Friedreich ataxia (FRDA) is an autosomal recessive, progressive degenerative disease caused by an expansion of a guanine-adenine-adenine (GAA) trinucleotide repeat in intron 1 of the FXN gene.1Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.L. Cocozza S. Koenig M. Pandolfo M. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Approximately 95% to 98% of affected individuals have expanded homozygous GAA trinucleotide repeats in intron 1 of the FXN gene.2Bartolo C. Mendell J.R. Prior T.W. Identification of a missense mutation in a Friedreich's ataxia patient: implications for diagnosis and carrier studies., 3Galea C.A. Huq A. Lockhart P.J. Tai G. Corben L.A. Yiu E.M. Gurrin L.C. Lynch D.R. Gelbard S. Durr A. Pousset F. Parkinson M. Labrum R. Giunti P. Perlman S.L. Delatycki M.B. Evans-Galea M.V. Compound heterozygous FXN mutations and clinical outcome in Friedreich ataxia., 4Anheim M. Mariani L.L. Calvas P. Cheuret E. Zagnoli F. Odent S. Seguela C. Marelli C. Fritsch M. Delaunoy J.P. Brice A. Durr A. Koenig M. Exonic deletions of FXN and early-onset Friedreich ataxia. The remaining individuals have one GAA expansion allele and a different pathogenic mutation in other exons of the FXN.1Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.L. Cocozza S. Koenig M. Pandolfo M. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.,5Delatycki M.B. Bidichandani S.I. Friedreich ataxia- pathogenesis and implications for therapies., 6Delatycki M.B. Williamson R. Forrest S.M. Friedreich ataxia: an overview., 7Durr A. Cossee M. Agid Y. Campuzano V. Mignard C. Penet C. Mandel J.L. Brice A. Koenig M. Clinical and genetic abnormalities in patients with Friedreich's ataxia., 8Filla A. De Michele G. Cavalcanti F. Pianese L. Monticelli A. Campanella G. Cocozza S. The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia., 9Monros E. Molto M.D. Martinez F. Canizares J. Blanca J. Vilchez J.J. Prieto F. de Frutos R. Palau F. Phenotype correlation and intergenerational dynamics of the Friedreich ataxia GAA trinucleotide repeat. Friedreich ataxia is estimated to affect 1 in 50,000 individuals (1 in 120 are carriers).10Gonzalez-Cabo P. Sanchez M.I. Canizares J. Blanca J.M. Martinez-Arias R. De Castro M. Bertranpetit J. Palau F. Molto M.D. de Frutos R. Incipient GAA repeats in the primate Friedreich ataxia homologous genes. Age of onset can be variable between ages 5 and 40 years.1Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.L. Cocozza S. Koenig M. Pandolfo M. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.,6Delatycki M.B. Williamson R. Forrest S.M. Friedreich ataxia: an overview.,11Friedreich's ataxia: a clinical and genetic study of 90 families with an analysis of early diagnostic criteria and intrafamilial clustering of clinical features.The expanded GAA trinucleotide repeat size correlates with the age of onset, severity of symptoms, and the rate of disease progression.7Durr A. Cossee M. Agid Y. Campuzano V. Mignard C. Penet C. Mandel J.L. Brice A. Koenig M. Clinical and genetic abnormalities in patients with Friedreich's ataxia.,12Erichsen A.K. Koht J. Stray-Pedersen A. Abdelnoor M. Tallaksen C.M. Prevalence of hereditary ataxia and spastic paraplegia in southeast Norway: a population-based study.,13Montermini L. Richter A. Morgan K. Justice C.M. Julien D. Castellotti B. Mercier J. Poirier J. Capozzoli F. Bouchard J.P. Lemieux B. Mathieu J. Vanasse M. Seni M.H. Graham G. Andermann F. Andermann E. Melancon S.B. Keats B.J. Di Donato S. Pandolfo M. Phenotypic variability in Friedreich ataxia: role of the associated GAA triplet repeat expansion. Larger repeat size is associated with earlier onset and more severe disease forms. The GAA repeats between 5 and 33 are considered normal alleles, although repeat numbers >27 are rare in unaffected individuals.14Bidichandani S.I. Delatycki M.B. Friedreich ataxia. In GeneReviews [Internet]. Repeat numbers between 34 and 65 GAA are identified as mutable normal (premutation) alleles,15Wedding I.M. Kroken M. Henriksen S.P. Selmer K.K. Fiskerstrand T. Knappskog P.M. Berge T. Tallaksen C.M. Friedreich ataxia in Norway - an epidemiological, molecular and clinical study. of which 44 to 65 GAA repeats are described as borderline alleles.16Bidichandani S.I. Delatycki M.B. The GAA repeats are considered expanded when the repeat numbers are between 66 and 1700,7Durr A. Cossee M. Agid Y. Campuzano V. Mignard C. Penet C. Mandel J.L. Brice A. Koenig M. Clinical and genetic abnormalities in patients with Friedreich's ataxia.,17Epplen C. Epplen J.T. Frank G. Miterski B. Santos E.J. Schols L. Differential stability of the (GAA)n tract in the Friedreich ataxia (STM7) gene. with most repeats falling between 600 and 1200.1Campuzano V. Montermini L. Moltò M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.L. Cocozza S. Koenig M. Pandolfo M. