scholarly journals Development of RB1 mutation detection method based on mRNA

2016 ◽  
Vol 14 (2) ◽  
pp. 209-214
Author(s):  
Vũ Phương Nhung ◽  
Lê Thúy Quỳnh ◽  
Nguyễn Đăng Tôn ◽  
Nguyễn Thị Xuân ◽  
Nguyễn Hải Hà
Keyword(s):  
Healthy Individual ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Reference Sequence ◽  
Coding Region ◽  
Blood Leukocytes ◽  
Standard Pattern ◽  
Rb1 Gene ◽  
Large Size ◽  
Pcr Products

Retinoblastoma (Rb) is a malignant tumor of the retina, occurring usually in children before age five. The heritable form acounting for about 40% of Rb making genetic analysis of RB1 gene is a important part of disease management. In previous study, we have successfully employed a method of direct sequencing for RB1 mutation screening from genomic DNA. However, given the large size of this gene and no reported mutation hotspots, the testing can be costly and time consuming method. To overcome this problem, we have developed a method to detect mutation from RB1 mRNA. Total RNA was isolated from blood leukocytes of a healthy individual and a Rb patient, cDNA was subsequently synthesized using reverse-transcriptase PCR. Whole cDNA of RB1 was amplified using six specific primer pairs and sequenced by Sanger method. The PCR products from healthy individual were showed as six specific bands on the agarose gel and could be used as the standard pattern of the normally spilced transcripts. Those of PCR products were sequenced successfuly and data can be aligned with the reference sequence of RB1 cDNA on the Genebanhk to identify nucleotide variants.  We also identified an upnormal splicing of RB1 gene in the Rb patient haboring a c. G1960>C mutation at the end of the exon 19 by amplification of  the RB1 cDNA fragments. Hence using RB1 mRNA test can reveal not only the silent/pathogenic mutations in the coding region of RB1, but also mutations that affect the normal spilcing of RB1 mRNA. This method reduces cost and time, can be used as the first step of RB1 analysis in Rb patients.

Mutations within the Protein Z-Dependent Protease Inhibitor (ZPI) Gene Are Associated with Venous Thromboembolic Disease: A New Form of Thrombophilia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 488-488 ◽  
Author(s):  
Paul L. Harper ◽  
Neil S. Van de Water ◽  
Fern Ashton ◽  
Tina Tan ◽  
Anne O Grady ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Stop Codon ◽  
Mutation Screening ◽  
Heteroduplex Analysis ◽  
Power Calculation ◽  
Coding Region ◽  
Protein Z ◽  
Stop Codons ◽  
Pcr Products ◽  
New Form

Abstract ZPI is a recently characterised inhibitory serpin present in plasma. In vitro studies have shown that ZPI inhibits both factor Xa (in the presence of protein Z) and factor XIa. Deficiency of ZPI is predicted to enhance coagulation and may be a risk factor for venous thrombosis. To test this hypothesis we carried out mutation screening within the coding region of the ZPI gene in a cohort of 150 patients with a history of DVT or PE (first event before the age of 60 years) and 150 matched controls. Five PCR products were produced for each subject. Heteroduplex analysis was performed using dHPLC and the PCR products were sequenced directly if a heteroduplex mismatch was identified. Sixteen mutations/polymorphisms were identified within the coding region of the ZPI gene. Two mutations were regarded as functionally significant as they produced stop codons at arginine 67 and tryptophan 303 and would be expected to result in the loss of ZPI activity. These stop codon mutations were found in 8 patients and 2 controls. Based on these results, a power calculation was performed that showed that an additional 100 patients and controls would be necessary to confirm that the stop codon mutations were statistically significant risk factors for thrombosis. Bidirectional allele specific PCR was developed to rapidly identify these stop codon mutations and a further 100 patients and 100 controls were examined using this technique. The stop codon mutations were found in 11 patients (W303X n=8: R67X n=3) and only 2 controls (R67X n=2) (two sided Fishers exact p=0.02) with an odds ratio = 5.7 (95%CI, 1.25 to 26.0). In addition two further non-conservative mutations were identified in two other thrombosis patients. These resulted in a serine to tyrosine (codon 122) and a phenylalanine to leucine (codon 124) change in the region homologous to the D helix of other heparin activated serpins. These changes, involving bulky aromatic amino acid residues, have the potential to disrupt the structure and function of the D helix. We identified a further 12 mutations/polymorphisms (C454G, C574T, A603G, A647G, G752A, A947T, C1276T, G1277A, G1438A, A1617C, G1789T and C1811T (italics: not previously described)). The significance of these mutations is uncertain. We have identified mutations causing stop codons, which will result in loss of ZPI function, in 4.4% of patients who present with venous thrombosis before the age of 60 years, compared with an incidence of only 0.8% in controls. Further studies are ongoing to measure the plasma concentration of ZPI in the affected individuals. Our results support an association between mutations in the ZPI gene and venous thrombosis. We propose that ZPI deficiency as a result of these mutations is potentially a new form of thrombophilia.

