scholarly journals Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

2008 ◽  
Vol 54 (6) ◽  
pp. 964-972 ◽  
Author(s):  
Clara R L Teo ◽  
Wen Wang ◽  
Hai Yang Law ◽  
Caroline G Lee ◽  
Samuel S Chong
Keyword(s):  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
False Positive Rate ◽  
Screening Method ◽  
Melting Curve ◽  
Single Step ◽  
Cag Repeat ◽  
Clinical Samples ◽  
Melting Curve Analysis

Abstract Background: Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps. Methods: We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (Tms) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (≤26 repeats), intermediate (27–35 repeats), reduced penetrance expanded (36–39 repeats), and fully penetrant expanded (≥40 repeats)]. We also measured well-to-well variation in Tm across the thermal block and validated cutoff Tms with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals. Results: We observed a strong correlation between CAG-repeat size and amplicon Tm among the reference DNA samples. Use of the Tm cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis. Conclusions: This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.

scholarly journals Library preparation and MiSeq sequencing for the genotyping-by-sequencing of the Huntington disease HTT exon one trinucleotide repeat and the quantification of somatic mosaicism

2020 ◽  
Author(s):  
Marc Ciosi ◽  
Sarah A. Cumming ◽  
Asma M. Alshammari ◽  
Efthymia Symeonidi ◽  
Pawel Herzyk ◽  
...  
Keyword(s):  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
Fragment Size ◽  
Somatic Mosaicism ◽  
Genotyping By Sequencing ◽  
Amplicon Sequencing ◽  
Cag Repeat ◽  
Miseq Sequencing ◽  
Flanking Sequence

Abstract Huntington disease \(HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the _HTT_ gene. Affected individuals inherit more than 40 repeats and the CAG repeat is genetically unstable in both the germline and soma. Molecular diagnosis and genotyping of the CAG repeat is traditionally performed by estimation of PCR fragment size. However, this approach is complicated by the presence of an adjacent polymorphic CCG repeat and provides no information on the presence of variant repeats, flanking sequence variants or on the degree of somatic mosaicism. To overcome these limitations, we have developed an amplicon-sequencing protocol that allows the sequencing of hundreds of samples in a single MiSeq run. The composition of the _HTT_ exon one trinucleotide repeat locus can be determined from the MiSeq sequencing reads generated. With sufficient sequencing depth, such MiSeq data can also be used to quantify the degree of somatic mosaicism of the _HTT_ CAG repeat in the tissue analysed.

scholarly journals A Case of Previously Unsuspected Huntington Disease Diagnosed at Autopsy

2017 ◽  
Vol 7 (1) ◽  
pp. 136-144
Author(s):  
Catherine R. Miller ◽  
Nobby C. Mambo ◽  
Jianli Dong ◽  
Gerald A. Campbell
Keyword(s):  
Genetic Testing ◽  
Huntington Disease ◽  
Clinical Symptoms ◽  
Neurodegenerative Disorder ◽  
Case Reports ◽  
Neuronal Loss ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Psychiatric Disturbances ◽  
Personality Changes

Huntington disease (HD) is a neurodegenerative disorder with a worldwide prevalence of four to ten per 100 000. It is characterized by choreiform movements, behavioral/psychiatric disturbances, and eventual cognitive decline. Symptoms usually present between 30 and 50 years of age and the diagnosis is based on the combination of clinical symptoms, family history, and genetic testing. A variation of HD, juvenile Huntington disease (JHD), presents earlier, with more severe symptoms and with a worse prognosis. Symptoms are different in JHD, with personality changes and learning difficulties being the predominant presenting features. Seizures are common in JHD, and chorea is uncommon; movement disorders at presentation of JHD are predominantly nonchoreiform. The inheritance pattern for both HD and JHD is autosomal dominant and the disease is caused by an elongation of the CAG repeat in the huntingtin gene. There are many published case reports of Huntington disease that were confirmed at autopsy, but to our knowledge, there are no reports in the literature where the diagnosis of Huntington disease was first made at autopsy. We present a case of a 28-year-old African-American male who was in a state of neglect due to a lifetime of abuse, cognitive difficulties, and seizures, whose cause of death was pneumonia. The gross autopsy findings included bilateral caudate nucleus atrophy and lateral ventricular dilation. Microscopically, severe bilateral neuronal loss and gliosis of the caudate and putamen nuclei were seen. Genetic testing for the number of CAG repeats confirmed the diagnosis and was consistent with JHD.

