Protocol for the Characterization of the Cytosine-Adenine-Guanine Tract and Flanking Polymorphisms in Machado-Joseph Disease

Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs. Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs. Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders of late onset. Although each of these genetic diseases has its own causative gene, they share a common etiology that consists of the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons.1Gatchel J.R. Zoghbi H.Y. Diseases of unstable repeat expansion: mechanisms and common principles.Nat Rev Genet. 2005; 6: 743-755Crossref PubMed Scopus (633) Google Scholar As a consequence, the disease-causing proteins display an expanded polyglutamine (polyQ) tract, which tends to accumulate in specific neuronal populations, ultimately leading to dysfunction and degeneration.1Gatchel J.R. Zoghbi H.Y. Diseases of unstable repeat expansion: mechanisms and common principles.Nat Rev Genet. 2005; 6: 743-755Crossref PubMed Scopus (633) Google Scholar, 2Taroni F. DiDonato S. Pathways to motor incoordination: the inherited ataxias.Nat Rev Neurosci. 2004; 5: 641-655Crossref PubMed Scopus (152) Google Scholar, 3Fan H.C. Ho L.I. Chi C.S. Chen S.J. Peng G.S. Chan T.M. Lin S.Z. Harn H.J. Polyglutamine (PolyQ) diseases: genetics to treatments.Cell Transplant. 2014; 23: 441-458Crossref PubMed Scopus (118) Google Scholar Symptoms frequently start in adulthood and include gait ataxia, limb incoordination, speech disturbances, and oculomotor abnormalities.4Rüb U. Schöls L. Paulson H. Auburger G. Kermer P. Jen J.C. Seidel K. Korf H.W. Deller T. Clinical features, neurogenetics and neuropathology of the polyglutamine spinocerebellar ataxias type 1, 2, 3, 6 and 7.Prog Neurobiol. 2013; 104: 38-66Crossref PubMed Scopus (218) Google Scholar,5Paulson H.L. Shakkottai V.G. Clark H.B. Orr H.T. Polyglutamine spinocerebellar ataxias—from genes to potential treatments.Nat Rev Neurosci. 2017; 18: 613-626Crossref PubMed Scopus (190) Google Scholar The considerable phenotypic overlapping observed in these disorders hinders the establishment of a definitive diagnosis based on clinical features. Hence, a differential diagnosis relies on genetic and molecular analysis. Sizing of CAG trinucleotide repeats in a specific causative gene has commonly involved the electrophoretic separation of PCR-amplified products by using polyacrylamide gel electrophoresis (PAGE).6Maciel P. Gaspar C. DeStefano A.L. Silveira I. Coutinho P. Radvany J. Dawson D.M. Sudarsky L. Guimaraes J. Loureiro J.E. Nezarati M.M. Corwin L.I. Lopez-Cendes I. Rooke K. Rosenberg R. LacLeod P. Farrer L.A. Sequeiros J. Roleau G.A. Correlation between CAG repeat length and clinical features in Machado-Joseph disease.Am J Hum Genet. 1995; 57: 54-61PubMed Google Scholar, 7Cancel G. Abbas N. Stevanin G. Dürr A. Chneiweiss H. Néri C. Duyckaerts C. Penet C. Cann H.M. Agid Y. Brice A. Marked phenotypic heterogeneity associated with expansion of a CAG repeat sequence at the spinocerebellar ataxia-3/Machado-Joseph disease locus.Am J Hum Genet. 1995; 57: 809-816PubMed Google Scholar, 8Maruyama H. Nakamura S. Matsuyama Z. Sakai T. Doyu M. Sobue G. Seto M. Tsujihata M. Oh-i T. Nishio T. Sunohara N. Takahashi R. Hayashi M. Nishino I. Ohtake T. Oda T. Nishimura M. Saida T. Matsumoto H. Baba M. Kawaguchi Y. Kakizuka A. Kawakami H. Molecular features of the CAG repeats and clinical manifestation of Machado-Joseph disease.Hum Mol Genet. 1995; 4: 807-812Crossref PubMed Scopus (168) Google Scholar, 9Maciel P. Costa M.C. Ferro A. Rousseau M. Santos C.S. Gaspar C. Barros J. Rouleau G.A. Coutinho P. Sequeiros J. Improvement in the molecular diagnosis of Machado-Joseph disease.Arch Neurol. 