scholarly journals Novel manifestations of Warburg micro syndrome type 1 caused by a new splicing variant of RAB3GAP1: a case report

BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Raziyeh Khalesi ◽  
Ehsan Razmara ◽  
Golareh Asgaritarghi ◽  
Ali Reza Tavasoli ◽  
Yasser Riazalhosseini ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cerebral White Matter ◽  
Global Developmental Delay ◽  
Spastic Diplegia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mutation System ◽  
Warburg Micro Syndrome

Abstract Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Exons sequencing identifies a novel mutation in GPR157 in a high myopia family

2020 ◽  
Author(s):  
Tao Tang ◽  
Mohan Liu ◽  
Ting Wei ◽  
Lin Deng ◽  
Yueyang Zhang ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
High Myopia ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Rt Pcr ◽  
Genetic Characteristics ◽  
Reverse Transcription Pcr ◽  
Whole Exome ◽  
Reduced Penetrance ◽  
Next Generation Sequencing Ngs

Abstract Background Next-generation sequencing (NGS) and whole exome sequencing (WES) have identified many potential disease-causing loci and genetic mutations of high myopia(HM). However, these known genes can only explain the heritability of a small proportion of HM patients. A large proportion of variants have yet to be discovered. Herein we aimed to investigate the genetic characteristics of HM through a Chinese HM family(the inheritance pattern unknown) . Methods We performed WES on the parent-offspring trio and identified mutations by Sanger sequencing. All the members in this family were sequenced to validate phenotype co-segregated with candidate genes via Sanger sequencing as well. Besides, mutations detected were further evaluated in a cohort of 110 sporadic high myopia controls and 200 unrelated ethically-matched controls. And reverse transcription PCR(RT-PCR) was applied to measure the mRNA expression levels of GPR157 in the 4-week-old KM mice. Results A novel heterozygous nonsense mutation, c.859C>T (p.Arg287*) of GPR157 gene, was detected in the proband and her father by WES. And this disease-associated mutation was not found in 310 control individuals. For the family under study, HM was classified as autosomal dominant inheritance with reduced penetrance. And RT-PCR results showed GPR157 was abundantly expressed in the eye. Conclusion The hybrid nonsense mutation of the GPR157 gene identified in this study may constitute a novel genetic cause of HM. Keywords :high myopia, WES, GPR157

scholarly journals Rapid Identification of Nine Microorganisms Causing Acute Respiratory Tract Infections by Single-Tube Multiplex Reverse Transcription-PCR: Feasibility Study

1999 ◽  
Vol 37 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Britta Gröndahl ◽  
Wolfram Puppe ◽  
Andrea Hoppe ◽  
Inka Kühne ◽  
Josef A. I. Weigl ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Reverse Transcription ◽  
Influenza A ◽  
Respiratory Tract Infections ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Tract Infections ◽  
Type 3

Acute respiratory tract infections (ARIs) are leading causes of morbidity and, in developing countries, mortality in children. A multiplex reverse transcription-PCR (RT-PCR) assay was developed to allow in one test the detection of nine different microorganisms (enterovirus, influenza A and B viruses, respiratory syncytial virus [RSV], parainfluenzaviruses type 1 and type 3, adenovirus,Mycoplasma pneumoniae, and Chlamydia pneumoniae) that do not usually colonize the respiratory tracts of humans but, if present, must be assumed to be the cause of respiratory disease. Clinical samples from 1,118 children admitted to the Department of Pediatrics because of an ARI between November 1995 and April 1998 were used for a first clinical evaluation. Detection of one of the microorganisms included in the assay was achieved for 395 of 1,118 (35%) clinical samples, of which 37.5% were RSV, 20% were influenza A virus, 12.9% were adenovirus, 10.6% were enterovirus, 8.1% were M. pneumoniae, 4.3% were parainfluenzavirus type 3, 3.5% were parainfluenzavirus type 1, 2.8% were influenza B virus, and 0.2% were C. pneumoniae. Seasonal variations in the rates of detection of the different organisms were observed, as was expected from the literature. The levels of concordance with the data obtained by commercially available enzyme immunoassays were 95% for RSV and 98% for influenza A. The results show that the multiplex RT-PCR–enzyme-linked immunosorbent assay is a useful and rapid diagnostic tool for the management of children with ARI. Studies of the overall benefit of this method with regard to the use of antibiotics, the use of diagnostic procedures including additional microbiological tests, and hospitalization rate and duration are warranted.

scholarly journals Multimethod Longitudinal HIV Drug Resistance Analysis in Antiretroviral-Therapy-Naive Patients

2017 ◽  
Vol 55 (9) ◽  
pp. 2785-2800 ◽  
Author(s):  
Aubin J. Nanfack ◽  
Andrew D. Redd ◽  
Jude S. Bimela ◽  
Genesis Ncham ◽  
Emmanuel Achem ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Antiretroviral Therapy ◽  
Sanger Sequencing ◽  
Resistance Testing ◽  
Amplification Refractory Mutation System ◽  
Hiv Drug Resistance ◽  
Arms Pcr ◽  
Mutation System ◽  
Major Mutation ◽  
Hiv 1

ABSTRACT The global intensification of antiretroviral therapy (ART) can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated and also in ART-naive patients. ART-naive HIV-1-infected patients from Cameroon were subjected to a multimethod HIVDR analysis using amplification-refractory mutation system (ARMS)-PCR, Sanger sequencing, and longitudinal next-generation sequencing (NGS) to determine their profiles for the mutations K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1-positive patients with highly diverse subtypes that underlined the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false-positive calls with ARMS-PCR. For 32/66 samples, we obtained NGS data and we observed two additional mismatches made up of minority variants (7% and 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus. HIVDR mutations can be sensitively detected by ARMS-PCR and sequencing methods with comparable performances. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.

