Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease
2013; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2012.09.005
ISSN1943-7811
AutoresMohamed Jama, Alison Millson, Christine E. Miller, Elaine Lyon,
Tópico(s)DNA Repair Mechanisms
ResumoDiagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout. Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout. CME Accreditation Statement: This activity ("JMD 2013 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2013 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2013 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)TM. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Abnormal expansions of the CAG repeat in the Huntingtin (HTT) gene on chromosome 4 are associated with Huntington disease (HD), an autosomal dominant neurodegenerative disorder.1Gusella J.F. Wexler N.S. Conneally P.M. Naylor S.L. Anderson M.A. Tanzi R.E. et al.A polymorphic DNA marker genetically linked to Huntington's disease.Nature. 1983; 306: 234-238Crossref PubMed Scopus (1654) Google Scholar, 2Ambrose C.M. Duyao M.P. Barnes G. Bates G.P. Lin C.S. Srinidhi J. et al.Structure and expression of the Huntington's disease gene: evidence against simple inactivation due to an expanded CAG repeat.Somat Cell Mol Genet. 1994; 20: 27-38Crossref PubMed Scopus (227) Google Scholar, 3The Huntington's Disease Collaborative Research GroupA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7052) Google Scholar Determination of the number of CAG trinucleotide repeats is routinely used in diagnostic and predictive testing of individuals symptomatic or at risk for HD.4International Huntington Association (IHA) and the World Federation of Neurology (WFN) Research Group on Huntington's ChoreaGuidelines for the molecular genetics predictive test in Huntington's disease.Neurology. 1994; 44: 1533-1536Crossref PubMed Google Scholar, 5The American College of Medical Genetics/American Society of Human Genetics Huntington Disease Genetic Testing Working GroupACMG/ASHG statement: laboratory guidelines for Huntington disease genetic testing.Am J Hum Genet. 1998; 62: 1243-1247Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar When a sample appears homozygous for a normal allele, additional testing is recommended to exclude the possibility that an expanded allele was not identified by PCR. This approach may include testing for heterozygosity in the adjacent polymorphic CCG loci and amplifying over the CCG+CAG region.6Holzmann C. Saecker A.M. Epplen J.T. Riess O. Avoiding errors in the diagnosis of (CAG)n expansion in the huntingtin gene.J Med Genet. 1997; 34: 264Crossref PubMed Google Scholar, 7Andrew S.E. Goldberg Y.P. Theilmann J. Zeisler J. Hayden M.R. A CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing.Hum Mol Genet. 1994; 3: 65-67Crossref PubMed Scopus (131) Google Scholar The polymorphic CCG repeat varies between 7 and 12 triplets in length and includes an apparent CCT site that is usually two or rarely three repeats in length.8Pecheux C. Mouret J.F. Durr A. Agid Y. Feingold J. Brice A. Dode C. Kaplan J.C. Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes.J Med Genet. 1995; 32: 399-400Crossref PubMed Scopus (20) Google Scholar The detection of a homozygous allele may also be due to PCR failure to amplify long CAG repeat expansions or a mutation under either of the two primers.7Andrew S.E. Goldberg Y.P. Theilmann J. Zeisler J. Hayden M.R. A CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing.Hum Mol Genet. 1994; 3: 65-67Crossref PubMed Scopus (131) Google Scholar, 9Gellera C. Meoni C. Castellotti B. Zappacosta B. Girotti F. Taroni F. DiDonato S. Errors in Huntington disease diagnostic test caused by trinucleotide deletion in the IT15 gene.Am J Hum Genet. 1996; 59: 475-477PubMed Google Scholar Further testing is not necessary if the CCG+CAG repeat is heterozygous. However, if the sample still appears to be homozygous, Southern blot analysis is performed. Southern blotting is expensive and labor intensive, requires high concentrations of DNA, and can delay turnaround time. Chimeric or triplet repeat primed PCR is defined as a PCR method that generates different sized amplicons due to multiple annealing sites on the template.10Tassone F. Pan R. Amiri K. Taylor A.K. Hagerman P.J. A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar It has been described in screening for fragile X premutations, screening for full mutations, and determination of mosaic fragile X samples.11Tassone F. Pan R. Amiri K. Taylor A.K. Hagerman P.J. A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 12Chen L. Hadd A. Sah S. Filipovic-Sadic S. Krosting J. Sekinger E. Pan R. Hagerman P.J. Stenzel T.T. Tassone F. Latham G.J. An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for southern blot analysis.J Mol Diagn. 