2001; 58: 1821-1827Crossref PubMed Scopus (108) Google Scholar However, apart from being a labor-intensive and time-consuming procedure, counting the number of CAGs through PAGE visualization is not always easy and accurate. Next-generation sequencing, however, still fails to detect these repeats efficiently. According to the best practice guidelines for molecular genetic testing of the SCAs described by the European Molecular Genetics Quality Network, capillary electrophoresis (CE) is currently the preferred methodology for this purpose.10Sequeiros J. Seneca S. Martindale J. Consensus and controversies in best practices for molecular genetic testing of spinocerebellar ataxias.Eur J Hum Genet. 2010; 18: 1188-1195Crossref PubMed Scopus (54) Google Scholar,11Sequeiros J. Martindale J. Seneca S. Giunti P. Kamarainen O. Volpini V. Weirich H. Christodoulou K. Bazak N. Sinke R. Sulek-Piatkowska A. Garcia-Planells J. Davis M. Frontali M. Hamalainen P. Wieczorek S. Zuhlke C. Saraiva-Pereira M.L. Warner J. Leguern E. Thonney F. Quintans Castro B. Jonasson J. Storm K. Andersson A. Ravani A. Correia L. Silveira I. Alonso I. Martins C. Pinto Basto J. Coutinho P. Perdigao A. Barton D. Davis M. European Molecular Quality Genetics Network: EMQN best practice guidelines for molecular genetic testing of SCAs.Eur J Hum Genet. 2010; 18: 1173-1176Crossref PubMed Scopus (29) Google Scholar Nonetheless, some reports have shown that GC-rich amplicons tend to migrate faster during CE compared with the internal size standards, leading to an underestimation of their lengths.12Larsen L.A. Grønskov K. Nørgaard-Pedersen B. Brøndum-Nielsen K. Hasholt L. Vuust J. High-throughput analysis of fragile X (CGG)n alleles in the normal and premutation range by PCR amplification and automated capillary electrophoresis.Hum Genet. 1997; 100: 564-568Crossref PubMed Scopus (48) Google Scholar, 13Williams L.C. Hegde M.R. Herrera G. Stapleton P.M. Love D.R. Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards.Mol Cell Probes. 1999; 13: 283-289Crossref PubMed Scopus (28) Google Scholar, 14Dorschner M.O. Barden D. Stephens K. Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis.J Mol Diagn. 2002; 4: 108-113Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar In trinucleotide disorders, this sizing discrepancy can ultimately lead to misdiagnosis, especially when dealing with alleles that are near the threshold of normal or intermediate sizes. As such, the use of CE for clinical diagnosis requires methods able to determine error margins and correct the referred sizing discrepancy.14Dorschner M.O. Barden D. Stephens K. Diagnosis of five spinocerebellar ataxia disorders by multiplex amplification and capillary electrophoresis.J Mol Diagn. 2002; 4: 108-113Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,15Ramos A. Raposo M. Milà M. Bettencourt C. Houlden H. Cisneros B. Magaña J.J. Bettencourt B.F. Bruges-Armas J. Santos C. Lima M. Verification of inter-laboratorial genotyping consistency in the molecular diagnosis of polyglutamine spinocerebellar ataxias.J Mol Neurosci. 2016; 58: 83-87Crossref PubMed Scopus (2) Google Scholar The current study focused on the specific case of SCA type 3 (SCA3), also known as Machado-Joseph disease (MJD), which is recognized as the most frequent form of autosomal dominant SCA in most populations.16Schöls L. Bauer P. Schmidt T. Schulte T. Riess O. Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis.Lancet Neurol. 