Association of plasminogen activator inhibitor-1 and angiotensin converting enzyme polymorphisms with recurrent pregnancy loss in Iranian women

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 flbrinolytic process, embryonic development and pregnancy success. The aim of this study was to investigate the relationship between RPL and common polymorphisms in ACE and PAI-1 genes. 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. 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. 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

[Identification of nucleotide changes of two known causative genes [BRCA2 and STK11] of hereditary breast cancer in an Iranian family using exome sequencing]

Since the identification of the two highly penetrant dominantly inherited genes, BRCA1/2, in the 1990s, a number of other genes have been identified which account for approximately 25% of the genetic basis for hereditary breast cancer. At least 75% are unidentified. The goal of this study is to investigate the presence or absence of a recessive pattern of inheritance in this heterogeneous disease whose possibility has been previously discussed by researchers. In this study we used exome sequencing as the most recent approach for identification of the genetic basis of any disease. The results of exome sequencing were confirmed by Sanger sequencing. Although we did not find any homozygous mutation in this family, however a heterozygous 4bp deletion that led to a frame shift mutation was identified in exon 11 of the BRCA2 gene. Also identified was a heterozygous single nucleotide polymorphism in exon 9 of the STK11 gene. The rs80359352 variation identified in this family is one of the frequent pathogenic mutations in the BRCA2 gene that has been reported in the BIC database. This variation has been previously observed in other ethnic populations such as Caucasians, Hispanics and the Chinese. In this study, for the first time, we report this mutation in Iranian population and its segregation in hereditary breast cancer

Investigation the role of gender on the HLA-DRB1 and -DQB1 association with type 1 diabetes mellitus in Iranian patients

Type 1 diabetes mellitus [T1D] is an autoimmune and multifactorial disorder. Subsequent analysis on human leukocyte antigen [HLA] region shows that HLA-DRB1 and -DQB1 genes have the strongest association with T1D. In this study, for the first time, we investigated the influence of gender on the HLA-DRB1 and -DQB1 association with type 1 diabetes mellitus in Iranian patients in order to determine gender dependent HLA heterogeneity in Iranian T1D patients. In this case control study, the HLA-DRB1 and -DQB1 typing were performed on 105 Iranian T1D patients and 100 healthy controls. The data were evaluated by using Fisher exact test. Our results indicate that DRB1*04:01, DQB1*03:02 alleles and DRB1*04:01-DQB1*03:02 haplotype were significantly more frequent in male T1D patients than females. Also, DRB1*03:01, DRB1*15:01, DQB1*06:01 alleles, DQB1*03:01/05:01 genotype, DRB1*03:01-DQB1*02:01 and DRB1*15:01-DQB1*06:01 haplotypes were significantly higher in female T1D group than males. Furthermore, our results showed that DRB1*04:01 and DQB1*03:02 alleles were significantly more frequent in male T1D patients 1-5 years old at onset than females with similar condition. The DRB1*03:01 allele and DRB1*03:01-DQB1*02:01 haplotype were significantly higher in female T1D patients 6-10 years old at onset than males with similar condition. The DRB1*15:01 allele and DRB1*15:01-DQB1*06:01 haplotype were significantly more frequent in female T1D patients 16-20 years old at onset than males with similar condition. Our findings suggest that gender has a significant influence on the distribution of HLA-DR and -DQ alleles, genotypes and haplotypes. Also, distribution of the HLA-DRB1 and -DQB1 alleles, genotypes and haplotypes vary based on the gender of T1D patients in different age at onset

association of -475 and -631 interleukin-2 gene polymorphism with multiple sclerosis in Iranian patients

Multiple sclerosis [MS] is a chronic autoimmune disease due to demyelination of the central nervous system. It is believed that cytokines are involved in the pathogenesis of MS. The interleukin-2 [IL2] gene is powerful functional candidate that is involved in immune regulation and operation. In this study, for the first time, we investigated the effect of -475 A/T and -631 G/A IL2 polymorphisms on MS disease in Iranian patients. In this case-control study, 100 MS patients [mean age: 32.95 +/- 6.51 years, age range: 20-42 years] selected according to McDonald criteria, and 100 ethnically, sex and age matched healthy controls [mean age: 29 +/- 7.8 years, age range: 20-52 years] with no personal or family history of autoimmune diseases were studied. The restriction fragment length polymorphism-polymerase chain reaction [RFLP-PCR] method was applied to define different alleles and genotypes of IL2 promoter single nucleotide polymorphism -475 A/T as well as -631 G/A among individuals. chi [2] was calculated and Fisher's exact test was applied to analyze the obtained data. The value of p <0.05 was considered significantly. Evaluation of the -475 IL2 revealed that T allele and A/T genotype are present in 2% and 4% of MS patients, respectively, whereas T allele was absent in control samples. The comparison between alleles and genotypes in MS patients and healthy controls was not significant [p=0.1]. For the -631 position, 1% and 2% of MS patients carried A allele and A/G heterozygote genotypes, respectively. All control samples had G allele and G/G genotype. The differences between patients and controls were not significant [p=0.4]. Moreover, our results showed a very low frequency of T at -475 and A at -631 IL2 position in each of the two groups. Both -475 and -631 IL2 polymorphisms were higher in MS patients as compared to controls, but the frequency differences were not significant. Based on these data, it is suggested that the -475 and -631 IL2 polymorphisms as functional promoter position may be involved in IL2 expression and regulation. To find out the exact effect of the mentioned SNPs on susceptibility to MS, study on a larger sample size is suggested