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.,8Filla A. De Michele G. Cavalcanti F. Pianese L. Monticelli A. Campanella G. Cocozza S. The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia.,17Epplen C. Epplen J.T. Frank G. Miterski B. Santos E.J. Schols L. Differential stability of the (GAA)n tract in the Friedreich ataxia (STM7) gene., 18Molecular basis of Friedreich ataxia., 19Montermini L. Andermann E. Labuda M. Richter A. Pandolfo M. Cavalcanti F. Pianese L. Iodice L. Farina G. Monticelli A. Turano M. Filla A. De Michele G. Cocozza S. The Friedreich ataxia GAA triplet repeat: premutation and normal alleles., 20Sharma R. De Biase I. Gomez M. Delatycki M.B. Ashizawa T. Bidichandani S.I. Friedreich ataxia in carriers of unstable borderline GAA triplet-repeat alleles. Individuals with GAA repeats of FXN gene to provide correct diagnosis and counseling to understand future generation risk.15Wedding I.M. Kroken M. Henriksen S.P. Selmer K.K. Fiskerstrand T. Knappskog P.M. Berge T. Tallaksen C.M. Friedreich ataxia in Norway - an epidemiological, molecular and clinical study.,20Sharma R. De Biase I. Gomez M. Delatycki M.B. Ashizawa T. Bidichandani S.I. Friedreich ataxia in carriers of unstable borderline GAA triplet-repeat alleles.,21O'Donnell D.M. Zoghbi H.Y. Trinucleotide repeat disorders in pediatrics. Interruptions in the GAA trinucleotide repeat pattern, such as GGAGAG, GAAGGA, or GAAGAAAA, make expanded allele detection challenging using triple-repeat primed PCR (TR-PCR).22McDaniel D.O. Keats B. Vedanarayanan V.V. Subramony S.H. Sequence variation in GAA repeat expansions may cause differential phenotype display in Friedreich's ataxia.An improved two-tier assay is described that rapidly genotypes GAA repeats in intron 1 of the FXN gene. The improved two-tier design allows for the detection of a broad range of alleles and increases sensitivity and accuracy of GAA repeat genotyping by detecting both the smallest (5 GAA repeats) and the largest (1700 GAA repeats) in the FXN gene. The triplet-repeat primed PCR/capillary electrophoresis protocol is used to detect and quantify alleles between 5 and 200 GAA repeats, and a second assay is used to detect expanded alleles beyond 200 repeats. This second assay is a gel-based long-range PCR (LR-PCR) protocol developed to quantify allele expansions of >200 repeats and confirm homozygous genotypes.23Barcia G. Rachid M. Magen M. Assouline Z. Koenig M. Funalot B. Barnerias C. Rotig A. Munnich A. Bonnefont J.P. Steffann J. Pitfalls in molecular diagnosis of Friedreich ataxia. Using this improved two-tier assay design will enhance patient care by reducing turnaround time and lowering costs to perform the assay. The TR-PCR assay can correctly genotype and classify normal, mutable normal, borderline, and expanded alleles in both homozygous and heterozygous states in a rapid, simplified analysis suitable for laboratory testing.Materials and MethodsSamplesA total of 1236 DNA samples were analyzed; 1215 patient DNA samples unrelated to FRDA diagnosis were collected at ARUP Laboratories (Salt Lake City, UT) (Table 1). Six previously FXN GAA-repeat genotyped samples were provided through collaboration, and 15 FRDA DNA reference samples were obtained from National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ): NA03665, NA03816, NA14519, NA15850, NA16203, NA16207, NA16209, NA16215, NA16216, NA16243, GM16220, GM16236, GM23443, NA15847, and NA15851. All ARUP Laboratories’ patient DNA samples were deidentified using University of Utah institutional review board protocol 00007275. Patient DNA samples collected at ARUP Laboratories were presumed to be normal or of carrier status for GAA repeats and considered representative of FDRA allele frequencies found in the US population.

