Ala67Thr Mutation in the Human Polio Virus Receptor (PVR) Gene inPost-Polio Syndrome Patients
30 Apr 2014-Journal of Microbial & Biochemical Technology (OMICS International)-Vol. 6, Iss: 4, pp 179-184
TL;DR: Clinical and demographic characteristics of the PPS individuals who were affected initially by poliomyelitis and later developed PPS are presented and changes in the PVR gene may result in slowly progressive cytopathic effects that may lead to progression of PPS.
Abstract: Poliovirus (PV) has been implicated in the etiology of poliomyelitis. Post-polio syndrome (PPS) is a condition that affects polio survivor years after recovery from an initial acute attack of the polio virus. DNA polymorphisms in the poliovirus receptor gene (PVR) are associated with persistent poliovirus infection. In the present study we have presented clinical and demographic characteristics of the PPS individuals who were affected initially by poliomyelitis and later developed PPS. We have also attempted to find out whether mutation in the PVR gene allows poliomyelitis patients to progress for PPS. PVR mutation was studied in 110 cases of PPS and 200 normal controls. In PVR exon 2, the Ala67Thr mutation was detected in 45.46% of progressive PPS and 10% of control subjects. The frequency of the mutation was significantly higher in patients with PPS than in controls. Changes in the PVR gene may result in slowly progressive cytopathic effects that may lead to progression of PPS.
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TL;DR: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene and will be useful for large scale screening of DNA samples.
Abstract: Background & objectives : It is important to understand the role of cell surface receptors in susceptibility to infectious diseases. CD155 a member of the immunoglobulin super family, serves as the poliovirus receptor (PVR). Heterozygous (Ala67Thr) polymorphism in CD155 has been suggested as a risk factor for paralytic outcome of poliovirus infection. The present study pertains to the development of a screening test to detect the single nucleotide (SNP) polymorphism in the CD155 gene. Methods: New primers were designed for PCR, sequencing and SNP analysis of Exon2 of CD155 gene. DNAs extracted from either whole blood (n=75) or cells from oral cavity (n=75) were used for standardization and validation of the SNP assay. DNA sequencing was used as the gold standard method. Results: A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150) of DNA samples tested by both SNP detection assay and sequencing. Interpretation & conclusions: The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR / CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.
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10 Jan 2020
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References
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TL;DR: A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure that yielded quantities comparable to those obtained from phenol-chloroform extractions, rendering the entire process of RFLP analysis free of toxic materials.
Abstract: One of the obstacles encountered when extracting DNA from a large number of samples is the cumbersome method of deprotein-izing cell digests with the hazardous organic solvents phenol and isochloroform. Several other non-toxic extraction procedures have been published, but require either extensive dialysis (1) or the use of filters (2). A rapid, safe and inexpensive method was developed to simplify the deprotein-ization procedure. This method involves salting out of the cellular proteins by dehydration and precipitation with a saturated NaCl solution. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl t 400 mM NaCl and 2 mM Na 2 EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10Z SDS and 0.5 ml of a protease K solution (1 mg protease K in 1Z SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pi TE buffer (10 mM Tris-HCl, 0.2 mM Na 2 EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. The DNA obtained from this simple technique yielded quantities comparable to those obtained from phenol-chloroform extractions. The 260/280 ratios were consistently 1.8-2.0, demonstrating good deproteinization. Restrictions were performed using a number of different enzymes requiring high, medium or low salt concentrations, all resulting in complete restriction. This procedure has been used in our laboratory on several thousand blood samples for parentage, population and forensic studies. This technique is used with our non-isotopic hybridization procedures (3) rendering the entire process of RFLP analysis free of toxic materials.
19,905 citations
"Ala67Thr Mutation in the Human Poli..." refers methods in this paper
...DNA from peripheral EDTA coated blood was extracted by a phenol-chloroform method [18]....
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TL;DR: Tight genetic linkage between FALS and a gene that encodes a cytosolic, Cu/Zn-binding superoxide dismutase (SOD1), a homodimeric metalloenzyme that catalyzes the dismutation of the toxic superoxide anion O–2 to O2 and H2O2 is reported.
Abstract: Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the cortex, brainstem and spinal cord. Its cause is unknown and it is uniformly fatal, typically within five years. About 10% of cases are inherited as an autosomal dominant trait, with high penetrance after the sixth decade. In most instances, sporadic and autosomal dominant familial ALS (FALS) are clinically similar. We have previously shown that in some but not all FALS pedigrees the disease is linked to a genetic defect on chromosome 21q (refs 8, 9). Here we report tight genetic linkage between FALS and a gene that encodes a cytosolic, Cu/Zn-binding superoxide dismutase (SOD1), a homodimeric metalloenzyme that catalyzes the dismutation of the toxic superoxide anion O2.- to O2 and H2O2 (ref. 10). Given this linkage and the potential role of free radical toxicity in other neurodenegerative disorders, we investigated SOD1 as a candidate gene in FALS. We identified 11 different SOD1 missense mutations in 13 different FALS families.
6,733 citations
"Ala67Thr Mutation in the Human Poli..." refers background in this paper
...The involvement of virus in the progression of motor neuron disorders has long been suspected, especially since when poliovirus (a member of enteroviral family) was known to affect motor neuron selectively [7]....
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TL;DR: The mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms was developed and SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers.
Abstract: We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.
3,887 citations
TL;DR: Northern hybridization analysis indicates that poliov virus receptor transcripts are expressed in a wide range of human tissues, in contrast to the limited expression of virus binding sites, which suggests that additional factors or modifications of the receptor protein are required to permit poliovirus attachment.
1,056 citations
TL;DR: It is found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor–related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor.
Abstract: We have isolated a novel actin filament–binding protein, named afadin, localized at cadherin-based cell–cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament–binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor–related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell–cell AJs in various tissues and cell lines. In E-cadherin–expressing EL cells, PRR was recruited to cadherin-based cell–cell AJs through interaction with afadin. PRR showed Ca2+-independent cell–cell adhesion activity. These results indicate that PRR is a cell–cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell–cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word “necto” meaning “to connect”).
537 citations
"Ala67Thr Mutation in the Human Poli..." refers background in this paper
...PVR is related to the nectin family of adhesion molecules and expressed in intercellular junctions [6]....
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