Clinical features, DYT1 mutation screening and genotype-phenotype correlation in patients with dystonia from Iran

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. DMA 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 D/H-positive patients, while mean age at onset in patients with no GAG deletion mutation was higher [15.7 +/- 11.5 years]. 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

Identification of alpha-globin chain variants: a report from Iran

This study was carried out to identify molecular and hematological features of alpha-globin chain variants and to evaluate their effects on the clinical and hematological characteristics in Iranian individuals suspected of having thalassemia trait. Analysis of red blood cell indices, hemoglobin [Hb] analysis and genomic DNA isolation were carried out according to standard methods. For identifying the alpha-thalassemia [alpha -thal] genotype, investigation of common Mediterranean alpha-globin gene deletions [-alpha[3.7] - alpha[4.2] - alpha[20.5] and -[MED]] was performed by Gap-FOR. To characterize chain variants the entire alpha1 and alpha2 genes that spanned from the promoter region to the poly Atail were amplified and directly sequenced. In this study, 19 members of 17 unrelated families showed alpha-chain variants. Among these cases ten alpha-chain variantsthat included Hb Setif, Hb Constant Spring [Hb CS], Hb Handsworth, Hb Icaria, Hb Evanston, Hb Val de Marne, Hb Utrecht, Hb Savaria, Hb Adana, and Hb Dartmouth were identified. The hematological profile and molecular basis of these ten alpha-chain variants and the phenotypic consequences of their interactions were discussed. The knowledge of the spectrum of alpha-globin variants present in the Iranian population is essential for the molecular diagnosis and prevention of hemoglobinopathies

Promoter hypermethylation of estrogen receptor alpha gene is correlated to estrogen receptor negativity in Iranian patients with sporadic breast cancer

Breast Cancer is the most common cancer in Iranian women. Breast tumors are classified based on the estrogen receptor alpha [ER alpha] expression status into ER negative and ER positive tumors. ER negative tumors tend to have worse prognosis and less likely to respond to endocrine therapy. Aberrant methylation of gene promoter is one of the mechanisms for gene silencing in breast tumors. Because of its reversible nature, promoter methylation is a good target for new therapeutic strategies. We aimed to evaluate the frequency of this epigenetic event in ER alpha gene and its association to clinicopathological features in Iranian breast cancer patients. In this case control study the patient series consisted of 100 sporadic primary breast cancer cases [51 ER negative and 49 ER positive tumors]. None of the participants had chemo or radiotherapy before surgery. In breast tumors ER alpha promoter methylation were assessed with methylation specific polymerase chain reaction [MSP]. Data was collected on clinicopathological features of the patients. Correlation between ER alpha methylation and clinicopathological characteristics of the patients was investigated by Pearson Chi-Square and Fisher's exact test. ER alpha methylation was detected in 98% of ER negative and 65% of ER positive breast tumors. A strong correlation was found between ER alpha methylation and ER negativity in tumors [p<0.0001]. Also, ER alpha methylation has associated to progesterone receptor negativity [p<0.008] and double receptor negative status [p<0.0001] in breast tumors. ER alpha methylation occurs with high frequency in the breast tumors of Iranian breast cancer patients and may play a considerable role in pathogenesis of ER alpha negative tumors as a poor prognosis and more aggressive category. The reversible nature of DNA methylation may provide new therapeutic possibilities in ER negative breast tumors