Table 1Summary of GAA-Repeat Genotypes for 1236 Samples

Samples were classified as normal or expanded FXN alleles. The 31 samples with expanded alleles were confirmed using the LR-PCR protocol.

GAA, guanine-adenine-adenine; LR-PCR, long-range PCR; TR-PCR, triple-repeat primed PCR.

Triplet-Repeat Primed PCR Primer DesignPrimers for TR-PCR were designed as described earlier for Huntington disease TR-PCR assays using a fluorophore-labeled forward primer but with two triplet-repeat reverse primers.23Barcia G. Rachid M. Magen M. Assouline Z. Koenig M. Funalot B. Barnerias C. Rotig A. Munnich A. Bonnefont J.P. Steffann J. Pitfalls in molecular diagnosis of Friedreich ataxia., 24Warner J.P. Barron L.H. Goudie D. Kelly K. Dow D. Fitzpatrick D.R. Brock D.J. A general method for the detection of large CAG repeat expansions by fluorescent PCR., 25Jama M. Millson A. Miller C.E. Lyon E. Triplet repeat primed PCR simplifies testing for Huntington disease., 26Fluorescence PCR and GeneScan analysis for the detection of CAG repeat expansions associated with Huntington's disease. The FXN TR-PCR primers were designed on the minus strand because of the DNA sequence complexity of FXN intron 1 region to contain a 21-bp sequence specific for the FXN gene and a 15 to 21 bp of (TTC)5&7 triplet repeats. Binding the full length of the TR-PCR primers allows for the amplification of entire repeat region to generate the major peaks. The nonspecific (TTC)5&7 triplet repeats permit the random annealing of the primer across the GAA repeat region. These amplification products result in a stuttering pattern (minor peaks) that represent amplicons differing in length by one triplet GAA repeat unit along the entire TTC repeats of the FXN gene. Mixing (TTC)5 and (TTC)7 TR-PCR reverse primers permits the assay to detect the smallest FXN repeat genotype (5 GAA) as well as improves the sensitivity of repeat length quantitation.PCR Amplification

Triplet-repeat primed PCR was performed using a SimpliAmp or GeneAmp 9700 thermal cycler (Applied Biosystems, Waltham, MA) in a 20-μL amplification reaction containing 2 μL of DNA (stock DNA at 15 to 25 ng/μL), 1× FailSafe Premix J containing PCR buffer, dNTPs, and MgCl2 (Lucigen Corp., Middleton, WI), 0.25 U of AccuStart II TaqDNA polymerase (QuantaBio, Beverly, MA), and 2 μL of the pooled, three TR-PCR primers mix at 5 μmol/L working stock.

