Genetics in the Third Millennium
Volume:9 Issue: 2, 2011

After the birth of the “cytogenetics” in 1956 and its application in medicine in 1959 as valuable tool for the diagnosis of congenital diseases caused by chromosomal abnormalities, its limitations persuades the investigators to discover and employ “banding” techniques. This method could overcome many pre-existing problems and limitations but was not diagnostic especially when tissue culture was not possible due to tissue necrosis or in microdeletions. Fortunately, Fluorescent In Situ Hybridization (FISH) could prevail over many of the aforementioned limitations but it could not evaluate necrosed tissues since it required a specific probe for each disease. Array Comparative Genomic Hybridization (aCGH) is a new technique which enables us to study both necrosed and viable tissues. Another advantage of this method is the ability to study the whole genome and assess any decrease or increase in the chromosome material. This technique is currently regarded as the best and most efficient. Since stillbirths and viable or necrosed curettage specimens comprise most of the referred specimens to Kariminejad & Najmabadi Pathology and Genetic Center and evaluation of the specimens with a special focus on chromosomal aberrations are required by clinicians, we have equipped our center with technical equipment and experienced cytogenetic technicians. In the end, two cases whose definite diagnoses were made using FISH and Array CGH are presented.

Quantitative fluorescence polymerase chain reaction (QF-PCR) has been introduced in a number of genetic laboratories as an inexpensive, rapid and reliable method for prenatal recognition of aneuploidy in chromosomes 13, 18, 21, X and Y. We have investigated the efficacy of QF-PCR for the prenatal recognition of common aneuploidy and compared our findings with cytogenetic results in Iran. A multiplex PCR involving 15 short tandem repeat (STR) sequences was established for aneuploidy screening and chromosomal study was performed for all samples as well. Finally, the results obtained from QF-PCR method and cytogenetic analysis were compared with each other. A total of 425 prenatal samples were analyzed including 393 amniotic fluid and 25 chorionic villus samples (CVS). The following abnormalities were detected in 13 individuals: 6 samples (1.41%) with Down syndrome, 2 samples with Edward syndrome (0.47%), and 3 samples with Patau syndrome, 47, XXY, and 47, XYY. All of the CVS samples were normal. In addition, 2 cases (0.47%) showed triploidy. We were able to detect all the aneuploidies of chromosomes 13, 18, 21, X and Y and this was confirmed by cytogenetic results. Contamination of the fetal specimens by maternal cells was present in 12 out of 425 samples (2.82%) and failed to obtain any results. Additional chromosomal abnormalities were detected in 3 cases by karyotyping: inversion 9, translocation t(9,14) and XX/XY mosaicism. Through the QF-PCR method, we were able to detect abnormalities in 96.47% of all referred families. We recommend the QF-PCR in combination with cytogenetic study to be performed on all prenatal cases to provide a rapid and reliable diagnosis in families at risk for aneuploidy. We also recommend all the families that are seeking prenatal diagnosis of single gene disorders a QF-PCR to be added to their work up.

Alzheimer’s Disease (AD) is the most common form of dementia in the elderly in which an interplay between genes and the environment is supposed to be involved. Mutations in mitochondrial DNA (mtDNA) have been suspected to be causally related since mitochondrial DNA is maternally inherited in a non-Mendelian way. All Mitochondrial tRNA genes of 24 AD patients and 50 healthy controls were investigated by sequencing method. Fifteen variations were found in different mitochondrial tRNA genes. Eleven variations were polymorphic mutations. Four variations {C1631A, T1633A (tRNA Val), T14704C and T14723C (tRNA Glu)} filled the criteria for pathogenicity of mutation because of their: a) heteroplasmic state, b) conserved nucleotide, c) absence in the literature and d) absence in healthy controls. A12308G polymorphic mutation (tRNA Leu (CUN)) was found in 8 patients. This mutation has been reported in different neurodegenerative diseases as well as controls. We believe that these variations may have pathogenic effects in AD or have secondary effects in the disease process. The percentage of hetroplasmy may play a role in signs/symptoms or onset age of the disease.

