A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes

doi:10.1006/mcpr.1993.1034

Molecular and Cellular Probes

Volume 7, Issue 3, June 1993, Pages 235-239

https://doi.org/10.1006/mcpr.1993.1034 Get rights and content

Abstract

The Huntington's Disease (HD) Collaborative Research Group has recently published sequence of a new cDNA, IT15, containing a polymorphic trinucleotide (CAG)_n repeat that is expanded and unstable on HD chromosomes. There is a correlation between the repeat size and the age of onset of symptoms. The suggested polymerase chain reaction (PCR) assay of the (CAG)_n repeat requires unusual reaction components and primer concentrations and the use of 5% polyacrylamide sequencing gels to resolve the amplification products. We present a simple PCR assay that produces a smaller product using standard reaction conditions. This gives better resolution of the (CAG)_n expansion observed on HD chromosomes by acrylamide gel electrophoresis and allows sufficient product to be obtained to perform assays using agarose gels. This will allow diagnostic labs to do rapid and accurate presymptomatic testing of HD in high risk families.

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Cited by (220)

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Genome-wide association studies (GWASs) of Huntington disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction by using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWASs. Combined with imputation with the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified additional modifier effects at the PMS1 and PMS2 loci and implicated a potential second locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.
CAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset
2019, Cell
Citation Excerpt :
These quality control filters generated a total of 10,986,607 imputed SNPs for 9,064 HD subjects with age at onset data for genetic association analysis. HTT uninterrupted CAG repeat size was estimated using a modified PCR amplification assay (Warner et al., 1993), adapted for the ABI3730XL DNA sequencer in a 96 well plate format. Each plate includes reactions for genomic DNA HTT CAG size standards, previously sequenced and known to have different uninterrupted CAG repeat lengths in the normal and expanded CAG repeat ranges.
Variable, glutamine-encoding, CAA interruptions indicate that a property of the uninterrupted HTT CAG repeat sequence, distinct from the length of huntingtin’s polyglutamine segment, dictates the rate at which Huntington’s disease (HD) develops. The timing of onset shows no significant association with HTT cis-eQTLs but is influenced, sometimes in a sex-specific manner, by polymorphic variation at multiple DNA maintenance genes, suggesting that the special onset-determining property of the uninterrupted CAG repeat is a propensity for length instability that leads to its somatic expansion. Additional naturally occurring genetic modifier loci, defined by GWAS, may influence HD pathogenesis through other mechanisms. These findings have profound implications for the pathogenesis of HD and other repeat diseases and question the fundamental premise that polyglutamine length determines the rate of pathogenesis in the “polyglutamine disorders.”
Leukocyte telomere shortening in Huntington's disease
2019, Journal of the Neurological Sciences
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded CAG repeat. Though symptom onset commonly occurs at midlife and inversely correlates with the CAG repeat expansion, age at clinical onset and progression rate are variable. In the present study we investigated the relationship between leukocyte telomere length (LTL) and HD development. LTL was measured by real-time PCR in manifest HD patients (HD, n = 62), pre-manifest HD patients (pre-HD, n = 38), and age-matched controls (n = 76). Significant LTL differences were observed between the three groups (p < .0001), with LTL values in the order: HD < pre-HD < controls. The relationship between LTL and age was different in the three groups. An inverse relationship between mean LTL and CAG repeat number was found in the pre-HD (p = .03). The overall data seem to indicate that after age 30 years, LT begins to shorten markedly in pre-HD patients according to CAG number and increasing age, up to the values observed in HD. This very suggestive picture allowed us to hypothesize that in pre-manifest HD, LTL could be a measure of time to clinical HD onset. The possible use of LTL as a reliable biomarker to track HD development and progression was evaluated and discussed.
Epidemiology of Huntington disease
2017, Handbook of Clinical Neurology
Huntington disease (HD) is an autosomal-dominant neurologic disorder caused by an expanded CAG trinucleotide repeat mutation in patients with characteristic motor signs and specific brain pathology. A repeat of 36 CAG or more can lead to the disease, with increased penetrance and decreased age of onset at longer CAG repeats. The epidemiology of HD thus depends on ascertainment of individuals with the expanded CAG mutation, and on examination of clinical signs to accurately assess disease onset. A larger number of individuals have an expanded CAG repeat than actively manifest the disease due to adult onset in the majority of cases. Because of incomplete penetrance at the lower end of the pathogenic CAG repeat range, the frequency of the expanded CAG repeat in the general population may be higher than previously thought. Genetic differences and changing demographics may account for geographic and ethnic variation in the prevalence of HD between populations and over time. There are gross differences in the prevalence of HD by ancestry, with a much higher rate of the disease in populations of European descent. Molecular studies have elucidated genetic causes for these population-specific differences, possibly resulting from differences in the HD new mutation rate.
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2015, Neurobiology of Disease
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American college of medical genetics and genomics standards and guidelines for clinical genetics laboratories, 2014 edition: Technical standards and guidelines for huntington disease
2014, Genetics in Medicine
Citation Excerpt :
PCR methods. Several sets of primers, polymerase chain reaction (PCR) conditions, and amplicon separation and detection techniques have been published.18,19,20,21,22 Regardless of the particular PCR-based strategy selected, it is important for assay conditions and post-PCR analyses to be optimized to ensure the accurate and unambiguous quantitation of repeat length (Figure 2a,b).
Huntington disease is an autosomal-dominant neurodegenerative disease of mid-life onset caused by expansion of a polymorphic trinucleotide (CAG) repeat. Variable penetrance for alleles carrying 36–39 repeats has been noted, but the disease appears fully penetrant when the repeat numbers are >40. An abnormal CAG repeat may expand, contract, or be stably transmitted when passed from parent to child. Assays used to diagnose Huntington disease must be optimized to ensure the accurate and unambiguous quantitation of CAG repeat length. This document provides an overview of Huntington disease and methodological considerations for Huntington disease testing. Examples of laboratory reports are also included.
Genet Med16 12, e2.

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Original ArticlesA new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes

Abstract

Original Articles
A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington's disease chromosomes