ABSTRACT
Background: Promyelocytic leukemia protein [PML] is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid [RA]. In addition, RA acts as a natural morphogen in neural development
Objectives: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development
Materials and Methods: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR [QRT-PCR] and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR [BSP] was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells [NT2.NPCs]
Results: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells [NT2.NCs]. Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell
Conclusions: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development
ABSTRACT
SLC26A4 gene mutations are the second identifiable genetic cause of autosomal recessive nonsyndromic hearing loss [ARNSHL] after GJB2 mutations and are currently investigated in molecular diagnosis. In databases, several potential STR markers related to this region have been introduced. In this investigation, the characteristics and informativeness of D7S2425 CA repeat STR marker in SLC26A4 gene region was examined in five ethnic groups of the Iranian population. The locus was genotyped in 165 individuals of five different ethnic groups including Fars, Azari, Turkmen, Gilaki, and Arab using polymerase chain reaction [PCR] followed by polyacrylamide gel electrophoresis [PAGE] and fluorescent capillary electrophoresis. In this study, the results were analyzed by GeneMarker HID, Human STR Identity software, GenePop program, and Microsatellite Tools. Analysis of the allelic frequency revealed the presence of 8 alleles for D7S2425 marker in the Iranian population, of which the 246bp allele at the D7S2425 locus with 0.30% frequency was the most frequent. The 93.9% observed heterozygosity of the Gilak ethnic group was the highest among all ethnic groups. Analysis of deviations of the Hardy-Weinberg equilibrium demonstrated that all the ethnic groups were in equilibrium [P > 0.05] for D7S2425 locus. Finally, analysis of PIC value revealed that the D7S2425 marker could be considered as a highly informative marker in each ethnic group of the Iranian population [PIC value above 0.7]. Our data suggested that D7S2425 could be introduced as a highly informative marker in molecular diagnosis of SLC26A4 based ARNSHL by Linkage analysis
ABSTRACT
Phenylketonuria [PKU] is the most common autosomal recessive disorder of amino acid metabolism. The disease is caused mainly by mutations in the phenylalanine hydroxylase [PAH] gene, encoding phenylalanine hydroxylase [PAH] enzyme. The PAH enzyme deficiency results in the elevation of phenylalanine in the blood, which may cause severe irreversible mental retardation in the affected individuals. More than 500 different disease causing mutations have been identified in the PAH gene. Direct and indirect molecular approaches have been developed for carrier detection and prenatal diagnosis of PKU disease. Population distribution of the PAH gene mutations and the PKU disease varies in different countries. In view of relatively high prevalence of the disease in Iranian population, investigations toward the elucidation of molecular aspects of the disease were required. In the present article, clinical and molecular basis of the PKU disease, with emphasis on the studies performed in Iranian population, were reviewed
ABSTRACT
The polymorphic variable number of tandem repeat [VNTR] genetic marker present at the 3' end of the phenylalanine hydroxylase [PAH] gene is commonly used for carrier detection among family members of phenylketonuria [PKU] patients. The utilization of this marker is solely dependent on the heterozygosity of its alleles [repeats] in the population. In the present study, we use this marker to investigate the carrier status of several PKU families in Isfahan. The VNTR marker was amplified using PCR with specific primers flanking the region. Evaluation of the length of the VNTR marker indicated the presence of 3, 7, 8, 9, 12 and 13 repeats with different frequencies. Alleles with 3 repeats had the highest [57.3%] and those with 13 repeats had the lowest [3%] frequency. Analysis of PKU patients and their families indicated that, the PAHVNTR marker was heterozygous among 78% of families. This study suggests that the PAHVNR marker could be a suitable marker in analyzing of carrier status among family members of PKU patients in Isfahan
ABSTRACT
In the present study, using anti-sense oligonucleotides the inhibition of expression of the PML protein has been investigated. The anti-sense oligonucleotides were designed against the translation initiation site of the PML gene, and their effects were investigated on cellular growth and DNA synthesis. Incubation of normal human fibroblast cells with the anti-sense oligonucleotides resulted in the complete inhibition of the PML protein expression. Inhibition of the PML protein expression by anti-sense oligonucleotides was found to be associated with an increase in cellular growth and doubling time. Furthermore, in cells treated with the anti-sense oligonucleotides, but not sense or scrambled oligonucleotides [control], the cellular DNA synthesis also showed a marked increase, confirming the induction of cellular growth upon inhibition of PML synthesis. These findings clearly demonstrated that the inhibition of the expression of the PML protein could be achieved using the anti-sense oligonucleotides, providing a model for better investigation of the biologic role of PML in the cell