scholarly journals Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations

2003 ◽  
Vol 49 (7) ◽  
pp. 1050-1057 ◽  
Author(s):  
Anthony A Killeen ◽  
John W Breneman ◽  
Arlene R Carillo ◽  
Jason Liu ◽  
Craig S Hixson
Keyword(s):  
Hereditary Hemochromatosis ◽  
Colorimetric Method ◽  
Rflp Analysis ◽  
Wild Type ◽  
Linear Amplification ◽  
Pcr Rflp ◽  
Homozygous Mutant ◽  
Allele Specific ◽  
Novel Method ◽  
The Common

Abstract Background: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations. Methods: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin–horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared. Results: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63. Conclusions: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.

NAT2 and CYP1B1 genetic polymorphisms in patients with genital endometriosis depending on tolerability of melatonin

2021 ◽  
Vol 70 (4) ◽  
pp. 35-42
Author(s):  
Tatyana E. Ivashchenko ◽  
Maria I. Yarmolinskaya ◽  
Saimat S. Tkhazaplizheva
Keyword(s):  
Genetic Polymorphism ◽  
Rflp Analysis ◽  
Acetylator Phenotype ◽  
Wild Type ◽  
Melatonin Administration ◽  
Pcr Rflp ◽  
Genes Encoding ◽  
Poor Tolerance ◽  
Nat2 Gene ◽  
Rapid Acetylator

BACKGROUND: Genital endometriosis is one of the most pressing problems of modern gynecology. Melatonin is a promising drug with a potentially curative effect on endometriosis. AIM: The aim of this study was to conduct a comparative analysis of the genetic polymorphism of some genes encoding enzymes involved in melatonin metabolism. MATERIALS AND METHODS: The genetic polymorphism in the NAT2 and CYP1B1 genes encoding enzymes involved in melatonin metabolism in patients with different tolerance to this drug was analyzed by PCR-RFLP analysis. RESULTS: In patients with genital endometriosis, the presence of a wild-type allele (N) of the NAT2 gene was associated with poor tolerance of melatonin. The NAT2 (N / N) rapid acetylator phenotype combined with the low catalytic activity of CYP1B1 (C / C) occurred more frequently in endometriosis patients having poor melatonin tolerability compared to the group of patients who tolerated the therapy well. CONCLUSIONS: For patients with genital endometriosis with the wild-type (N) allele of the NAT2 gene, melatonin administration is inappropriate due to numerous side effects during the drug use.

scholarly journals Molecular Characterization of Pathogenicity Locus (PaLoc) and tcdC Genetic Diversity Among tcdA+B+ Clostridioides Difficile Clinical Isolates in Tehran, Iran

2020 ◽  
Author(s):  
Mansoor Kodori ◽  
Zohreh Ghalavand ◽  
Abbas Yadegar ◽  
Gita Eslami ◽  
Masoumeh Azimirad ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Molecular Characterization ◽  
Disease Severity ◽  
Genetic Variants ◽  
Rflp Analysis ◽  
Clostridioides Difficile ◽  
Pcr Rflp ◽  
The Common ◽  
Healthcare Associated

Abstract Background: Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs).Methods: Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene.Results: Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene.Conclusions: The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/ toxinotype 0 and the RT 126/ toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

Genetic variation and epidemiology of Echinococcus granulosus in Argentina

Parasitology ◽  
1999 ◽  
Vol 118 (5) ◽  
pp. 523-530 ◽  
Author(s):  
M. C. ROSENZVIT ◽  
L.-H. ZHANG ◽  
L. KAMENETZKY ◽  
S. G. CANOVA ◽  
E. A. GUARNERA ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Rflp Analysis ◽  
Santa Fe ◽  
Cystic Hydatid Disease ◽  
Rio Negro ◽  
Pcr Rflp ◽  
Control Programmes ◽  
The Common ◽  
Cystic Hydatid ◽  
Sheep Strain