Von Willebrand Disease Type 2M “Vicenza” in Italian and German Patients: Identification of the First Candidate Mutation (G3864A; R1205H) in 8 Families

2000 ◽  
Vol 83 (01) ◽  
pp. 136-140 ◽  
Author(s):  
Augusto Federici ◽  
Ulrich Budde ◽  
Giancarlo Castaman ◽  
Elke Drewke ◽  
Sonja Krey ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Mutation Screening ◽  
Normal Subjects ◽  
Disease Type ◽  
Von Willebrand Disease ◽  
Heteroduplex Analysis ◽  
Molecular Defect ◽  
Coding Region ◽  
Candidate Mutation ◽  
Von Willebrand

SummaryVon Willebrand disease type 2M “Vicenza” (VWD 2M V) is characterised by autosomal dominant inheritance, low von Willebrand factor (VWF) and the presence of “supranormal” multimers in plasma. This specific phenotype has been described in Italian and recently also in German patients. The molecular defect is linked to the VWF gene. However, no specific mutations have been identified until now. We analysed the complete coding region and adjacent intron sequences of the VWF gene in Italian families in comparison to German families with VWD 2M V by a PCR-based mutation screening, combined with SSC-and heteroduplex-analysis of exons 2 through 52, followed by direct sequencing. We identified the first heterozygous candidate mutation (G3864A; R1205H) in all affected members of the 7 Italian families and in 1 German patient but not in the unaffected family members nor on 100 chromosomes of normal subjects, suggesting a causal relationship between the mutation and the phenotype. Haplotype identity, with minor deviations in one Italian family, suggests a common but not very recent genetic origin of R1205H.

scholarly journals The association of CYP2D6 gene polymorphisms in the full-length coding region with higher recurrence rate of vivax malaria in Yunnan Province, China

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Herong Huang ◽  
Ying Dong ◽  
Yanchun Xu ◽  
Yan Deng ◽  
Canglin Zhang ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Gene Mutation ◽  
Dna Sequences ◽  
Vivax Malaria ◽  
Yunnan Province ◽  
Reference Sequence ◽  
Radical Cure ◽  
Coding Region ◽  
Cyp2d6 Gene ◽  
Genotype 4

Abstract Background Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene. Methods Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation “reference” sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed. Results Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104–4.252, P < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, *10 and *39, were confirmed by comparison, and CYP2D6*10 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (Χ2 = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group. Conclusion Mutation of CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D6*2 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.

scholarly journals Gene mapping in an anophthalmic pedigree of a consanguineous Pakistani family opened new horizons for research

2016 ◽  
Vol 19 (1) ◽  
pp. 77-84
Author(s):  
S Saleha ◽  
M Ajmal ◽  
S Zafar ◽  
A Hameed
Keyword(s):  
Linkage Mapping ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Ocular Tissue ◽  
Inherited Disease ◽  
Pakistani Family ◽  
New Horizons ◽  
Family Pedigree ◽  
Environmental Causes ◽  
Short Tandem