Induction of fetal hemoglobin (HbF) is one of the most promising therapies of β-thalassemia and sickle cell disease, as HbF permanent induction can substantially ameliorate the clinical symptoms of these genetic disorders. Several classes of therapeutics have been implemented to induce γ-globin expression in beta-thalassemia patients and subsequently to raise total hemoglobin levels. Sodium butyrate and Talidomide are two of the known agents. This study was conducted on erythroid cells differentiated from cord blood CD133 + stem cells. Four groups were designed and gamma globin was induced by thalidomide and sodium butyrate treatment (100 μM concentrations). Gamma gene expression was evaluated using Quantitative Real Time PCR techniques. Among the treated groups with sodium butyrate and thalidomide, it was shown that although these two agents both affected the induction of gamma and beta genes, thalidomide had a greater potency on beta cluster genes than sodium butyrate. Our results showed that the pharmaceutical regime of thalidomide and sodium butyrate could be useful for the treatment of β-Thalassemia and sickle cell patients because of the induction of the beta gene cluster.

Charcot-Marie-Tooth (CMT) is the commonest neurogenetic disorder with phenotypic and genotyping heterogeneity. CMT1A encompasses approximately 60% of all types of CMT and has AD inheritance. CMT1A maps to chromosome17 p11.2 and is majorly caused by 1.5 Mb DNA duplication that includes the peripheral protein 22 (PMP) genes. The severity, onset and progression of CMT1A vary markedly within and between families. Patients usually develop symptoms in the first two decades of life and onset after 30 years is rare. Patients develop a slowly progressive atrophy and weakness of the distal muscles of the leg and feet but rarely lose the ability to walk later in the disease course; most patients develop weakness and wasting in the hands. We investigated 67 patients compatible with electeroclinical presentations of CMT1A for the PMP22 gene duplication by PCR-RFLP method. Moreover, the RFLP dosage test is uninformative if the patient is homozygous and an alternative test is required. In this study, PMP22 duplication was found in 36% of our patients. However, up to 30% of the cases with a CMT1 phenotype who had a strand exchange event outside the hotspot region could have remained undetected by our method. Therefore, we used this method as a screening approach to detect patients with crossovers in the hotspot and then performed MLPA to identify the remaining 30% of the patients with crossovers outside the hotspot. Key Note: The problems of genetic diagnosis of CMT are: a) many genes are involved in the same phenotype b) many phenotypes are caused by the same gene. So, a battery of genetic diagnosis needs to clarify the cause of the disease such as MPZ, PMP22 and EGR2 by PCR-Sequencing method to diagnose the point mutations.

GST tagging is a valuable tool for quick detection, isolation, and analysis of protein-protein interactions, without prior knowledge of the target protein. The GST tag, as the most widely used tag with 27 kD weight, can be used for detection and/or purifying unknown interacting partners of bait protein. Previous studies have indicated that the peroxisomal protein (PEP), a recently characterized protein, is somehow involved in neural differentiation of embryonic stem cells. The aim of this study was efficient production and purification of the recombinant peroxisomal protein fused with the GST tag in bacterial competent Ecoli cells for further analysis to understand the special proteins which directly interact with PEP. Recombinant prokaryotic expression vector (pGEX6P2) encompassing Glutathione S-transferase (GST) cDNA fused with peroxisomal protein cDNA was transformed in BL21 Escherichia coli cells. E. coli cells, separately expressing GST and GST-PEP fusion protein were examined for efficient protein production. At first, various concentrations of isopropyl-b-D-thiogalactoside (IPTG) were added to the culture medium to optimize the induction condition. Next, cells were allowed to culture in different temperatures post induction. Finally, cells were ruptured in 600 µl of chilled Tris-HCl (pH: 7.5) containing 1 mM phenylmethylsulfonyl Fluoride (PMSF), in the presence of different concentrations of detergents (Triton X-100 or NP40), and sonicated. Sonication was done using different strengths of the ultrasonic probe. The lysate cells were centrifuged. The supernatant fraction was collected and incubated with 15 µl of 50% slurry glutathione-Sepharose at 4 °C for 3h. Glutathione-Sepharose beads were washed three times and resuspended in the same buffer for SDS-PAGE electrophoresis. Sepharose beads linked with glutathione were then used for binding and pull down of GST-PEP fusion protein. Fifteen µl of elution subjected to the gel and gel was stained with commasie brilliant blue (CBB) in order to detect the protein bands. The optimum gene expression was found when IPTG was added to the final concentration of 0.1 µg/ml bacterial cell culture overnight. We found no difference between Triton X-100 and NP40 usage as solublizing detergents. However, the best concentration for detergent was set to be 1% (V/V).