Polymerase chain reaction–ribosomal ITS-1 DNA (rDNA) restriction fragment length polymorphism (PCR–RFLP) analysis and sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) genes were used to characterize 33 Echinococcus granulosus isolates collected from different regions and hosts in Argentina, and to determine which genotypes occurred in humans with cystic hydatid disease. The results of the study demonstrated the presence of at least 4 distinct genotypes; the common sheep strain (G1) in sheep from Chubut Province and in humans from Río Negro Province, the Tasmanian sheep strain (G2) in sheep and 1 human from Tucumán Province, the pig strain (G7) in pigs from Santa Fe Province and the camel strain (G6) in humans from Río Negro and Buenos Aires Provinces. The finding that pigs harboured the pig strain and the occurrence of the Tasmanian sheep strain has considerable implications for the implementation of hydatid control programmes due to the shorter maturation time of both strains in dogs compared with the common sheep strain. Furthermore, this is the first report of the presence of the G2 and G6 genotypes in humans which may also have important consequences for human health.

scholarly journals Mean Stable Warfarin Doses Versus CYP2C9*2 and VKORC11639G>A Genotypes in Sudanese Population

2016 ◽  
Vol 9 (1) ◽  
pp. 34
Author(s):  
Azza A. M. H. Swar Aldahab ◽  
Abdalla O. Elkhawad ◽  
Ahmed S. A. Elsayed ◽  
Hanan B. Eltahir
Keyword(s):  
Warfarin Dose ◽  
Therapeutic Index ◽  
Target Range ◽  
Wild Type ◽  
Genotype 3 ◽  
Pcr Rflp ◽  
Homozygous Mutant ◽  
Variant Genotype ◽  
The Mean ◽  
Warfarin Dosing

Warfarin is a potent anticoagulant with a confirmed effectiveness when anticoagulation targets are attained, an issue, that is troublesome to reach due to the fact that warfarin has a narrow therapeutic index (NTI), that means they have a narrow window between their effective doses and those at which they produce adverse toxic effects. However, oral anticoagulation throughout genetics recommended a genotype guided dosing, but is it favourable over clinical based dosing? Objectives: To analyze the mean stable warfarin doses attained clinically within CYP2C9*2 and VKORC11639G>A wild-type and variant genotype status in Sudanese patients. Method: Genotyping for the CYP2C9*2 and VKORC1-1639G>A polymorphisms were accomplished with polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique. The mean stable warfarin dose per genotype was defined as the mean stable warfarin dose related to the stable INR within target range within a genotype of each of CYP2C9*2 or VKORC11639G> Agenes, using Analysis of Variance (ANOVA) as the statistical method. Results: Fifty-three stable patients with wild-type CYP2C9*1*1 genotype had a mean stable warfarin dose of 4.9 ± 2.1 mg, 5 patients who were heterozygous CYP2C9*1*2 genotype, had a mean stable warfarin dose of 5.0 ± 0.71mg, and 2 patients were homozygous mutant CYP2CP*2*2 genotype had a mean stable warfarin dose of 4.8 ± 0.3 mg. The results were statistically insignificant, P = 0.992.Sixteen unstable patients were of wild-type CYP2C9*1*1 genotype, 40 patients with heterozygous CYP2C9*1*2 genotype, and 4 with homozygous mutant CYP2CP*2*2 genotype, had mean stable doses of 5.32 ± 2.9, 6.5 ± 2.7 and 4.5 ± 0.71 mg respectively. The result was statistically insignificant, P = 0.508. Fifty-two stable patients were having wild-type VKORC1G/G genotype, 3 patients had heterozygous VKORC1G/A genotype and 3 patients had homozygous mutant VKORC1/AA genotype, these patients had mean stable doses of 5.41 ± 1.63, 4.8 ± 2.19 and 5.4 ± 0.99 mg respectively. The mean warfarin stable dose among homozygous mutant VKORC1A/A genotype was lower than among wild-type and heterozygous genotype profiles. This result was statistically not significant, P = 0.729.In the unstable group, 8 patients of wild-type VKORC1G/G genotype, had a mean stable warfarin dose of 7.34 ± 3.9 mg, 40 patients of heterozygous VKORC1G/A genotype had a mean stable warfarin dose of 5.21 ± 2.76 mg, and 10 patients of homozygous VKORC1A/A genotype had a mean stable warfarin dose of 4.3 ± 1.83 mg. The result was statistically insignificant, P = 0.067.Conclusion: In our study, as there were no significant differences between warfarin mean stable doses related to different CYP2C9*2 and VKORC11639G>A genotypes, the evidence is not satisfactory to conclude that the conventional use of genotype guided warfarin dosing will correct stable warfarin dose among Sudanese patients.