2010; 12: 589-600Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 13Lyon E. Laver T. Yu P. Jama M. Young K. Zoccoli M. Marlowe N. A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis.J Mol Diagn. 2010; 12: 505-511Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar With a similar approach, a chimeric primer is used for the detection of an expanded allele in HD samples. The HD workflow can be streamlined by use of this chimeric PCR, where expanded alleles and apparent homozygous alleles are easily distinguished by the presence or absence of continuous stuttering. This approach greatly reduces the need for additional PCR over the adjacent repeats and the number of Southern blots performed. In addition, stuttering generated by the chimeric PCR can be used as an allelic ladder for correct CAG repeat sizing in HD samples. A total of 246 HD samples were used in this study, including 14 HD DNA reference samples obtained from the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, New Jersey): NA20206, NA20207, NA20208, NA20209, NA20210, NA20245, NA20246, NA20247, NA20248, NA20249, NA20250, NA20251, NA20252, and NA20253 The Coriell samples were tested by multiple laboratories and were sequenced to confirm the CAG repeats.14Kalman L. Johnson M.A. Beck J. Berry-Kravis E. Buller A. Casey B. Feldman G.L. Handsfield J. Jakupciak J.P. Maragh S. Matteson K. Muralidharan K. Richie K.L. Rohlfs E.M. Schaefer F. Sellers T. Spector E. Richards C.S. Development of genomic reference materials for Huntington disease genetic testing.Genet Med. 2007; 9: 719-723Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Three 2002 College of American Pathologists (CAP) survey proficiency samples were tested. Two hundred twenty-nine samples were obtained from the ARUP clinical laboratory. These samples were chosen from previously tested samples from 2005 to 2009 to represent a range of CAG repeat sizes but enriched for expanded alleles. The ARUP samples included those that were both homozygous (11 samples) and heterozygous and had CAG repeats ranging from 9 to 60. They were then deidentified as per our institutional review board protocol, and results were blinded for validation purposes. The CAP samples included two samples within the normal range and one with expanded alleles of >200 repeats. The ARUP samples were genotyped using the following HD workflow. The CAG alleles were genotyped using standard PCR.15Riess O. Noerremoelle A. Soerensen S.A. Epplen J.T. Improved PCR conditions for the stretch of (CAG)n repeats causing Huntington's disease.Hum Mol Genet. 1993; 2: 1523Crossref PubMed Scopus (72) Google Scholar If the sample was heterozygous, no further testing was required. All homozygous samples underwent additional testing by amplifying the CCG allele and over the CCG+CAG (and CCT) loci as reported.15Riess O. Noerremoelle A. Soerensen S.A. Epplen J.T. Improved PCR conditions for the stretch of (CAG)n repeats causing Huntington's disease.Hum Mol Genet. 1993; 2: 1523Crossref PubMed Scopus (72) Google Scholar, 16Andrew S.E. Goldberg Y.P. Kremer B. Telenius H. Theilmann J. Adam S. Starr E. Squitieri F. Lin B. Kalchman M.A. et al.The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease.Nat Genet. 1993; 4: 398-403Crossref PubMed Scopus (885) Google Scholar, 17Snell R.G. MacMillan J.C. Cheadle J.P. Fenton I. Lazarou L.P. Davies P. MacDonald M.E. Gusella J.F. Harper P.S. Shaw D.J. Relationship between trinucleotide repeat expansion and phenotypic variation in Huntington's disease.Nat Genet. 1993; 4: 393-397Crossref PubMed Scopus (604) Google Scholar Further testing was stopped if the CCG+CAG repeat was heterozygous.15Riess O. Noerremoelle A. Soerensen S.A. Epplen J.T. Improved PCR conditions for the stretch of (CAG)n repeats causing Huntington's disease.Hum Mol Genet. 1993; 2: 1523Crossref PubMed Scopus (72) Google Scholar If homozygous, a Southern blot was performed. The distributions of samples in each HD category (normal, intermediate, reduced, and full penetrance) are listed in Table 1. Classification categories are based on recommendations by the American College of Medical Genetics (ACMG)/American Society of Human Genetics (ASHG) guidelines for HD genetic testing.5The American College of Medical Genetics/American Society of Human Genetics Huntington Disease Genetic Testing Working GroupACMG/ASHG statement: laboratory guidelines for Huntington disease genetic testing.Am J Hum Genet. 