2004; 3: 291-304Abstract Full Text Full Text PDF PubMed Scopus (810) Google Scholar,17de Araújo M.A. Raposo M. Kazachkova N. Vasconcelos J. Kay T. Lima M. Trends in the epidemiology of spinocerebellar ataxia type 3/Machado-Joseph disease in the Azores Islands, Portugal.JSM Brain Sci. 2016; 1Google Scholar The disease locus was initially mapped to the long arm of chromosome 14 (14q32.12) in 1993.18Takiyama Y. Nishizawa M. Tanaka H. Kawashima S. Sakamoto H. Karube Y. Shimazaki H. Soutome M. Endo K. Ohta S. Kagawa Y. Kanazawa I. Mizuno Y. Yoshida M. Yuasa T. Horikawa Y. Oyanagi K. Nagai H. Kondo T. Inusuka T. Onodera O. Tsuji S. The gene for Machado-Joseph disease maps to human chromosome 14q.Nat Genetics. 1993; 4: 300-304Crossref PubMed Scopus (315) Google Scholar However, the disease mutation was only identified in the following year, consisting of the abnormal repetition of CAGs in exon 10 of the human ATXN3 gene.19Kawaguchi Y. Okamoto T. Taniwaki M. Aizawa M. Inoue M. Katayama S. Kawakami H. Nakamura S. Nishimura M. Akiguchi I. Kimura J. Narumiya S. Kakizuka A. CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1.Nat Genetics. 1994; 8: 221-228Crossref PubMed Scopus (1556) Google Scholar In accordance with the currently accepted guidelines, normal chromosomes carry between 11 and 44 repeats, whereas repeat lengths ranging from 61 to 87 have been related to the development of MJD/SCA3, displaying full penetrance.10Sequeiros J. Seneca S. Martindale J. Consensus and controversies in best practices for molecular genetic testing of spinocerebellar ataxias.Eur J Hum Genet. 2010; 18: 1188-1195Crossref PubMed Scopus (54) Google Scholar Although rare, alleles with intermediate sizes (45 to 60 repeats) have also been reported, however, their pathological outcome is still unclear.20Bettencourt C. Lima M. Machado-Joseph disease: from first descriptions to new perspectives.Orphanet J Rare Dis. 2011; 6: 35Crossref PubMed Scopus (111) Google Scholar An important feature observed in polyQ SCAs is the correlation between the number of CAG repeats and both the initiation and severity of clinical symptoms.21Zoghbi H.Y. Orr H.T. Glutamine repeats and neurodegeneration.Annu Rev Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1101) Google Scholar In the case of MJD/SCA3, the number of CAGs explains 50% to 75% of the variation observed in the age of onset, and an association between longer repeats and more severe symptoms has also been suggested.6Maciel P. Gaspar C. DeStefano A.L. Silveira I. Coutinho P. Radvany J. Dawson D.M. Sudarsky L. Guimaraes J. Loureiro J.E. Nezarati M.M. Corwin L.I. Lopez-Cendes I. Rooke K. Rosenberg R. LacLeod P. Farrer L.A. Sequeiros J. Roleau G.A. Correlation between CAG repeat length and clinical features in Machado-Joseph disease.Am J Hum Genet. 1995; 57: 54-61PubMed Google Scholar, 7Cancel G. Abbas N. Stevanin G. Dürr A. Chneiweiss H. Néri C. Duyckaerts C. Penet C. Cann H.M. Agid Y. Brice A. Marked phenotypic heterogeneity associated with expansion of a CAG repeat sequence at the spinocerebellar ataxia-3/Machado-Joseph disease locus.Am J Hum Genet. 1995; 57: 809-816PubMed Google Scholar, 8Maruyama H. Nakamura S. Matsuyama Z. Sakai T. Doyu M. Sobue G. Seto M. Tsujihata M. Oh-i T. Nishio T. Sunohara N. Takahashi R. Hayashi M. Nishino I. Ohtake T. Oda T. Nishimura M. Saida T. Matsumoto H. Baba M. Kawaguchi Y. Kakizuka A. Kawakami H. Molecular features of the CAG repeats and clinical manifestation of Machado-Joseph disease.Hum Mol Genet. 1995; 4: 807-812Crossref PubMed Scopus (168) Google Scholar In addition, a gene dosage effect seems to be present in MJD/SCA3, as homozygosity of expanded ATXN3 alleles leads to aggravated clinical manifestations, with a harsh progression and early age of onset (reported onset extreme of a 4-year–old).22Carvalho D.R. La Rocque-Ferreira A. Rizzo I.M. Imamura E.U. Speck-Martins C.E. Homozygosity enhances severity in spinocerebellar ataxia type 3.Pediatr Neurol. 2008; 38: 296-299Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,23Lang A.E. Rogaeva E.A. Tsuda T. Hutterer J. St George-Hyslop P. Homozygous inheritance of the Machado-Joseph disease gene.Ann Neurol. 