scholarly journals Assessment of Poliovirus Eradication in Japan: Genomic Analysis of Polioviruses Isolated from River Water and Sewage in Toyama Prefecture

2000 ◽  
Vol 66 (11) ◽  
pp. 5087-5091 ◽  
Author(s):  
Kumiko Matsuura ◽  
Mitsuhiro Ishikura ◽  
Hiromu Yoshida ◽  
Takashi Nakayama ◽  
Sumiyo Hasegawa ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
River Water ◽  
Viral Genome ◽  
Fragment Length Polymorphism ◽  
Genomic Analysis ◽  
Rflp Analysis ◽  
Wild Type ◽  
Pcr Rflp ◽  
Nucleotide Divergence ◽  
Restriction Fragment Length

ABSTRACT Seventy-eight poliovirus strains isolated from river water and sewage in Toyama Prefecture, Japan, during 1993 to 1995 were characterized by the PCR-restriction fragment length polymorphism (RFLP) method and by partially sequencing the VP3 and VP1 regions of the viral genome. Of these isolates, 36 were identified as Sabin vaccine strains, and 42 were identified as vaccine variant strains that had less than 1.4% nucleotide divergence from the Sabin strains, including 7 isolates with patterns different from those of Sabin strains as determined by PCR-RFLP analysis. These findings suggest that wild-type poliovirus was not circulating in Toyama Prefecture.

scholarly journals REAL-TIME PCR TECHNOLOGY FOR CATTLE GENOTYPING BY A AND B KAPPA-CASEIN GENE ALLELES

2021 ◽  
pp. 126-132
Author(s):  
R. R. Vafin ◽  
Kh. Kh. Gilmanov
Keyword(s):  
Real Time ◽  
Fluorescence Detection ◽  
Real Time Pcr ◽  
Bos Taurus ◽  
Rflp Analysis ◽  
Casein Gene ◽  
Analysis Technique ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Fluorescence Quencher

The main goal of the study was to develop and test an effective technology for cattle genotyping by the CSN3 gene based on real-time PCR with hybridization-fluorescence detection. There was developed a method for real-time PCR for cattle genotyping by A and B alleles of the CSN3 gene in the format of hybridization-fluorescence detection, involving the use of two 5/-fluorescence-labeled forward allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher at the 3/-end of the oligonucleotide. As a result of practical studies aimed at testing the developed method, we obtained the technical result provided by the proposed technology, expressed in the effective identification of the desired genotypes due to correct interpretation of these curves of increasing fluorescence intensity, the results reliability of which was also confirmed by the well-known PCR-RFLP analysis technique for Bos taurus genotyping for similar allelic variants of the kappa-casein gene.

scholarly journals The 1B vaccine strain of Chlamydia abortus produces placental pathology indistinguishable from a wild type infection

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242526
Author(s):  
Sergio Gaston Caspe ◽  
Morag Livingstone ◽  
David Frew ◽  
Kevin Aitchison ◽  
Sean Ranjan Wattegedera ◽  
...  
Keyword(s):  
Vaccine Strain ◽  
Animal Health ◽  
Small Ruminants ◽  
Rflp Analysis ◽  
Whole Genome Sequence ◽  
Wild Type ◽  
Typing Method ◽  
Chlamydia Abortus ◽  
Vaccine Type ◽  
Pcr Rflp

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals.

scholarly journals Haptoglobin Polymorphism and Iron Homeostasis

2002 ◽  
Vol 48 (12) ◽  
pp. 2232-2235 ◽  
Author(s):  
Ernest Beutler ◽  
Terri Gelbart ◽  
Pauline Lee
Keyword(s):  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Wild Type ◽  
C282y Mutation ◽  
Ferritin Concentration ◽  
Denatured Protein ◽  
Oligonucleotide Hybridization ◽  
Allele Specific ◽  
Haptoglobin Polymorphism

