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Khordadpoor-Deilamani F.,Islamic Azad University at Tehran | Khordadpoor-Deilamani F.,Tehran Medical Genetics Laboratory | Akbari M.T.,Tarbiat Modares University | Akbari M.T.,Tehran Medical Genetics Laboratory
Bratislava Medical Journal | Year: 2015

OBJECTIVES: To use the PCR-RFLP-based linkage analysis for non-invasive prenatal diagnosis of β-thalassemia. BACKGROUNDS: Thalassemia is a prevalent genetic disorder occurring throughout the world. Cell-free fetal DNA (cffDNA) in the maternal plasma during pregnancy has been used to develop non-invasive prenatal screening and diagnostic tests. METHODS: PCR-RFLP for six SNPs in the β-globin gene was executed on paternal and maternal DNA as well as DNA extracted from CVS of the fetuses in seven β-thalassemic families. Based on the results, two families in which the paternal inherited SNPs in specific loci were different from the maternal one were selected and PCR-RFLP was performed on cffDNA extracted from the maternal plasma. RESULTS: Paternal SNPs in cffDNA were distinguished and the inheritance of paternally normal or mutant β globin allele was predicted by linkage analysis. CONCLUSION: The use of PCR-RFLP on cffDNA as a simple and inexpensive method was capable to provide similar results achieved by studying CVS of the fetuses. However, there is a limiting factor in this approach, namely that there is the little amount of cffDNA in maternal plasma. The PCR yield was improved either by adding BSA to PCR reaction or increasing the PCR cycles. Source

Nezhat N.,Islamic Azad University at Tehran | Akbari M.T.,Tarbiat Modares University | Akbari M.T.,Tehran Medical Genetics Laboratory
Genetic Testing and Molecular Biomarkers | Year: 2012

Although Iran is one of the countries with a high frequency of thalassemia, few studies have been carried out on characterization of different mutations in the α-globin gene cluster. There are a proportion of patients suspected of having α-thalassemia according to hematological profile with no abnormalities identified by gap-PCR for the most common α-thalassemia deletions and no point mutations detected by amplification refractory mutation system and sequencing of the α-globin genes. So the aim of the present study was to identify the mutations at the α-globin cluster using the multiplex ligation-dependent probe amplification (MLPA) method in patients who were suspected to be carrier of α-globin gene mutations, but in whom no mutations were found by conventional techniques. Twenty patients whose mutations were not identified were selected. In addition, 10 and 5 samples were chosen as positive and negative controls, respectively. MLPA results demonstrated mutations in 15% of the cases undetected by the conventional methods. One case showed the deletion of regulatory element HS-40 and in two other cases α-triplication in α-genes was determined. The simplicity and high accuracy of MLPA make this method a complementary method along with gap-PCR for detecting common known and unknown deletions and duplications in the α-globin gene cluster. © Copyright 2012, Mary Ann Liebert, Inc. Source

Khordadpoor-Deilamani F.,Islamic Azad University at Tehran | Akbari M.T.,Tarbiat Modares University | Akbari M.T.,Tehran Medical Genetics Laboratory | Nafissi S.,Tehran University of Medical Sciences | Zamani G.,Tehran University of Medical Sciences
Genetic Testing and Molecular Biomarkers | Year: 2011

Duchenne's muscular dystrophy and Becker muscular dystrophy (BMD) are X-linked recessive disorders caused by mutations in the dystrophin gene. In this project, 100 unrelated male patients were initially screened for deletions in the dystrophin gene by multiplex polymerase chain reaction, of whom 52 were positive. We performed the multiplex ligation-dependent probe amplification (MLPA) method on 43 of the remaining 48 patients, as well as 12 females suspected to be carriers, to detect deletions and duplications of their dystrophin gene. The MLPA method found deletions and duplications in 8 unidentified male patients. Sequencing revealed that in one case the deletion detected was a point mutation. One of 12 females was heterozygous for deletion of exons 49 and 50. In conclusion, the MLPA method proved to be reliable for studying affected males as well as female carriers. © Copyright 2011, Mary Ann Liebert, Inc. Source

Asgari N.,Islamic Azad University at Tehran | Akbari M.T.,Tarbiat Modares University | Akbari M.T.,Tehran Medical Genetics Laboratory | Zare S.,Tehran Medical Genetics Laboratory | And 2 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2013

Problem: Numerous lines of evidence implicate Apolipoprotein E (Apo E) in lipid metabolism during pregnancy. Hence, a role for its polymorphism has been envisaged in recurrent pregnancy loss (RPL) considering major structural and functional differences between different Apo E genotypes. Method of study: A case control study of 81 women with two or more pregnancy losses that did not have any other known risk factors including anatomic anomalies of the reproductive system, infections, immunologic factors, hormonal imbalances, chromosomal abnormalities and environmental factors was carried out. The control group consisted of 81 women with at least two healthy children and no RPL in their reproductive history. DNA was extracted from the peripheral blood following written consent and Apo E genotyping was carried out by amplifying exon 4 of the gene and subjecting it to digestion by HhaI restriction enzyme. Results: Genotyping was concluded by analyzing different fragment sizes produced, which resulted in finding significantly higher frequency of combined E3/E4 and E4/E4 genotypes in the patients (about 20 %) compared to the normal controls (2.4 %). The genotypes were confirmed by DNA sequencing. Conclusion: Allelic frequency for E4 was 13.5 % in the patients and only 1 % in the non-RPL group. Our findings confirm and are in line with a number of similar studies carried out on other populations. Therefore, Apo E4 polymorphism seems to be contributing to the thrombophilic risk factors as a background to RPL. © 2012 Springer Science+Business Media New York. Source

Shakarami F.,Guilan University | Akbari M.T.,Tarbiat Modares University | Akbari M.T.,Tehran Medical Genetics Laboratory | Karizi S.Z.,Islamic Azad University at Varamin
Iranian Journal of Reproductive Medicine | Year: 2015

Background: Recurrent pregnancy loss (RPL) defined by two or more failed pregnancies before 20 weeks of gestation. Several factors play a role in RPL including thrombophilic conditions which can be influenced by gene polymorphisms. Plasminogen activator inhibitor-1 (PAI-1) and angiotensin converting enzyme (ACE) genes are closely related to fibrinolytic process, embryonic development and pregnancy success. Objective: The aim of this study was to investigate the relationship between RPL and common polymorphisms in ACE and PAI-1 genes. Materials and Methods: In this case control study, 100 women with recurrent abortions (at least two) were selected as cases and 100 healthy women with two or more normal term deliveries without a history of abortion as controls. Total genomic DNA was isolated from blood leukocytes. The status of the PAI-1 4G/5G and ACE (D/I) polymorphism was determined by PCR-RFLP. Results: Homozygosity for PAI-1 4G polymorphism was seen in 17 cases (17%), and 5 controls (5%) (p=0.006) so patients with homozygote 4G mutation were significantly more prone to RPL in contrast to control group (OR: 4.63, % 95 CI: 1.55-13.84). In addition, 7 patients (7 %), and no one from the control group, were homozygote (I/I) for ACE polymorphism (p=0.034), suggesting no significant associations between ACE D allele or DD genotype and RPL. Conclusion: Considering these results, because 4G/4G polymorphism for PAI-1 gene could be a thrombophilic variant leading to abortion, analysis of this mutation and other susceptibility factors are recommended in patients with RPL. © 2015, Research and Clinical Center for Infertitlity. All rights reserved. Source

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