1994; 36: 443-447Crossref PubMed Scopus (60) Google Scholar No treatment to stop or delay the progression of this disorder is currently available. In all cases, MJD/SCA3 is progressive and ultimately fatal. One of the most promising therapeutic strategies relies on the reduction of mutant protein levels because its expression initiates disease pathogenesis.24Matos C.A. Carmona V. Vijayakumar U.G. Lopes S. Albuquerque P. Conceição M. Nobre R.J. Nóbrega C. Pereira de Almeida L. Gene therapies for polyglutamine diseases.in: Nóbrega C. de Almeida L.P. Polyglutamine Disorders. Springer, Cham2018: 395-438Crossref Scopus (13) Google Scholar In particular, allele-specific reduction of the mutant protein has been successfully achieved in in vitro25Miller V.M. Xia H. Marrs G.L. Gouvion C.M. Lee G. Davidson B.L. Paulson H.L. Allele-specific silencing of dominant disease genes.Proc Natl Acad Sci U S A. 2003; 100: 7195-7200Crossref PubMed Scopus (348) Google Scholar and in vivo26Alves S. Nascimento-Ferreira I. Auregan G. Hassig R. Dufour N. Brouillet E. Pedroso de Lima M.C. Hantraye P. Pereira de Almeida L. Déglon N. Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease.PLoS One. 2008; 3: e3341Crossref PubMed Scopus (121) Google Scholar, 27Nóbrega C. Nascimento-Ferreira I. Onofre I. Albuquerque D. Hirai H. Déglon N. de Almeida L.P. Silencing mutant ataxin-3 rescues motor deficits and neuropathology in Machado-Joseph disease transgenic mice.PLoS One. 2013; 8: e52396Crossref PubMed Scopus (88) Google Scholar, 28Conceição M. Mendonça L. Nóbrega C. Gomes C. Costa P. Hirai H. Moreira J.N. Lima M.C. Manjunath N. Pereira de Almeida L. Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado-Joseph disease neurological phenotype.Biomaterials. 2016; 82: 124-137Crossref PubMed Scopus (71) Google Scholar models of the disease using RNA interference strategies directed against a single nucleotide polymorphism (SNP) located at the 3′ end of the expanded CAG tract (C987GG/G987GG: rs12895357). The nucleotide C in this position has been described as being in linkage disequilibrium with the disease-causing expansion in 72% of patients with MJD/SCA3.29Stevanin G. Cancel G. Didierjean O. Dürr A. Abbas N. Cassa E. Feingold J. Agid Y. Brice A. Linkage disequilibrium at the Machado-Joseph disease/spinal cerebellar ataxia 3 locus: evidence for a common founder effect in French and Portuguese-Brazilian families as well as a second ancestral Portuguese-Azorean mutation.Am J Hum Genet. 1995; 57: 1247-1250PubMed Google Scholar, 30Gaspar C. Lopes-Cendes I. DeStefano A.L. Maciel P. Silveira I. Coutinho P. MacLeod P. Sequeiros J. Farrer L.A. Rouleau G.A. Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins.Hum Genet. 1996; 98: 620-624Crossref PubMed Scopus (23) Google Scholar, 31Gaspar C. Lopes-Cendes I. Hayes S. Goto J. Arvidsson K. Dias A. Silveira I. Maciel P. Coutinho P. Lima M. Zhou Y.X. Soong B.W. Watanabe M. Giunti P. Stevanin G. Riess O. Sasaki H. Hsieh M. Nicholson G.A. Brunt E. Higgins J.J. Lauritzen M. Tranebjaerg L. Volpini V. Wood N. Ranum L. Tsuji S. Brice A. Sequeiros J. Rouleau G.A. Ancestral origins of the Machado-Joseph disease mutation: a worldwide haplotype study.Am J Hum Genet. 