Abstract Background: There is a marked difference in the degree of expression of the homozygous C282Y HFE genotype that is associated with hereditary hemochromatosis. It has been reported that individuals with the haptoglobin 2-2 type manifest increased iron concentrations, including serum iron, transferrin saturation, and ferritin. Methods: We studied 232 patients, 115 homozygous for the c.845G→A (C282Y) mutation and 117 matched controls with the wild-type HFE genotype, for haptoglobin phenotypes. Haptoglobin types were determined by electrophoresis of the denatured protein. The HFE genotype was determined by allele-specific oligonucleotide hybridization. Ferritin and transferrin saturation were measured by standard methods. Results:There was no relationship between haptoglobin type and ferritin concentration or transferrin saturation. Conclusions: The effect of haptoglobin type on iron homeostasis cannot account for the marked phenotypic variation that is seen in patients homozygous for the HFE C282Y mutation.

Frequency of the JAK2V617F Mutation in Platelets from Essential Thrombocythemia (ET) Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4972-4972
Author(s):  
Paula G. Heller ◽  
Paola R. Lev ◽  
Juan P. Salim ◽  
Laura I. Kornblihtt ◽  
Patricia S. Vassallu ◽  
...  
Keyword(s):  
Colony Growth ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Myeloid Metaplasia ◽  
Pcr Rflp ◽  
In The Wild ◽  
Allele Specific ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract The JAK2V617F mutation has been recently reported in patients with polycythemia vera (PV) and a proportion of patients with ET and myelofibrosis with myeloid metaplasia. This acquired point mutation constitutively activates JAK2 tyrosine kinase and is believed to underlie growth factor hypersensitivity displayed by hematopoietic progenitors in these disorders. Identification of this genetic abnormality may be useful for diagnostic purposes although its prognostic implication remains to be determined. The prevalence of this mutation in granulocytes from ET patients ranges from 23 to 57% depending on the detection technique used. ET is characterized by predominant involvement of the megakaryocytic lineage and increased platelet production. In order to evaluate whether the frequency of the V617FJAK2 mutation in platelets from ET patients differs from that previously reported in granulocytes, we amplified a fragment of JAK2 cDNA from leukocyte-depleted platelets by RT-PCR followed by digestion with BsaXI restriction endonuclease (PCR-RFLP). The JAK2V617F mutation abolishes a BsaXI restriction site present in the wild-type sequence and generates a different band pattern. In addition, allele-specific RT-PCR (ASPCR) was performed with a primer pair common to both wild-type and mutant alleles and a third primer complementary to the mutant allele. Forty four patients with ET diagnosed according to the PVSG criteria were included, median age 39 years old (14–81), 10 were men; 21 were studied before treatment, 10 during treatment with anagrelide or hydroxyurea and 13 both before and during treatment. Twenty-two (50%) patients were found to harbour the JAK2V617F mutation by both PCR-RFLP and ASPCR, 3 were homozygous. No change in genotype was found in repeated samples from patients studied both before and during treatment. Hemoblobin levels and the frequency of thrombosis were significantly higher in patients with the mutation respect to those without (141 ±14 g/L vs. 129 ± 11 g/L, p=0.009, and 41% vs. 4.5%, p=0.004, respectively). In addition, patients carrying the mutation were significantly older (51 vs. 33 years old, p=0.002) and had lower platelet counts at diagnosis (1.070 x 109/L vs. 1.500 x 109/L, p=0.02). Interestingly, 2 patients harbouring the mutation developed PV on follow-up. In conclusion, the prevalence of the JAK2V617F mutation in platelets from ET patients did not differ from that previously reported in granulocytes. The increase in the frequency of thrombosis found in patients carrying the mutation should be confirmed in a larger study. Besides, the finding of higher hemoglobin levels in this group of patients is of interest since JAK2V617F-positive ET patients have been reported to display PV markers such as endogenous erythroid colony growth and PRV-1 overexpression, suggesting they may have larger involvement of the erythroid lineage. Further studies will help clarify whether the risk of developing PV is higher in this subset of patients.

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