frequency of DYT1 [GAG deletion] mutation in primary dystonia patients from Iran

To determine the frequency of DYT1 mutation in Iranian patients affected with primary dystonia. in this study, we investigated 60 patients with primary dystonia who referred to the Tehran Medical Genetics Laboratory [TMGL] to determine the deletional mutation of 904-906 del GAG in the DYT1 gene, DNA extracted from patients' peripheral blood was subjected to PCR-sequencing for exon 5 of the DYT1 gene. The collection of samples was based on random sampling. The deletional mutation of 904-906 del GAG in the DYT1 gene [15099 to 15101 based on reference sequence: NGJ308049.1] was identified in 11 patients [18.33%]. The average age of affected patients with this mutation was 13.64 +/- 7.4 years. Conclusion: It can be concluded that the DYT1 deletional mutation of 904-906 del GAG has a high frequency in Iranian patients in comparison with other non-Jewish populations. Therefore, this particular mutation may be the main representative of pathogenic DYT1 gene for a large proportion of Iranian patients with primary dystonia

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

To describe hematological and molecular features of a 13-bp deletion in the 3' untranslated region[3' UTR] of the beta-globin gene in carrier individuals and a compound heterozygous patient. 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. beta-Globin gene amplification and DNA sequencing were performed. One patient had a 13-bp deletion in the 3' UTR of the beta-globin gene that causes the beta-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 beta-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. This case showed a patient with a 13-bp deletion in the 3' UTR of beta-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 beta-chain synthesis in trans is compromised

[Study of the association between estrogen receptor alpha gene expression in the levels of RNA and protein in primary breast tumors]

Estrogen receptor alpha protein status is determined by routine immunohistochemistry analysis in all malignant breast tumors. This assay has its limitations. RNA based techniques are potential complements for immunohistochemistry but it must be noticed that gene silencing may occur at different levels from RNA to protein. The aim of this study was the comparison of the results from these two assays and characterizing the tumors subgroup in which gene expression occurs at RNA level but the target protein is absent. 92 primary breast tumors including their clinical and IHC results were collected before treatment. Estrogen receptor gene expression of tumors was studied by Reverse Transcription Polymerase Chain Reaction [RT PCR]. In this assay, GAPDH was used as a reference gene. 36.6% of tumors with negative estrogen receptor protein showed gene expression at mRNA level. In this subgroup most of the patient were older than 50 years and in stages 3 or 4 of breast cancer and had poor prognosis according to Nottingham prognostic index. Most cases of the perineural invasion have been seen in this subgroup. It seems that RT-PCR assay would enable us to recognize a subgroup of breast tumors with poor prognosis which expresses RNA but not protein

[ influence of HLA-DRB1,-DQB1 genes on age at onset of type 1 diabetes in Iranian T1D patients]

Survey of the influence of HLA-DRB1,-DQB1 alleles, genotypes and haplotypes on age at onset of type 1 diabetes [T1D] in an Iranian population 105 Iranian T1D patients of different ethnic group and 100 ethnically, age and sex matched individuals were selected from Tehran's hospitals and HLA-DRB,-DQB typing was performed. According to the age at onset of T1D, the patients were divided into 4 groups [1-5, 6-10, 11-15, 16-20 years]. The frequency of susceptible and protective alleles, genotypes and haplotypes was calculated in each group. The data were evaluated by using fisher's exact test. Odds Ratio or relative Risk was measured for all samples. The results illustrated that the frequency of the HLA-DRB1*0401 allele decreased with increasing age, whereas the frequency of the HLA-DQB1*0201 allele increased with increasing age. The HLA-DRB1*0301 and HLA-DQB1*0302 alleles demonstrated the highest frequency in the 6-11 and 1-5 years age at onset group, respectively. HLA-DRB1*0401-DQB1*0302 haplotype had the most frequency among the 1-5 years age at onset group [p: 2x10-7, OR: 69.919] and the frequency of HLA-DRB1*0301-DQB1*0201 haplotype was the highest in the 6-11 years age at onset group among others [p: 2x10-6, OR: 6.243]. The current study indicated that HLA-DRB1,-DQB1 alleles, genotypes and haplotypes are associated with age at onset of type1 Diabetes in Iranian T1D patients. The individuals carrying alleles that are associated with younger age at onset should take care under preventive treatment

Transient expression assay of [A]gamma-588 [A/G] mutations in the k562 cell line

In the previous study, we have shown that the presence of A allele at position -588 in [A]gamma -globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele [A allele at -588] could result in an increase in [A]gamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients. Three constructs containing ji locus control region, [A]gamma -globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of, [A]gamma -globin gene [A and G alleles at -588]. A construct with T to C base substitution at -175 of, [A]gamma -globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of, [A]gamma -globin gene was determined by quantitative real-time reverse transcription-PCR. There was not a significant increase in the expression of, [A]gamma -globin gene in the construct containing A allele comparing the one with G allele at -588. -588 [A>G] mutation does not play a major role in regulation of, [A]gamma -globin gene, suggesting that other factors may be involved