1998; 62: 1243-1247Abstract Full Text Full Text PDF PubMed Scopus (71) Google ScholarTable 1Categories of 246 HD Samples Analyzed in This Study and Their Size ComparisonHD sample typeNo. of alleles correctly sized by both assayAlleles sizes within ±1 repeat, new HD vs old HD assay∗Allele sizes are in CAG repeats.Total No.Normal ( 39 CAG repeats)1406†Six samples that were within ±1 CAG repeats had chimeric HD assays versus current HD assays as follows: 17/51 versus 17/50, 18/60 versus 18/59, 15/55 versus 15/54, 19/43 versus 19/42, 20/55 versus 20/54, and 17/41 versus 17/42.146Total2406246∗ Allele sizes are in CAG repeats.† Six samples that were within ±1 CAG repeats had chimeric HD assays versus current HD assays as follows: 17/51 versus 17/50, 18/60 versus 18/59, 15/55 versus 15/54, 19/43 versus 19/42, 20/55 versus 20/54, and 17/41 versus 17/42. Open table in a new tab The 11 true homozygous HD samples were determined by amplification across the CCG+CAG and adjacent CCG loci and, when the CCG was also apparently homozygous, by Southern blot analysis. The 14 HD reference samples from Coriell cell repositories (http://wwwn.cdc.gov/dls/genetics/rmmaterials/pdf/Huntington.pdf; accessed June 30, 2012) were previously genotyped by PCR, sequencing, and Southern blot analysis and were used for precision studies, with each sample amplified five times to assess intrarun and interrun variability. The assay's diagnostic capabilities are due to the primer design (Figure 1). The unique design of the reverse primer enables it to have two or more annealing sites (chimeric) to anneal anywhere within the CAG repeats and generate stutters (minor peaks) and allows amplification over the entire CAG repeat (major peak). The first 15 bp of the reverse primer from the 5′ end are specific for the HD site (underlined), and the next 15 bp are chimeric (shown in bold): 5′-CGGTGGCGGCTGTTGCTGCTGCTGCTGCTG-3′. This introduces nonspecific DNA amplification by random annealing within the CAG repeats. The result is a stuttering pattern that represents PCR products differing in size by one triplet repeat of the length of CAG repeats contained in the samples. A similar design has been put forward by Warner et al18Warner 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.J Med Genet. 1996; 33: 1022-1026Crossref PubMed Google Scholar and Vnencak-Jones19Vnencak-Jones C.L. Fluorescence PCR and GeneScan analysis for the detection of CAG repeat expansions associated with Huntington's disease.Methods Mol Biol. 2003; 217: 101-108PubMed Google Scholar with denaturing polyacrylamide gel, which is less sensitive in detecting stuttering peaks when compared with capillary electrophoresis. Our design differs from their forward primer, which has a known benign variant at its 3′ end.20Margolis R.L. Stine O.C. Callahan C. Rosenblatt A. Abbott M.H. Sherr M. Ross C.A. Two novel single-base-pair substitutions adjacent to the CAG repeat in the huntington disease gene (IT15): implications for diagnostic testing.Am J Hum Genet. 1999; 64: 323-326Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Five fully complementary CAG repeats found on the 3′ end of our reverse primer amplify the CAG repeats in such a way that a peak is produced starting from the fifth complete CAG repeat, enhancing the counting and binning of repeats using GeneMarker software version 1.85 (SoftGenetics, State College, PA), and GeneMapper software version 4.0 (Applied Biosystems, Foster City, CA). Our PCR primer design uses two primers for the triplet repeat primed PCR but generates similar triplet repeats.21Lyon E. Laver T. Yu P. Jama M. Young K. Zoccoli M. Marlowe N. A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis.J Mol Diagn. 2010; 12: 505-511Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22Filipovic-Sadic S. Sah S. Chen L. Krosting J. Sekinger E. Zhang W. Hagerman P.J. Stenzel T.T. Hadd A.G. Latham G.J. Tassone F. A novel FMR1 PCR method for the routine detection of low abundance expanded alleles and full mutations in fragile X syndrome.Clin Chem. 2010; 56: 399-408PubMed Google Scholar, 23Chen L. Hadd A. Sah S. Filipovic-Sadic S. Krosting J. Sekinger E. Pan R. Hagerman P.J. Stenzel T.T. Tassone F. Latham G.J. An information-rich CGG repeat primed PCR that detects the full range of fragile X expanded alleles and minimizes the need for Southern blot analysis.J Mol Diagn. 2010; 12: 589-600Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 24Ciotti P. Di Maria E. Bellone E. Ajmar F. Mandich P. Triplet repeat primed PCR (TP PCR) in molecular diagnostic testing for Friedreich ataxia.J Mol Diagn. 