scholarly journals Gene expression profiles complement the analysis of genomic modifiers of the clinical onset of Huntington disease

2019 ◽  
Author(s):  
Galen E.B. Wright ◽  
Nicholas S. Caron ◽  
Bernard Ng ◽  
Lorenzo Casal ◽  
Xiaohong Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Huntington Disease ◽  
Neurodegenerative Disorder ◽  
Age Of Onset ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cag Repeat ◽  
Biologically Relevant ◽  
Clinical Onset ◽  
Genome Wide Data

ABSTRACTHuntington disease (HD) is a neurodegenerative disorder that is caused by a CAG repeat expansion in the HTT gene. In an attempt to identify genomic modifiers that contribute towards the age of onset of HD, we performed a transcriptome wide association study assessing heritable differences in genetically determined expression in diverse tissues, employing genome wide data from over 4,000 patients. This identified genes that showed evidence for colocalization and replication, with downstream functional validation being performed in isogenic HD stem cells and patient brains. Enrichment analyses detected associations with various biologically-relevant gene sets and striatal coexpression modules that are mediated by CAG length. Further, cortical coexpression modules that are relevant for HD onset were also associated with cognitive decline and HD-related traits in a longitudinal cohort. In summary, the combination of population-scale gene expression information with HD patient genomic data identified novel modifier genes for the disorder.

scholarly journals A Multiplex Snapback Primer System for the Enrichment and Detection ofJAK2V617F andMPLW515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Zhiyuan Wu ◽  
Yunqing Zhang ◽  
Xinju Zhang ◽  
Xiao Xu ◽  
Zhihua Kang ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Amplification Efficiency ◽  
Primer Sets ◽  
Low Ionic Strength ◽  
Multiplex System

A multiplex snapback primer system was developed for the simultaneous detection ofJAK2V617F andMPLW515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets forJAK2andMPLmutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.

scholarly journals Nonisotopic method for accurate detection of (CAG)n repeats causing Huntington disease

1996 ◽  
Vol 42 (10) ◽  
pp. 1601-1603 ◽  
Author(s):  
M Muglia ◽  
O Leone ◽  
G Annesi ◽  
A L Gabriele ◽  
E Imbrogno ◽  
...  
Keyword(s):  
Silver Nitrate ◽  
Molecular Diagnosis ◽  
Huntington Disease ◽  
Trinucleotide Repeat ◽  
Neurodegenerative Disorder ◽  
The Novel ◽  
Direct Visualization ◽  
Pcr Products ◽  
Radioactive Background ◽  
Presymptomatic Diagnosis

Abstract Huntington disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG)n located at the 5' end of the novel IT15 gene. Discovery of this expansion allows the molecular diagnosis of HD by measuring repeat length. We applied a simple nonisotopic method to detect (CAG)n repeats, avoiding both radioactive and Southern transfer analysis. The assay is based on direct visualization of electrophoresed PCR products, after silver nitrate gel staining. Its accurate sizing of HD alleles allows presymptomatic diagnosis of at-risk persons. By avoiding isotopic manipulations, the method is safe and accurate, with no radioactive background bands. Furthermore, because it permits direct allele visualization after gel staining, the method is simple and rapid, allowing allele sizing within hours rather than days.

scholarly journals Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1019-1029 ◽  
Author(s):  
Seyed A. Ghorashi ◽  
Amir H. Noormohammadi ◽  
Philip F. Markham
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Trinucleotide Repeat ◽  
Melting Curve ◽  
Mycoplasma Gallisepticum ◽  
Chronic Respiratory Disease ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Specific Pathogen ◽  
The Usa