Platelets have a crucial role in homeostasis and thrombosis. Their haemostatic role is to prevent from bleeding after activation in the platelet aggregation pathway. In pathologic cases, they contribute to atherothrombosis. Antiplatelet drugs inhibit platelet aggregation so they are used successfully in treatment courses. Clopidogrel, an antiplatelet drug, is an ADP analogue. It inhibits ADP coupling with its receptor and prevents atherothrombosis. Despite its great success, some patients do not display an adequate antiplatelet response due to genetic resistance. Genetic investigations have displayed CYP2C19 polymorphism as the most prevalence factor of genetic resistance. Use of higher doses of Clopidogrel, combined antiplatelet therapies or new potent drugs is a potential alternative strategy.

The p75 neurotrophin receptor (p75NTR) is a 75-kDa glycoprotein and the first identified member of the TNF receptor superfamily. p75NTR is initially expressed very early in embryogenesis in cells derived from all three germinal layers but becomes progressively restricted to specific cell populations such as CNS and PNS. The p75NTR expression is induced after pathologic conditions in a wide variety of cell types. p75NTR is a versatile protein, but its function in different cell types has remained enigmatic. One of the reasons for the lack of clarity regarding the function of p75NTR is that it does not signal by traditional pathways; it has no kinase activity nor is it linked to a G-protein-coupled pathway. p75NTR has no intrinsic enzymatic activity so it depends on its interaction with several adaptor proteins to signal the cascade for neuronal survival or death. The various functions of p75 depend on the type of ligand bound to it (neurotrophins or pro- neurotrophins), the cell type in which it is expressed and the presence or absence of Trk receptor and Interaction with other receptors. This mini-review explores the function, gene expression, interactions with co-receptors and signaling pathways of p75NTR in the nervous system.

Inflammation is the normal response of the body to injuries to remove dead cell debris and infections at injury sites and repair the tissue. However, excessive inflammation is harmful to the body because of its positive feedback and disruption of normal cells. Thus, recruitment of the regulatory mechanisms for modulating the inflammation is crucial for the body. Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated transcription factors and members of the nuclear receptor super family. Among these superfamily members, PPARγ ia involved in the regulation of numerous cellular processes such as inflammation. Several activators of PPARγ have been known to exert possible roles in immune function through mechanisms such as transactivation of genes that suppress the inflammation, trans-repression of inflammatory genes, interaction with other receptors that lead to effective suppression of the inflammatory agents. Here in this review article, we describe the molecular events which are triggered by these molecules in elaboration of inflammation process.

Cold stress, as an environmental tension, is one of the important limiting factors of survival and growth. It has a crucial role in the ecological disperse of organisms. All living organisms, especially plants, have developed many different mechanisms at physiological, biochemical and molecular levels for adaptation to cold stress. The main mechanisms of these responses require the presence and direct, indirect effects of the content and type of proteins. The cold stress influences the membrane fluidity and cell division and changes the types and content of proteins. Cold shock proteins (Csps) play a role in inducing cold resistance in organisms by increasing their expression, synthesis and accumulation. These proteins are produced as conserved molecules in different parts of the cell among various organisms. Csps are protein structures that bind to nucleic acids and have a substantial function in RNA conservation and regulation of the different levels of gene expression patterns in stress conditions. They are also present in normal conditions to regulate physiological, developmental and metabolic activities. In this review, the structure and function of different kinds of Csps in response to cold stress are described.

Miller-Dieker Syndrome is one of reasons of profound neuro-developmental delay with lissencephaly. A contiguous gene deletion in 17p13.3 is documented. It is characterized by microcephaly, bitemporal narrowing, a small nose with anteverted nostrils, a prominent upper lip, micrognathia, low set ears, hypotonia, seizures, brain malformation, lissencephaly and profound mental retardation. We report a two-year-old boy with microcephaly, profound mental retardation, bitemporal narrowing, low set ears and lissencephaly. CGH array documented the disease.