ABSTRACTClinical anophthalmia is a rare inherited disease of the eye and phenotype refers to the absence of ocular tissue in the orbit of eye. Patients may have unilateral or bilateral anophthalmia, and generally have short palpebral fissures and small orbits. Anophthalmia may be isolated or associated with a broader syndrome and may have genetic or environmental causes. However, genetic cause has been defined in only a small proportion of cases, therefore, a consanguineous Pakistani family of the Pashtoon ethnic group, with isolated clinical anophthalmia was investigated using linkage mapping. A family pedigree was created to trace the possible mode of inheritance of the disease. Blood samples were collected from affected as well as normal members of this family, and screened for disease-associated mutations. This family was analyzed for linkage to all the known loci of clinical anophthalmia, using microsatellite short tandem repeat (STR) markers. Direct sequencing was performed to find out disease-associated mutations in the candidate gene. This family with isolated clinical anophthalmia, was mapped to the SOX2 gene that is located at chromosome 3q26.3-q27. However, on exonic and regulatory regions mutation screening of the SOX2 gene, the disease-associated mutation was not identified. It showed that another gene responsible for development of the eye might be present at chromosome 3q26.3-q27 and needs to be identified and screened for the disease-associated mutation in this family.

scholarly journals Detection of erythromycin-resistant determinants by PCR.

1996 ◽  
Vol 40 (11) ◽  
pp. 2562-2566 ◽  
Author(s):  
J Sutcliffe ◽  
T Grebe ◽  
A Tait-Kamradt ◽  
L Wondrack
Keyword(s):  
Direct Sequencing ◽  
Pcr Primers ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Gene Products ◽  
Mechanisms Of Resistance ◽  
Erythromycin Resistance ◽  
Surveillance Studies ◽  
Resistance Determinants ◽  
Pcr Products

Erythromycin resistance determinants include Erm methylases, efflux pumps, and inactivating enzymes. To distinguish the different mechanisms of resistance in clinical isolates, PCR primers were designed so that amplification of the partial gene products could be detected in multiplex PCRs. This methodology enables the direct sequencing of amplified PCR products that can be used to compare resistance determinants in clinical strains. Further, this methodology could be useful in surveillance studies of erythromycin-resistant determinants.

scholarly journals Screening of Three Different Alleles of mtDNA (G709A, G3496T, A3537G) in Subpopulation of UKM Students

2018 ◽  
Vol 5 (1) ◽  
pp. 37-40
Author(s):  
Seri Mirianti Ishar ◽  
Jeyaganesan Pillay a/l Balaraman ◽  
Muhammad Jefri Mohd Yusof ◽  
Khairul Osman ◽  
Lee Loong Chuen
Keyword(s):  
Uv Light ◽  
Forensic Genetics ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Documentation System ◽  
Single Nucleotide ◽  
National University ◽  
Pcr Products ◽  
Malay Population ◽  
Mutation Allele

Human DNA consists of nucleus DNA (nDNA) and mitochondrial DNA (mtDNA). Both are valuable in medicine and forensic genetics but in this project, single nucleotide polymorphisms (SNPs) in mtDNA are used to trace the mutation occurred. Mutations in the sequence of alleles can lead to haplogroup variation and also certain diseases. The purpose of this study is to screen of mutations on alleles G709A, G3496T, and A3537G in Malay population of The National University of Malaysia (UKM) students. These SNPs lie in the ND1 (nitrogen dehydrogenase subunit 1) coding region, and the reports state that these three alleles are prone to mutate. From MitoMap Web site, the mutations of these alleles are reported to have potential in causing several diseases with the collaboration of other SNPs mutation. Allele G709A is reported to have an association with hearing loss and Leber Hereditary Optic Neuropathy (LHON) while allele G3496T is associated to LHON only. Allele A3537G is related to diabetes. A total of 100 DNA samples were collected from Malay students of UKM and preserved on FTA card to be purified later. The concentration of the DNA on the purified FTA card was between 10μM to 20μM. An attempt was made by amplifying those three loci from the genomic DNA. The amplified product was detected and separated using 1% gel electrophoresis. Before sequencing, the PCR products were visualized under UV light using gel documentation system. All PCR products were sequenced to detect the mutation on every single position chosen. From the alignment of sequencing results, allele G709A and allele G3496T showed no mutation. Meanwhile four samples from alleles A3537G has the mutation. From the results obtained, it seems that mutations are rare in all selected alleles. It is recommended to increase the sample size and alleles selected in the future to increase the strength of the study. This study also should be applied to other populations in Malaysia such as Chinese and Indian.  

