%) as well as ACA>GCA and CAC>AAC are polymorphisms with frequencies of 14% and 22%, respectively in the population of Southwest Iran.

Clinical Features, DYT1 Mutation Screening and Genotype-Phenotype Correlation in Patients with Dystonia from Iran

Objective: To test Iranian patients with primary torsion dystonia to determine the frequency of 904–906 del GAG mutation in the DYT1 (TOR1A) gene and to investigate the genotype-phenotype association for this disease. Subjects and Methods: Sixty-three patients with primary dystonia were investigated. DNA was extracted from peripheral blood and these samples were subjected to PCR-sequencing for exon 5 of the DYT1 gene. Results: Of the 63 patients, 10 (15.9%) carried the triplet GAG deletion mutation; this is a high DYT1-positive rate in comparison with other populations and the type of dystonia in this positive group was generalized in all except 1. In our patients, limbs were the most severely involved site at the time of onset and in most cases it developed to generalized form. The majority of DYT1-positive cases showed higher leg onset (5 patients, 62.5%) in comparison with higher arm onset in negative patients (20 patients, 50%). Also, the progression to generalized dystonia in DYT1-positive patients was significantly higher than in DYT1-negative patients. The mean age at onset was 8.6 ± 1.6 years (7–12 years) in DYT1-positive patients, while mean age at onset in patients with no GAG deletion mutation was higher (15.7 ± 11.5 years). Conclusions: The DYT1 904–906 del GAG mutation is responsible for some of Iranian dystonia patients, and screening for the DYT1 deletion is significant in cases with the generalized type of primary dystonia. Also, patients with leg or arm onset at a younger age are more likely to be DYT1-positive among primary torsion dystonia cases.

Determination of exon 7 SMN1 deletion in Iranian patients and heterozygous carriers by quantitative real-time PCR.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by the degeneration of motor neurons of the spinal cord anterior horns, leading to progressive atrophy of proximal muscles, paralysis, respiratory failure, and even infant death. Patients with SMA are classified into three types, based on the age at onset and clinical severity (Munsat and Davies 1992; Wirth et al. 1995): type I (MIM 253300) is the most severe form, type II (MIM 253550) is the intermediate form, and type III (MIM 253400) is the mildest form. With an incidence of 1/6000 to 1/10000 and a carrier frequency of 1/40 to 1/50, SMA is the second most frequent autosomal recessive disease in Europeans (Pearn 1980). The SMA determining gene called the ‘survival motor neuron’ gene (SMN) is present on 5q13 in two copies, SMN1 and SMN2, which differ by only five nucleotide exchanges within their 3′ ends (Lefebvre et al. 1995). Two of these base-pair exchanges, located in exons 7 and 8 (which allow SMN1 to be distinguished from SMN2) currently are used for direct diagnosis of SMA. Therefore, the first step in this test is to amplify specifically SMN1 exon 7 or exon 8 and later on subjecting the PCR product to restriction digestion, a PCR-RFLP approach (Scheffer et al. 2001). In a study of 1122 patients with type I, type II, or type III SMA it was shown that in about 94% of SMA patients (in all three types) homozygous absence of SMN1 is responsible for this disease (Scheffer et al. 2000). Approximately half of the remaining patients were identified as compound heterozygote (with deletions and intragenic SMN1mutations), whereas the other half, without SMN1 mutations, were considered as distinct genetic entities (Wirth et al. 1999). The carrier test for

Impact of the BCR-ABL1 fusion transcripts on different responses to Imatinib and disease recurrence in Iranian patients with Chronic Myeloid Leukemia

BACKGROUND One genomic breakpoint can result in variable BCR-ABL1 transcript types due to alternative splicing. The influence of different BCR-ABL1 transcript types on clinical outcome is still controversial. AIM OF THE STUDY The objective of this analysis was to determine the impact of transcript type on response, clinical outcome, recurrence risk after treatment with Imatinib mesylate in Chronic Myeloid Leukemia (CML) patients. METHODS Sixty CML patients in chronic phase were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and banding standard protocols. RESULTS There was a significant difference in collective incidence of complete cytogenetic response (CCR) between the e14a2 and e13a2 groups (P=0.04). The median time to achieve CCR was shorter in e14a2 patients than to e13a2 (P=0.01). This finding is paralleled by the molecular response where the median of the BCR-ABL1/ABL1 expression levels were significantly lower in e14a2 transcript compared to e13a2 type at 3, 6, 9 and 12months from the start of therapy (P<0.01). The probability of recurrence after treatment discontinuation was 9.33 fold higher in e13a2 transcript, that is reported here for the first time (χ2=5.49; P=0.01; OR: 9.33; 95% CI: 1.59, 54.67). No significant difference was observed regarding overall survival (OS), although Patients with e14a2 transcript displayed a significant tendency toward a higher event free survival (EFS) ratio (P=0.03). CONCLUSION We found that patients with the e14a2 transcript achieved better and faster responses to Imatinib mesylate. In this study, parallel data regarding molecular and cytogenetic responses, impact of transcript type on the probability of recurrence might suggest a general outcome that the type of transcript can be used as a prognostic marker at diagnosis.