2001; 68: 523-528Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar This finding highlights the importance of SNP characterization in the target population for the development and use of new therapies. Our aim was to design an experimental protocol, not only for the accurate determination of the number of CAG repeats, but also for the identification of intronic and exonic flanking polymorphisms in the ATXN3 gene of patients with MJD/SCA3. Blood samples from unaffected control individuals and patients with MJD/SCA3 (a total of 6 and 13, respectively) were collected at the Hospital Center of the University of Coimbra, following receipt of informed consent. Genomic DNA was extracted with the All-in-One Purification kit (Norgen Biotek Corp., Thorold, ON, Canada), serving as a template for the subsequent step of PCR amplification. As a positive quality control for the determination of the number of CAGs in the ATXN3 gene using the established methodology, genomic DNA extracted from MJD/SCA3 fibroblasts obtained from the Coriell Institute for Medical Research (GM06153; Camden, NJ) was included. The available information for this sample reports a normal allele with 23 CAGs and a second expanded allele bearing 71 repetitions. The CAG tract and flanking region of the ATXN3 gene was PCR amplified by using a pair of primers designed and validated in our laboratory (Table 1). The design was based on the annotated reference sequence for the ATXN3 gene at the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/gene, accession number NG_008198.2), using the online Primer-BLAST design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast).Table 1Primer Sequences Used for PCR Amplification of the Flanking Region of the CAG Repeat Motif of the Human ATXN3 GeneGenePrimer sequenceAmplicon size in bp (reference gene with 14 CAGs)ATXN3Forward5′-TTTCCTAAGATCAGCACTTCCA-3′473 bpReverse5′-ACTGCTCCTTAATCCAGGGAA-3′CAG, cytosine-adenine-guanine. Open table in a new tab CAG, cytosine-adenine-guanine. Briefly, each PCR reaction (50 μL) was prepared on ice, using the following components: 10 μL of 5X Phusion GC Buffer (Thermo Fisher Scientific, Waltham, MA), 1 μL of 10 mmol/L deoxynucleotide triphosphates (Thermo Fisher Scientific), forward and reverse primers (Table 1) with a final concentration of 0.5 μmol/L each, 10 to 20 ng of DNA template, 2.5 μL (5%) of dimethyl sulfoxide (Thermo Fisher Scientific), and 0.5 μL (one unit) of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR amplification was performed in a Veriti 96-Well Thermal Cycler (Thermo Fisher Scientific) with the following protocol: 1 cycle at 98°C for 1 minute (initial denaturation) and 35 cycles at 98°C for 30 seconds (denaturation), 60°C for 10 seconds (annealing), and 72°C for 30 seconds (extension), with a final extension at 72°C for 10 minutes. PCR amplicons mixed with the appropriate amount of loading buffer (6X Orange DNA Loading Dye; Thermo Fisher Scientific) were electrophoresed in 2% agarose (MilliporeSigma, Burlington, MA) gels, adjacent to a DNA ladder (GeneRuler 100 bp DNA Ladder; Thermo Fisher Scientific). Agarose gels were prepared with 1× tris-acetate-EDTA (MilliporeSigma) buffer and resolved at 90 V for 1 hour. Unaffected control individuals, whose corresponding PCR amplicons exhibited bands of molecular weight ranging from 464 to 563 bp, were characterized by Sanger sequencing (Figure 1A) after enzymatic clean-up. Briefly, Exonuclease I (Thermo Fisher Scientific) and thermosensitive alkaline phosphatase (FastAP; Thermo Fisher Scientific) were added directly to the PCR product. The mixture was incubated at 37°C, allowing the digestion of excess primer and the dephosphorylation of nucleotides, followed by a second incubation at 80°C, promoting the inactivation of the specified enzymes. Sanger sequencing was performed by GATC Biotech (Konstanz, Germany). PCR amplicons corresponding to MJD/SCA3 blood samples present at least one band with size ranging from 614 to 692 bp after the electrophoretic resolution. In these samples, the characterization of the amplified region was achieved through the use of two different yet complementary methods (Figure 1B). In method I, PCR cloning and DNA sequencing were