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145–352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.

scholarly journals Early white matter pathology in the fornix of the limbic system in Huntington disease

2021 ◽  
Vol 142 (5) ◽  
pp. 791-806
Author(s):  
Sanaz Gabery ◽  
Jing Eugene Kwa ◽  
Rachel Y. Cheong ◽  
Barbara Baldo ◽  
Costanza Ferrari Bardile ◽  
...  
Keyword(s):  
Transcription Factor ◽  
White Matter ◽  
Limbic System ◽  
Huntington Disease ◽  
Neurodegenerative Disorder ◽  
Cag Repeat ◽  
Polycomb Repressive Complex 2 ◽  
Transcription Factor Target ◽  
Motor Component ◽  
White Matter Pathology

AbstractHuntington disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The typical motor symptoms have been associated with basal ganglia pathology. However, psychiatric and cognitive symptoms often precede the motor component and may be due to changes in the limbic system. Recent work has indicated pathology in the hypothalamus in HD but other parts of the limbic system have not been extensively studied. Emerging evidence suggests that changes in HD also include white matter pathology. Here we investigated if the main white matter tract of the limbic system, the fornix, is affected in HD. We demonstrate that the fornix is 34% smaller already in prodromal HD and 41% smaller in manifest HD compared to controls using volumetric analyses of MRI of the IMAGE-HD study. In post-mortem fornix tissue from HD cases, we confirm the smaller fornix volume in HD which is accompanied by signs of myelin breakdown and reduced levels of the transcription factor myelin regulating factor but detect no loss of oligodendrocytes. Further analyses using RNA-sequencing demonstrate downregulation of oligodendrocyte identity markers in the fornix of HD cases. Analysis of differentially expressed genes based on transcription-factor/target-gene interactions also revealed enrichment for binding sites of SUZ12 and EZH2, components of the Polycomb Repressive Complex 2, as well as RE1 Regulation Transcription Factor. Taken together, our data show that there is early white matter pathology of the fornix in the limbic system in HD likely due to a combination of reduction in oligodendrocyte genes and myelin break down.

scholarly journals McSpoligotyping, a One-Step Melting Curve Analysis-Based Protocol for Spoligotyping of Mycobacterium tuberculosis

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Xiaohong Zeng ◽  
Ye Xu ◽  
Yang Zhou ◽  
Hui Li ◽  
Rongrong Zheng ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Melting Curve ◽  
Direct Repeat ◽  
Single Step ◽  
Ease Of Use ◽  
Melting Curve Analysis ◽  
Nucleotide Polymorphisms ◽  
Content Type ◽  
One Step ◽  
Epidemiology Studies

ABSTRACT The direct repeat (DR) region in the Mycobacterium tuberculosis (MTB) genome is composed of highly polymorphic direct variant repeats, which are the basis of spacer oligonucleotide typing (spoligotyping) to study the population structure and epidemiology of M. tuberculosis. However, the membrane hybridization-based detection format requires various post-PCR manipulations and is prone to carryover contamination, restricting its wide use in high-TB-burden and resource-limited countries. We developed a one-step spoligotyping protocol, termed McSpoligotyping, based on real-time PCR. The typing results can be generated within 3 h by a single step of DNA addition. When evaluated with a collection of 1,968 isolates of MTB, McSpoligotyping agreed 97.71% (1,923/1,968) by sample and 99.93% (84,568/84,624) by spacer with traditional spoligotyping. Sequencing results showed that McSpoligotyping was even more accurate than spoligotyping (99.34% versus 98.37%). Further exploration of the false results of McSpoligotyping revealed the presence of single-nucleotide polymorphisms in the DR region. We concluded that McSpoligotyping could be used in epidemiology studies of tuberculosis by taking advantage of the shortened procedure, ease of use, and compatibility of results with standard spoligotyping.

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