Development of genomic reference materials for Huntington disease genetic testing

Background: Accurate determination of the CAG repeat number is crucial for diagnostic and predictive testing for Huntington disease (HD). Current PCR-based assays can accurately size up to ∼110 HTT CAG repeats. Objective: To develop an improved assay capable of detecting larger CAG repeat expansions. Methods: A triplet-primed PCR (TP-PCR) assay was optimized and validated on 14 HD reference DNAs, including a sample carrying a large expansion of ∼180 CAG repeats. Results: All 14 HD reference samples showed 100% concordance with the previously verified allele sizes. For alleles under 45 CAGs, identical repeat sizes were obtained, while alleles larger than 46 CAGs were sized to within ±1 CAG. The improved TP-PCR assay successfully detected the ∼180 CAG repeat allele in an affected sample. Conclusion: This method extends the detection limit of large expanded alleles to at least ∼175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample.

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“…The results were completely concordant with the verified allele sizes of all samples [3,4] (fig. 1).…”

Section: Resultssupporting

confidence: 75%

“…RFU = Relative fluorescence units; NL = normal allele; IA = intermediate allele; EX = expanded allele; NA = not applicable. Sample IDs and their verified sizes [3] are shown in the right top corner. Sizes obtained from this study are indicated by arrows.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Detection and Sizing of the HTT CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay

Zhao

Lee²,

Law³

et al. 2016

Neurodegener Dis

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“…Among the 90 participants and 270 challenges, there were three "serious genotyping errors" (1.1%, 95% CI: 0.2-3.2%). Given the general availability of specific HD reference material for sizing, 14 it is unclear why these international laboratories participating in the CAP/ACMG survey are not performing to a higher standard. Information was not collected to determine whether these international participants were licensed by CLIA, and certified by CAP, New York State Department of Health, or an equivalent governmental/professional organization.…”

Section: Discussionmentioning

confidence: 99%

Assessing the analytic validity of molecular testing for Huntington disease using data from an external proficiency testing survey

Palomaki

Richards

2012

Genetics in Medicine

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original research article Purpose: Documenting high analytic validity of the molecular diagnostic test for Huntington disease is important because of counseling implications. This dominantly inherited adult onset disorder (prevalence of three or more per 100,000) is characterized by chorea, ataxia, and personality changes. The molecular basis is excessive CAG repeats in the HTT gene. methods:External proficiency testing survey results for Huntington disease were extracted (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010). Analytic interpretations and CAG repeat lengths were compared with published performance criteria.Results: Between 2008 and 2010, 33 US participating laboratories reported clinical test interpretations. Analytic validity was high (sensitivity: 99.5%, 95% confidence interval: 97.1-99.9%; specificity: 99.2%, 95% confidence interval: 97.1-99.9%). Repeat length errors occurred in 2.6% (95% confidence interval: 1.8-3.8%) of 1,060 allelic challenges, with most being minor or from a single participant. Past performance (2003)(2004)(2005)(2006)(2007) was similar. The 23 international participants had more total repeat length errors (17.5%, 95% confidence interval: 14.6-20.7%). Further analyses indicated that assessment criteria can be relaxed without jeopardizing analytic validity.conclusion: Analytic validity is high for Huntington disease testing among US laboratories. International survey participants had lower analytic validity and a higher proportion of poorly performing laboratories. The reasons for this are unclear.Genet Med 2012:14(1):69-75

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“…These include materials for various inherited genetic disorders, such as cystic fibrosis, 7 Huntington disease, 8 Fragile X, 9 and genetic conditions with relatively high prevalence in the Ashkenazi Jewish population 10 (for example, Bloom syndrome, Canavan disease, Fanconi anemia, Tay Sachs disease, familial dysautonomia, Gaucher disease, glycogen storage disease type 1a, Mucolipidosis IV, and Niemann-Pick disease). The program is currently developing new cell lines and conducting RM (on the lower level of the hierarchy) commutability and genotype characterization studies for Duchenne muscular dystrophy, pharmacogenetic loci and disorders included in the American College of Medical Genetics newborn screening panel.…”

Section: Discussionmentioning

confidence: 99%

“…Though sponsored by the CDC, much of the work performed by the GeT-RM, including RM priority decisions, specimen collection, material development, and molecular genetic characterization, occurs through voluntary collaborations with various clinical genetic laboratories. To date, the GeT-RM program has coordinated the development and/or commutability/genotype characterization of RMs for cystic fibrosis, 7 Huntington disease, 8 fragile X syndrome, 9 and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population 10 ; and has also provided information for several pharmacogenetic markers, including members of the CYP450 gene family, VKORC1, and UGT1A1.…”

mentioning

confidence: 99%

Development and Characterization of Reference Materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 Genetic Testing

Barker

Bale²,

Booker

et al. 2009

The Journal of Molecular Diagnostics

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Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control , monitoring of test performance, test validation , and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date , the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SER-PINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.

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Development of genomic reference materials for Huntington disease genetic testing

Abstract: These DNA materials, which are available from Coriell Cell Repositories, will facilitate accurate and reliable Huntington genetic testing.

Cited by 19 publications

References 12 publications

Enhanced Detection and Sizing of the <b><i>HTT</i></b> CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay

Enhanced Detection and Sizing of the <b><i>HTT</i></b> CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay

Assessing the analytic validity of molecular testing for Huntington disease using data from an external proficiency testing survey

Development and Characterization of Reference Materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 Genetic Testing

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