The forward TR-PCR primer had a carboxyfluorescein fluorescent label on the 5′ end: sequence 5′-6-FAM_CAACATGGTGAAACCCAGTATCTA-3 (Integrated DNA Technologies, Coralville, IA). The two unlabeled reverse TR-PCR primers differ at their 3′ ends in the number of (TTC)n repeats and are pooled together. The 5GAA_R primer has five TTC repeats on the 3′ end with a sequence of 5′-CCCGGCTAACTTTTCTTTATT(TTC)5-3′. The 7GAA_R primer has seven TTC repeats on the 3′ end with a sequence of 5′-CCCGGCTAACTTTTCTTTATT(TTC)7-3′. The 5GAA_R and 7GAA_R primers were mixed at a 5:1 ratio (respectively) at a stock 100 μmol/L concentration with 0.01 mol/L TE buffer, pH 8.0. This reverse primer mixture was then combined 1:1 with the labeled TR-PCR forward primer at a stock 100 μmol/L concentration, and this final TR-PCR primer mix was diluted with 0.01 mol/L TE buffer, pH 8.0, to 5 μmol/L working stock. The TR-PCR primer mix is stable at 4°C for 1 month when covered with aluminum foil. Amplification was performed with an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 59.5°C for 30 seconds, extension at 72°C for 60 seconds, and a final one cycle of 30-minute extension at 72°C.

Capillary Electrophoretic Analysis

The generated PCR products (2 μL) were added to a mixture of 1.0 μL of MapMarker 1000 internal size standard (BioVentures Inc., Murfreesboro, TN) and 8 μL of HiDi formamide (ThermoFisher, Waltham, MA). The mixture was heated at 95°C for 2 minutes and cooled on a cold block for 2 minutes. The PCR fragments were resolved by capillary electrophoresis on an automated ABI Prism 3730 Genetic Analyzer (solid-state laser; Windows 10 OS) using performance-optimized polymer, with a 50-cm array (ThermoFisher). Samples were electrokinetically injected using a modified GS1200LIZ-50_POP7-v2 run module, where samples were injected at 1.0 kV for 15 seconds and electrophoresed at 15 kV for 3600 seconds at 60°C under filter set D.

Statistical AnalysisRaw data were analyzed with GeneMarker software 3.0 (Soft Genetics, State College, PA) with a defined FXN Bin and Panel, where macros were set for automated bin calling. The FXN Bin and Panel was generated by the stutter peak pattern using DNA sequencing confirmed samples with various genotypes. These samples represented genotypes that are classified as normal, mutable normal, borderline, and expanded alleles.25Jama M. Millson A. Miller C.E. Lyon E. Triplet repeat primed PCR simplifies testing for Huntington disease. Each bin represents a stutter peak of a GAA repeat. With the FXN chimeric TR-PCR primers, the GAA trinucleotide stutter pattern begins with the fifth GAA repeat and is detectable until the 200th GAA repeat of an expanded allele. Each GAA bin was calculated from the mean generated from multiple replicated assays. Two SDs were used for setting the upper and lower boundary of each GAA bin size to minimize inclusion of stray alleles into the wrong bin interval.Long-Range PCRThe 20 μL LR-PCR contained 2 μL DNA (stock DNA at 15 to 25 ng/μL), 1× AccuStart Long Range SuperMix (Quanta Bio), and 0.5 mmol/L for each LR-PCR primer [forward LR-PCR primer, 5′-TTGTGTTTGAAGAAACTTTGGGATTGG-3′; and reverse LR-PCR primer, 5′-GCTTTCCTAGAGGAGATCTAAGGACC-3′ (Integrated DNA Technologies, Coralville, IA)]. Amplification was performed with an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of denaturation at 94°C for 10 seconds, annealing and extension at 68°C for 5 minutes 30 seconds, and a final 15-minute extension at 72°C. The expected amplicon product was 501 bp for a sample with six GAA repeats (GRCh38: chromosome 9:69037073-69037573). The PCR products (6 μL) were mixed with 1 μL of 6× Orange DNA loading dye (ThermoFisher) and loaded onto a 2% agarose gel27Lee P.Y. Costumbrado J. Hsu C.Y. Kim Y.H. Agarose gel electrophoresis for the separation of DNA fragments. (2% precast gels; Lonza, Salisbury, MD) for 60 minutes at 80 V. The LR-PCR products were sized by two molecular weight DNA standard ladders: Low Biomarker 50 to 1000 bp (catalog number MI; BioVentures Inc.) and 10-kb Biomarker (catalog number M10 kb; BioVentures Inc.). Both Biomarker ladder mixes were diluted as per manufacturer’s recommendations. The size of the LR-PCR products was estimated by the two molecular weight DNA standard ladders28Johnson P.H. Grossman L.I. Electrophoresis of DNA in agarose gels: optimizing separations of conformational isomers of double- and single-stranded DNAs., 29Separation and size determination of circular and linear single-stranded DNAs by alkaline agarose gel electrophoresis., 30Helling R.B. Goodman H.M. Boyer H.W. Analysis of endonuclease R-EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. run on either side of the PCR products, and visualized with AlphaImager Gel Imaging System (Alpha HP 3.4.0 build 0728; ProteinSimple, San Jose, CA) using the gel imaginer sizing function to select, define, and size the LR-PCR products. The cubic spline analysis option was selected to give a better sizing of the PCR products31Gauthier J. Wu Q.V. Gooley T.A. Cubic splines to model relationships between continuous variables and outcomes: a guide for clinicians., 32Gariepy C.E. Lomax M.I. Grossman L.I. SPLINT: a cubic spline interpolation program for the analysis of fragment sizes in one-dimensional electrophoresis gels., 33Russell P.J. Crandall R.E. Feinbaum R. GELYSIS: Pascal-implemented analysis of one-dimensional electrophoresis gels., 34Gray A.J. Beecher D.E. Olson M.V. Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments. (Figure 1). GAA repeat size was determined by subtracting 483 bp from the size of the amplicon and dividing by 3 (Table 2).