scholarly journals Human Rhinovirus Molecular Epidemiology and Genotyping in Iranian Military Trainees with Acute Respiratory Symptoms

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Fahimeh Safarnezhad Tameshkel ◽  
Ali Salimi Jeda ◽  
Ahmad Tavakoli ◽  
Mohammad Hadi Karbalaie Niya ◽  
Morteza Izadi ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Clinical Symptoms ◽  
Respiratory Infections ◽  
Direct Sequencing ◽  
Human Rhinovirus ◽  
P Value ◽  
Rt Pcr ◽  
Respiratory Problems ◽  
Pcr Products ◽  
Tract Infections

Background: Human rhinovirus (HRV) is still the most prevalent viral infection in humans and a significant cause of acute respiratory tract infections (ARTIs) in many communities, including military personnel undergoing basic training. Objectives: In this research, we assessed the molecular epidemiology, genotyping, and phylogenetic classification of HRVs in Iranian military trainees with respiratory infections (RI). Methods: For HRV identification and genotyping, respiratory specimens were obtained, and RT-PCR was conducted for genotyping and phylogenetic analysis of HRV utilizing primers for the 5-UTR region. Results: Among 400 Iranian military trainees (average age of 21 ± 4 years, the range of 18 - 57 years) with respiratory infections, HRV was detected in 29 patients (7%) using RT-PCR. The direct sequencing of PCR products from 10 specimens showed that the incidence of type A (n = 5, 50%) was higher than that of type B (n = 4, 40%) and type C (n = 1, 10%). There were no significant associations between HRV and respiratory and clinical symptoms, blood group, and indoor or outdoor conditions (P-value > 0.05). Conclusions: This research was the first to record HRV as a significant cause of respiratory problems among military trainees in Iran, with a frequency of 7%. The most prevalent genotype was HRV-A, which may be applicable in epidemiological and clinical studies, as well as vaccination plans.

scholarly journals ANALYSIS OF THE ASSOCIATION OF THE METHYLATION LEVELS OF MIR10B AND MIR21 GENES IN BLOOD LEUKOCYTES WITH ADVANCED CAROTID ATHEROSCLEROSIS

2018 ◽  
Vol 33 (2) ◽  
pp. 77-82
Author(s):  
Iu. A. Koroleva ◽  
A. A. Zarubin ◽  
A. V. Markov ◽  
A. N. Kazancev ◽  
O. L. Barbarash ◽  
...  
Keyword(s):  
Risk Factors ◽  
Dna Methylation ◽  
Methylation Level ◽  
Carotid Atherosclerosis ◽  
Gene Promoter ◽  
Control Group ◽  
Coding Region ◽  
Blood Leukocytes ◽  
Atherosclerosis Risk Factors

Complications of atherosclerosis remain the leading cause of morbidity and mortality worldwide. MiRNAs are short regulatory molecules that are involved in all processes of pathogenesis. Expression of miRNAs is regulated by DNA methylation. Methylation and/or expression of MIR10B and MIR21 genes are known to vary in atherosclerotic tissues of the arteries, but there is no data about the changes in the methylation levels of these genes in blood leukocytes and their association with atherosclerosis risk factors.Objective.To evaluate the association of methylation levels of MIR10B and MIR21 genes in the blood leukocytes with risk factors and pathogenetically significant traits of carotid atherosclerosis.Material and Methods. DNA for the study was extracted from the samples of blood leukocytes of 122 patients with advanced carotid atherosclerosis as well as from blood leukocytes of 135 individuals in the control group. The DNA methylation level was analyzed by bisulfite pyrosequencing.Results.The methylation level of the MIR10B and MIR21 genes in leukocytes of patients with atherosclerosis is higher than in the leukocytes of the control group. In leukocytes of patients with carotid atherosclerosis the methylation level of the MIR21 gene promoter was correlated with type 2 diabetes and serum cholesterol level, and the methylation level of the coding region of the MIR10B gene was correlated with smoking.Conclusions.The level of DNA methylation in the regions of MIR10B and MIR21 genes in blood leukocytes is associated with the risk of advanced atherosclerosis of the carotid arteries. 