combined; in method II, PCR and CE were used. Each of the methods is described in further detail in the following sections. PCR amplicons of MJD/SCA3 samples (normal and mutant alleles) were separately excised from the agarose gel with a clean scalpel and further purified with a QIAquick Gel Extraction Kit (Qiagen, Venlo, the Netherlands), following the manufacturer's instructions. Each purified PCR fragment was then inserted into a plasmid vector, making use of the CloneJET PCR Cloning Kit (Thermo Fisher Scientific). Briefly, 30 ng of each PCR product was mixed with 10 μL of 2× reaction buffer, 1 μL pJET1.2/blunt cloning vector, 1 μL T4 DNA ligase, and nuclease-free water (up to 20 μL). The reaction was gently mixed, incubated for 5 minutes at 25°C, and placed on ice before proceeding to the transformation of competent cells. From each of the previously prepared cloning reactions, 5 μL was added into a vial of One Shot Top 10 chemically competent Escherichia coli (Thermo Fisher Scientific) and mixed gently. After incubation for 30 minutes on ice, competent cells underwent a heat-shock step for 30 seconds at 42°C and then immediately transferred to ice (2 minutes). Aseptic super optimal broth with catabolic repressor (S.O.C. Medium, Thermo Fisher Scientific) was added, and competent bacteria were incubated at 37°C for 1 hour at 225 rpm in a shaking incubator. Subsequently, each transformation was spread on a prewarmed selective agar plate (100 μg/mL ampicillin; Enzo Life Sciences, Farmingdale, NY) and incubated overnight at 37°C. To confirm the presence of the intended PCR amplicon in plasmid constructs, 5 to 10 colonies were cultured overnight in lysogeny broth (Lennox; Fisher Scientific, Hampton, NH) containing 100 μg/mL of ampicillin and further analyzed by means of colony PCR. Briefly, 5 μL of each bacterial growth was diluted in water (45 μL) and lysed with a short heating step (95°C for 5 minutes). Plasmid DNA released from bacterial cells was subsequently amplified by PCR. Briefly, 2 μL of the plasmid DNA was amplified by using 1 μL of 10X Taq buffer (Thermo Fisher Scientific), 0.2 μL deoxynucleotide triphosphates 10 mmol/L (Thermo Fisher Scientific), ATXN3 forward and reverse primers (Table 1) with a final concentration of 0.5 μmol/L each, and 0.04 μL (one unit) of Dream Taq Polymerase (Thermo Fisher Scientific) in a total reaction volume of 10 μL. The PCR amplification was performed in a Veriti 96-Well thermal cycler (Thermo Fisher Scientific) with the following protocol: 1 cycle at 95°C for 3 minutes (initial denaturation) and 25 cycles at 95°C for 30 seconds (denaturation), 58°C for 30 seconds (annealing), and 72°C for 1 minute (extension), with a final extension at 72°C for 10 minutes. The generated PCR products were analyzed by electrophoresis in 2% agarose gels (1× tris-acetate-EDTA), resolved at 90 V, for 1 hour, allowing a fast screening of the positive colonies. Plasmid DNA was isolated from each bacterial culture by using a NZYMiniprep kit (NZYTech, Lisbon, Portugal) according to the manufacturer's instructions. A total of one to three clones (normal alleles) and three to five clones (mutant alleles) of each sample were sequenced at GATC Biotech and the resultant electropherograms analyzed with SnapGene software version 1.1.3 (GSL Biotech, San Diego, CA). The determination of the number of CAGs in each tract was achieved following the best practice guidelines for molecular testing of SCAs, which defines the repeat in SCA3 locus as (CAG)2 CAA AAG CAG CAA (CAG)n.10Sequeiros J. Seneca S. Martindale J. Consensus and controversies in best practices for molecular genetic testing of spinocerebellar ataxias.Eur J Hum Genet. 