Triplet Repeat Primed PCR (TP PCR) in Molecular Diagnostic Testing for Friedreich Ataxia

Ciotti, Paola; Maria, Emilio Di; Bellone, Emilia; Ajmar, Franco; Mandich, Paola

doi:10.1016/s1525-1578(10)60523-5

Cited by 48 publications

(41 citation statements)

References 16 publications

(23 reference statements)

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“…Visual acuity was assessed with low letter visual acuity test with the Monoyer vision chart or finger counting. The presence and length of GAA-TRE were assessed essentially as described elsewhere [5,8]; for the present study, all patients' GAA-TRE were reassessed and directly compared in one assay. In patients heterozygous for GAA-TRE, the entire coding and exon flanking sequences of FXN were sequenced using standard procedures.…”

Section: Methodsmentioning

confidence: 99%

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

Ygland

Taroni

Gellera

et al. 2014

Parkinsonism & Related Disorders

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Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis.Ygland, Emil; Taroni, Franco; Gellera, Cinzia; Caldarazzo, Serena; Duno, Morten; Soller, Maria; Puschmann, Andreas Link to publication Citation for published version (APA): YGLAND, E. M. I. L., Taroni, F., Gellera, C., Caldarazzo, S., Duno, M., Soller, M., & Puschmann, A. (2014). Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis. Parkinsonism & Related Disorders, 20(8), 919-923. DOI: 10.1016/j.parkreldis.2014 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Results: p.R165P mutation carriers became wheelchair bound early, but had retained reflexes, better arm function, milder dysarthria, and were more independent in activities of daily living.One p.R165P mutation carrier developed psychosis. Frataxin levels were higher than in homozygous trinucleotide expansion patients. One patient with homozygous trinucleotide repeat expansions and comorbid hemochromatosis had more severe FRDA symptoms than his sibling without hemochromatosis. Conclusion:p.R165P patients progress to a less disabling disease state than typical FRDA.Comorbid hemochromatosis may worsen FRDA symptoms through additive effects on iron metabolism.

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Section: Methodsmentioning

confidence: 99%

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

Ygland

Taroni

Gellera

et al. 2014

Parkinsonism & Related Disorders

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“…39,40 Figure 2 shows the location of FMR1F and FMR1R primers and the concept of the method. Two reverse primers were tested, each harboring almost four CCG-units; the difference between FMR1R and FMR1CCGR is that the FMR1R primer included FMR1 sequences downstream of the CGG repeat tract, with the concept that presence of both triplet CCG sequences and downstream sequences might facilitate unwinding of the CGG secondary structure at the junction point and could also provide better specificity for FMR1 sequences.…”

Section: Assay Developmentmentioning

confidence: 99%

Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: Implications for fragile X syndrome carrier and newborn screening

Hantash

Goos

Tsao

et al. 2010

Genetics in Medicine

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Purpose: Fragile X syndrome is caused by expansion and subsequent methylation of a CGG trinucleotide repeat in the FMR1 5Ј-untranslated region. Southern blot analysis is typically required to determine expansion size for triplet repeat lengths Ͼ200. We describe a triplet-primed polymerase chain reaction-based method using automated capillary electrophoresis detection for qualitative assessment of expanded CGG repeats. Methods: The assay uses triplet-primed polymerase chain reaction in combination with GC-melting reagents and substitution of 7-deaza-2-deoxyGTP for dGTP. Amplicons are resolved by capillary electrophoresis. Results: A distinctive pattern of tapering or "stutter" polymerase chain reaction amplification was evident on capillary electrophoresis in male and female patients harboring all expanded allele lengths examined (up to 2000 CGG repeats) and could be used to differentiate normal, intermediate, premutation, and full mutation alleles. Full mutation alleles exhibited an additional late-migrating amplicon on capillary electrophoresis. Mixing experiments demonstrated sensitivity as low as 1% for detection of the full mutation allele. In a 1275-sample concordance study against our existing polymerase chain reaction platform (with Southern blot analysis for repeat lengths Ն55), the triplet-primed polymerase chain reaction method exhibited 100% concordance for normal, intermediate, expanded, and full mutation alleles. This method also detected the full mutation alleles in DNA isolated from blood spots. Conclusion: This assay provides an accurate assessment of FMR1 repeat status and holds promise for use in carrier and newborn screening. The method distinguishes normal homozygous females from full mutation carrying females. Although the method is not useful for accurate sizing, it supplements the classic polymerase chain reaction method and results in significant reduction in the number of Southern blot analyses required to be performed in the laboratory to accurately assess the FMR1 genotype in all individuals. Genet Med 2010:12(3):162-173.

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“…A volume of 2 μL of diluted PCR product (1/10 to 1/20) was loaded on an ABI 3100 Sequencer (Applied Biosystems) and electrophoresed for 1 hour for fluorescent detection using a MapMarker1000 Rox internal size standard (BioVentures Inc.). Large repeats in FRDA, SCA7 and SCA8 were investigated using a TPPCR which features a gene-specific forward primer and a repeat-specific reverse primer [2]. The PCR reaction was conducted in a 10 μL reaction with 60 ng DNA, under the following cycling conditions: 1 cycle at 95 °C for 15 min, 35 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min and a final extension at 72 °C for 10 min.…”

Section: ■ Statistical Methodologymentioning

confidence: 99%

The genetic aetiology of late-onset chronic progressive cerebellar ataxia

et al. 2009

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SCA6 and DRPLA were the most frequent genetic diagnoses identified. Patterns of high-normal allele frequency in this UK population were distinct compared to other ethnic groups but this was poorly predictive of the distribution of disease in this region. The relative contribution of new mutation formation and founder effects to the prevalence of familial ataxia is uncertain, and further exploration of these factors will require detailed analysis of disease allele haplotypes and meiotic instability of intermediate length alleles.

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Triplet Repeat Primed PCR (TP PCR) in Molecular Diagnostic Testing for Friedreich Ataxia

Cited by 48 publications

References 16 publications

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: Implications for fragile X syndrome carrier and newborn screening

The genetic aetiology of late-onset chronic progressive cerebellar ataxia

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