Mutation Profiling in Haemophilia A

2001 ◽  
Vol 85 (04) ◽  
pp. 577-579 ◽  
Author(s):  
J. Oldenburg
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Mutation Screening ◽  
Screening Method ◽  
Coagulation Factor ◽  
Causative Mutation ◽  
Haemophilia A ◽  
Coding Region ◽  
Systematic Analysis ◽  
Intron 22 Inversion ◽  
Gradient Gel Electrophoresis

SummaryHaemophilia A is a X-linked recessive bleeding disorder caused by deficiency or absence of coagulation factor VIII (FVIII) due to heterogeneous defects in the FVIII gene. The large size of the FVIII gene (26 exons spanning 186 kb) has hampered mutation analysis for many years. In 1991 the first systematic analysis of the complete coding region of the FVIII gene was performed by Higuchi et al. using Denaturing Gradient Gel Electrophoresis (DGGE) as a mutation screening method (1, 2). Notably, the causative mutation was not found in about half of the severely affected patients (1). This mystery was solved in 1993, when the intron 22 inversion was discovered (3, 4) that accounts for about 50% of the severe haemophilia A cases. The inversion mutation can be easily detected by Southern Blot. A recently described PCR-based method is more sophisticated, however once established, it allows rapid and convenient detection of the intron 22 inversion (5).

Abstract 562: Association Of The Heteroplasmic Level Of The Mitochondrial Gene Nd2 5178c>A Variant With Essential Hypertension

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Tong Yin ◽  
Yusong Zhang ◽  
Xi Chen ◽  
Jie Yang ◽  
Yuxiao Zhang ◽  
...  
Keyword(s):  
Essential Hypertension ◽  
Protective Effect ◽  
Total Cholesterol ◽  
Mitochondrial Gene ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Health Examination ◽  
Coding Region ◽  
Chinese Han ◽  
Blood Leukocytes

BACKGROUND: The mitochondrial genetic variant of nicotinamide adenine dinucleotide dehydrogenase subunit-2 (ND2) 5178C>A (Leu237Met) was previously found to have a protective effect against essential hypertension (EH) in Chinese and Japanese populations. AIM: The present study aimed to determine whether the protective effect of ND2 5178C>A against EH is associated with the mitochondrial heteroplasmy level of the variant. METHODS: This case-control study involved 109 unrelated Chinese-Han matched pairs of essential hypertension and healthy normotensive controls. All consented subjects were recruited consecutively from Institute of Geriatric Cardiology and Health Examination Center in General Hospital of Chinese People’s Liberation Army from February 2013 to January 2014. Demographic and clinical characteristics were recorded. Mitochondrial DNA was isolated from the blood leukocytes for each subject. The level of heteroplasmy for 5178C>A variant occurring in the coding region of ND2 was quantified using real-time polymerase chain reaction (PCR) method. RESULTS: The ND2 5178C>A heteroplasmy level ranged between 27% and 77%, with a median level of 44%. Between the upper and lower quartiles of 5178C>A heteroplasmy distribution, significant differences were observed for systolic blood pressure (116±17 vs. 129±25 mmHg, P=0.004) , body mass index (BMI) (24.6±3.3 vs.26.5±3.6, P=0.008) , total cholesterol (4.0±0.8 vs. 3.4±0.9 mmol/L, P<0.001) , low density lipoprotein (2.5±0.7 vs. 1.5±0.7 mmol/L, P<0.001) and triglyceride levels (1.8±1.6 vs. 4.0±1.6 mmol/L, P<0.001). ND2 5178C>A heteroplasmic level was negatively correlated with the presence of hypertension (r = -0.38, P < 0.001), BMI (r = -0.19, P = 0.007), and the level of total cholesterol (r = -0.18, P = 0.01). Logistic regression analysis showed that participants with higher ND2 5178C>A heteroplasmy level (≥44%) were found to have significantly lower prevalence of essential hypertension (29%, 32 in 109), as compared to those with lower (< 44%) ND2 5178C>A heteroplasmy level (71%, 77 in 109) (OR: 0.18, 95% CI: 0.10-0.31, P<0.001). CONCLUSION: The protective effect of mitochondrial ND2 5178C>A against EH is associated with the heteroplasmy level of the variant.

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