scholarly journals Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern

2022 ◽  
Author(s):  
Dongyan Xiong ◽  
Xiaoxu Zhang ◽  
Mengjuan Shi ◽  
Nuo Wang ◽  
Ping He ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Cost Effective ◽  
Amplification Refractory Mutation System ◽  
Pcr Assay ◽  
High Quality ◽  
Reverse Transcription Pcr ◽  
Mutation System ◽  
The Common ◽  
Current Stage

The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed.

scholarly journals Ποσοτικοποίηση των στελεχών του ιού της ηπατίτιδας Β (HBV) με τις μεταλλαγές που προσδίδουν αντοχή στην αντιϊκή θεραπεία

2011 ◽  
Author(s):  
Φωτεινή Ντζιώρα
Keyword(s):  
Deep Sequencing ◽  
Molecular Beacon ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Mutation System

ΕΙΣΑΓΩΓΗ: Η ανάπτυξη αντοχής στην αντιϊκή θεραπεία έχει σχετισθεί με την εμφάνιση μεταλλαγών και αποτελεί ένα βασικό πρόβλημα στην αντιμετώπιση της λοίμωξης του ιού της ηπατίτιδας Β (HBV). Οι συμβατικές μέθοδοι αδυνατούν να ανιχνεύσουν ιϊκούς υποπληθυσμούς με τις μεταλλαγές αντοχής αν αντιπροσωπεύουν λιγότερο από το 20% του συνολικού πληθυσμού του ιού στο υπό εξέταση δείγμα. ΣΚΟΠΟΣ: Η δημιουργία μιας μεθόδου για την ποσοτικοποίηση των ιϊκών υποπληθυσμών του HBV με τις κυριότερες μεταλλαγές που προσδίδουν αντοχή στην αντιϊκή θεραπεία (V173L, L180M, A181V, T184A, M204I/V και N236T).ΥΛΙΚΟ-ΜΕΘΟΔΟΣ: Η μεθοδολογία του συστήματος αναστολής της γονιδιακής επέκτασης του μεταλλαγμένου τύπου με PCR πραγματικού χρόνου (amplification refractory mutation system, ARMS RT-PCR) συνδυάστηκε με την τεχνολογία του μοριακού φάρου (molecular beacon) χρησιμοποιώντας το σύστημα του LightCycler®. ΑΠΟΤΕΛΕΣΜΑΤΑ: Η μέθοδος ARMS rt-PCR με μοριακό φάρο έχει ευαισθησία 10 αντιγράφων ανά αντίδραση και αναλυτική ευαισθησία 95% για το μεταλλαγμένο τύπο και 100% για το φυσικό τύπο. Η διακριτική ικανότητα της μεθόδου είναι 0.01%-0.25%. Ιϊκοί υποπληθυσμοί με τις μεταλλαγές αντοχής του HBV V173L, L180M, A181V, T184A, M204I και N236T βρέθηκαν σε υψηλή συχνότητα (38/60, 63.3%), σε δείγματα ασθενών με ηπατίτιδα Β, που δεν είχαν λάβει ποτέ θεραπεία με νουκλεοσ(τ)ιδικά ανάλογα. Σε 41 δείγματα ασθενών με HBV λοίμωξη, που είχαν λάβει θεραπεία, εκτός από τις μεταλλαγές αντοχής με επικρατούντα ιϊκό υποπληθυσμό, ανιχνεύθηκαν και ιϊκοί υποπληθυσμοί με μικρότερους λόγους, που πιθανόν να είχαν συμβάλλει στη συνολική εκδήλωση αντοχής για τον κάθε ασθενή.ΣΥΜΠΕΡΑΣΜΑΤΑ: Η μέθοδος ΑRMS rt-PCR με μοριακό φάρο παρουσιάζει συγκρίσιμη ευαισθησία, πιστότητα και διακριτική ικανότητα με άλλες εξαιρετικά ευαίσθητες μοριακές μεθόδους, όπως είναι το ultra-deep sequencing, αλλά πλεονεκτεί σε θέματα κόστους για την ανίχνευση των ιϊκών υποπληθυσμών του ιού της ηπατίτιδας Β με τις κυριότερες μεταλλαγές αντοχής.

scholarly journals The genotyping of Infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction

2014 ◽  
Vol 26 (6) ◽  
pp. 721-733 ◽  
Author(s):  
Shr-Wei Huang ◽  
Chia-Fang Ho ◽  
Kun-Wei Chan ◽  
Min-Chung Cheng ◽  
Jui-Hung Shien ◽  
...  
Keyword(s):  
Amplification Refractory Mutation System ◽  
Wild Type ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Infectious Bronchitis ◽  
Melting Temperatures ◽  
Qrt Pcr ◽  
Multiplex Amplification ◽  
Mutation System ◽  
Polymerase Chain

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China.

scholarly journals Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

2019 ◽  
Vol 11 (3) ◽  
Author(s):  
Narutchala Suwannakhon ◽  
Tanapat Pangeson ◽  
Teerapat Seeratanachot ◽  
Khwanruedee Mahingsa ◽  
Arunee Pingyod ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Beta Thalassemia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mutation System ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Thalassemia Mutation

We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT.

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