Molecular diagnosis of congenital adrenal hyperplasia in Iran: focusing on CYP21A2 gene

Congenital adrenal hyperplasia [CAH] is characterized by impaired biosynthesis of cortisol. 21-hydroxylase deficiency is the most common cause of CAH affecting 1 in 10000-15000 live births over the world. The frequency of the disorder is very high in Iran due to frequent consanguineous marriages. Although biochemical tests are used to confirm the clinical diagnosis, molecular methods could help to define accurate diagnosis of the genetic defect. Recent molecular approaches such as polymerase chain reaction based methods could be used to detect carriers and identify different genotypes of the affected individuals in Iran which may cause variable degrees of clinical expression of the condition. Molecular tests are also applied for prenatal diagnosis, and genetic counseling of the affected families. Here, we are willing to delineate mechanisms underlying the disease, genetic causes of CAH, genetic approaches being used in the country and recommendations for health care improvement on the basis of the molecular and clinical genetics to control and diminish such a high prevalent disorder in Iran. Also, the previous studies on CAH in Iran are gathered and a diagnostic algorithm for the genetic causes is proposed

[Development and application of real-time quantitative polymerase chain reaction technique using SYBR green I in the diagnosis of Down syndrome]

Rapid diagnosis of Trisomy 21 Syndrome [Down Syndrome] patients using Real-Time quantitative Polymerase Chain Reaction [Real-Time qPCR] in order to establish a novel method for prenatal diagnosis in the future. A total of 5 ml of peripheral blood was obtained from each patient and normal controls [NR]. Then, genomic DNA from lymphocytes was extracted using the salting out procedure. Gene dosage levels of DSCAM and [PMP22, DSCAM] in Down Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/ PMP22 ratio was calculated according to the 2-delta delta Ct formula for all samples. Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 +/- 0.18 and 1.01 +/- 0.10 [p<0.001] in Down Syndrome and normal samples, respectively, demonstrating three copies of the target [DSCAM] gene in Trisomy 21 Syndrome. DSCAM/PMP22 ratio is increased significantly in Down Syndrome patients than NR [1.5 times]. Therefore, the real-time quantitative PCR technique can be used as a sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy 21 Syndrome

Mutations of RAS gene family in specimens of bladder cancer

Studies have shown different types of RAS mutations in human bladder tumors with a wide range of mutation frequencies in different patient populations. This study aimed to assess the frequency of specific-point mutations in the RAS gene family of a group of Iranian patients with bladder cancer. We examined the tumor specimens of 35 consecutive patients with transitional cell carcinoma. The DNA samples were evaluated for the occurrence of HRAS, KRAS, and NRAS activation using a polymerase chain reaction-restriction fragment length polymorphism technique. None of the patients had mutations in the RAS gene family "hot spots" including codons 12, 13, and 61. We failed to find RAS mutations in our bladder tumor samples. These observations may reflect the involvement of different etiological factors in the induction of bladder tumor of which RAS mutation might not be present in all populations

Comparison of inosine triphate pyrophosphatase gene expression in chronic myelogenous leukemia patients and controls

One of the most significant damages to the genetic material is oxidative deamination of DNA and free nucleotides in the cell pool. Incorporation of deaminated purine nucleotides such as inosine triphosphate [ITP, dITP] into the DNA can increase the frequency of base substitution mutation. It has been suggested that presence and accumulation of these rough nucleotides can lead to genetic instability which is the perquisite of different types of diseases or cancers. Inosine triphosphaate pyrophosphates [ITPase/] encoded by ITPA gene, is responsible for protecting the cells by omitting deaminated purines from the free nucleotide pool. The objective of this study was to examine the possible dysfunction of ITPA gene activity as an important factor in genetic background predisposing to chromosomal disorders and malignancies such as CML. ITPA gene expression study performed on 23 CML patients and 21 controls using semi-quantitative RT-PCR technique and the expression level of GAPDH gene was used as an internal control. Unusual variants obtained from cDNA amplification of ITPA gene were clones. Our results showed a significant reduction of ITPA gene expression in CML patients in comparison to the controls. Two types of transcripts were produced in addition of expected transcript in some samples. One of them had a 123 nucleotide and the other had 77 nucleotide deletions in their open reading frame. Due to decreased expression of ITPA gene; it seems that the function of ITPase is not normal in CML patients. Therefore the altered expression of this gene can be considered as an additive factor for genetic instability in these patients