Figure 1Agarose gel from nine preselected samples. Sample order is as follows: 1, 10-kb molecular DNA ladder; 2, NTC; 3, 5/expanded; 4, 131/expanded; 5, heterozygous expanded; 6, 9/expanded; 7, homozygous expanded; 8, 188/expanded; 9, 9/37; 10, 17/33; 11, homozygous 7/7; and 12, lower DNA molecular marker. All nine samples amplified well and included both small to large guanine-adenine-adenine (GAA) repeats without preferential amplification of one allele over the other. Sticky DNA phenomena were seen in samples 5 (heterozygous expanded), 6 (9/expanded), and 8 (188/expanded) and appear as shadow bands in PCR products, as indicated by white arrows.

Table 2Results Obtained by Both TR-PCR and LR-PCR from Nine Preselected Samples of Various GAA Repeats

The results of LR-PCR and TR-PCR in samples with previously known genotypes.

GAA, guanine-adenine-adenine; LR-PCR, long-range PCR; TR-PCR, triple-repeat primed PCR.

FXN Allele Sanger SequencingSamples with various GAA repeats were amplified and sequenced for precise sizing. Amplicon DNA bands were excised from the agarose gel under low UV (transilluminator) and gel purified for Sanger sequencing using the GeneJet Gel Extraction kit (ThermoFisher). Sanger sequence data for the FXN intron 1 fragment were compared with the National Center for Biotechnology Information reference sequence NG_008845.2 (https://www.ncbi.nlm.nih.gov/nuccore/NG_008845.2?from=5190&to=48514&report=genbank, last accessed April 25, 2022) using Mutation Surveyor software version 5.1.2 (Soft Genetics). The samples with known GAA repeat sizes were used to set up, improve, and refine GAA repeat binning in the Gene Marker software and electrophoretic gel analysis.DiscussionBiallelic GAA trinucleotide repeat expansion within intron 1 of FXN gene in chromosome 9 is the main cause of Friedreich ataxia. Molecular diagnostic tests are used for carrier detection, clinical data, and prenatal diagnosis. Traditionally, FXN gene repeat expansion detection has utilized PCR amplification over the repeat region, followed by agarose gel electrophoresis, Southern blotting, Sanger sequencing, melting curve analysis, or TR-PCR.37Advanced technologies for the molecular diagnosis of fragile X syndrome., 38Teo C.R. Law H.Y. Lee C.G. Chong S.S. Screening for CGG repeat expansion in the FMR1 gene by melting curve analysis of combined 5' and 3' direct triplet-primed PCRs., 39Loomis E.W. Eid J.S. Peluso P. Yin J. Hickey L. Rank D. McCalmon S. Hagerman R.J. Tassone F. Hagerman P.J. Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. The GAA repeat number in the FXN gene is varied, ranging from 5 to >1700 GAA repeats.7