2010; 18: 1188-1195Crossref PubMed Scopus (54) Google Scholar PCR amplicons from patients with MJD/SCA3 were obtained as already described (see PCR Amplification of the Region Surrounding the CAG Repeat Motif in the Human ATXN3 Gene from Human Blood Samples). In this case, the forward primer was labeled with a fluorescent dye, 6-carboxyfluorescein, and the annealing temperature adjusted to 58°C. All PCR amplicons were analyzed by automated CE (Eurofins Genomics, Ebersberg, Germany) in an ABI 3130 XL sequencing machine (Thermo Fisher Scientific). The preparation of loading samples for fragment length analysis was undertaken at Eurofins. This process involved the addition of 10 μL Hi-Di formamide loading buffer (Thermo Fisher Scientific) and 0.5 μL internal size standard (GeneScan 1200LIZ size standard; Thermo Fisher Scientific) to 2 μL of each PCR product. Amplicon length was calculated by comparison with the size standard, using GeneMapper software version 5 (Thermo Fisher Scientific). The size correction formula was calculated by regression analysis of a pair-wise comparison of the number of CAGs determined by PCR cloning (method I) versus PCR-CE (method II). Calculations were performed by using GraphPad Prism software version 6.00 (GraphPad Software, La Jolla, CA). A set of customized primers was initially designed to amplify the vicinity of exon 10 of the ATXN3 gene, using Primer-BLAST software. The objective was not only to amplify the entire exon 10, which comprises the CAG repeat motif and the exonic polymorphism C987GG/G987GG (rs12895357),19Kawaguchi Y. Okamoto T. Taniwaki M. Aizawa M. Inoue M. Katayama S. Kawakami H. Nakamura S. Nishimura M. Akiguchi I. Kimura J. Narumiya S. Kakizuka A. CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1.Nat Genetics. 1994; 8: 221-228Crossref PubMed Scopus (1556) Google Scholar but also the intronic region where two polymorphisms (TAA1118/TAC1118: rs7158733; and C1178/A1178: rs3092822) can be encountered9Maciel P. Costa M.C. Ferro A. Rousseau M. Santos C.S. Gaspar C. Barros J. Rouleau G.A. Coutinho P. Sequeiros J. Improvement in the molecular diagnosis of Machado-Joseph disease.Arch Neurol. 2001; 58: 1821-1827Crossref PubMed Scopus (108) Google Scholar,32Goto J. Watanabe M. Ichikawa Y. Yee S.B. Ihara N. Endo K. Igarashi S. Takiyama Y. Gaspar C. Maciel P. Tsuji S. Rouleau G.A. Kanazawa I. Machado-Joseph disease gene products carrying different carboxyl termini.Neurosci Res. 1997; 28: 373-377Crossref PubMed Scopus (66) Google Scholar,33Costa M.C. Sequeiros J. Maciel P. Identification of three novel polymorphisms in the MJD1 gene and study of their frequency in the Portuguese population.J Hum Genet. 2002; 47: 205-207Crossref PubMed Scopus (4) Google Scholar (Figure 2A). The predicted size of the generated amplicon is 473 bp, considering the reference sequence with 14 CAG repeats used for primer design and available at the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/gene, accession number NG_008198.2). Considering the currently accepted guidelines10Sequeiros J. Seneca S. Martindale J. Consensus and controversies in best practices for molecular genetic testing of spinocerebellar ataxias.Eur J Hum Genet. 2010; 18: 1188-1195Crossref PubMed Scopus (54) Google Scholar and the primers designed in the context of the present study, it is estimated that in normal alleles (ie, 11 to 44 CAG repeats) the size of the generated amplicons can vary from 464 to 563 bp, whereas in mutant alleles (61 to 87 CAGs), the expected size is in the range of 614 to 692 bp. A single PCR and subsequent electrophoretic resolution of the PCR amplicons in a 2% agarose gel allows a rapid identification of healthy individuals and patients with disease (2Taroni F. DiDonato S. Pathways to motor incoordination: the inherited ataxias.Nat Rev Neurosci. 200