2004; 6: 285-289Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The forward and reverse primers were designed to avoid all reported variants and deletions within exon 1 of the HTT gene (Figure 1), resulting in the ability to provide results when polymorphisms or deletions in the region between the CAG and CCG tracts9Gellera C. Meoni C. Castellotti B. Zappacosta B. Girotti F. Taroni F. DiDonato S. Errors in Huntington disease diagnostic test caused by trinucleotide deletion in the IT15 gene.Am J Hum Genet. 1996; 59: 475-477PubMed Google Scholar, 25Margolis R.L. Ross C.A. Diagnosis of Huntington disease.Clin Chem. 2003; 49: 1726-1732Crossref PubMed Scopus (82) Google Scholar, 26Yu S. Fimmel A. Fung D. Trent R.J. Polymorphisms in the CAG repeat: a source of error in Huntington disease DNA testing.Clin Genet. 2000; 58: 469-472Crossref PubMed Scopus (17) Google Scholar are present. PCR was performed using the GeneAmp 9700 thermal reaction cycler (Applied Biosystems) in a 20-μL PCR mixture containing 1.0 μL of 15 to 25 ng/μL of extracted DNA, 0.5 μmol/L each of forward primer 5′-ATGAAGGCCTTCGAGTCCCTCAAGTCC-3′, with its 5′ end fluorescently labeled with carboxyfluorescein, and the reverse oligonucleotide primer 5′-CGGTGGCGGCTGTTGCTGCTGCTGCTGCTG-3′ (Integrated DNA Technologies, Coralville, IA); 1X FailSafe Premix J containing PCR buffer, dNTPs, and MgCl2 (Epicenter, Madison, WI); and 1.0 U of Platinum TaqDNA polymerase (Invitrogen Corporation, Carlsbad, CA). Amplification was performed with an initial denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 64°C for 1 minute, extension at 72°C for 2 minutes, and a final 15-minute extension at 72°C. A total of 1 μL of the generated PCR products was added to a mixture of 1.0 μL of MapMarker1000 internal size standard (BioVentures Inc., Murfreesboro, TN) and 9 μL of HiDi formamide (Applied Biosystems). 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 electrophoresis on an automated ABI Prism 3100 Genetic Analyzer using performance optimized polymer (POP-6), with a 50-cm array (Applied Biosystems). Samples were electrokinetically injected at 15 kV for 10 seconds and electrophoresed at 15 kV for 5400 seconds at 60°C under filter set D. Raw data were analyzed with GeneMarker software where macros were set for automated bin calling, generated from the stuttering pattern obtained from Coriell sample NA20253. With the HD chimeric primer, the first CAG trinucleotide stutter began at the fifth CAG repeat and continued until the 110th CAG repeat past the 101st CAG allele of the expanded allele. Each stutter represents a CAG repeat that is binned. Each CAG bin is calculated from the means generated from within and between reproducibility runs. Two SDs were used for setting the upper and lower boundary of each CAG bin size to minimize inclusion of stray alleles into the wrong bin interval27Ghosh S. Karanjawala Z.E. Hauser E.R. Ally D. Knapp J.I. Rayman J.B. Musick A. Tannenbaum J. Te C. Shapiro S. Eldridge W. Musick T. Martin C. Smith J.R. Carpten J.D. Brownstein M.J. Powell J.I. Whiten R. Chines P. Nylund S.J. Magnuson V.L. Boehnke M. Collins F.S. FUSION (Finland-US Investigation of NIDDM Genetics) Study GroupMethods for precise sizing, automated binning of alleles, and reduction of error rates in large-scale genotyping using fluorescently labeled dinucleotide markers.Genome Res. 1997; 7: 165-178Crossref PubMed Scopus (94) Google Scholar, 28Idury R.M. Cardon L.R. A simple method for automated allele binning in microsatellite markers.Genome Res. 1997; 7: 1104-1109PubMed Google Scholar (Figure 2). Assay specificity and sensitivity were determined by combining and comparing unaffected categories (normal and normal mutable) to affected categories (reduced penetrance and full penetrance). All calculations were performed using SAS statistical software version 9.2 of the SAS System (SAS Institute Inc., Cary, NC). A total of 246 HD samples were characterized within ±1 CAG repeat in this study, giving a perfect concordance (Spearman ρ = 1), 100% (95% CI, 96.1% to 100%) specificity, and 100% (95% CI, 97.6% to 100%) sensitivity among the genotypes determined by this HD chimeric assay and a previous HD method used to characterize the same samples. Six affected samples with >40 CAG repeats were within the ±1 CAG4International Huntington Association (IHA) and the World Federation of Neurology (WFN) Research Group on Huntington's ChoreaGuidelines for the molecular genetics predictive test in Huntington's disease.Neurology. 1994; 44: 1533-1536Crossref PubMed Google Scholar, 25Margolis R.L. Ross C.A. Diagnosis of Huntington disease.Clin Chem. 2003; 49: 1726-1732Crossref PubMed Scopus (82) Google Scholar (Table 1). The 14 HD reference samples from Coriell cell repositories were amplified five times each for intrarun and interrun variability. Alleles ranging from 15 to 101 CAG repeats were accurately sized within ±1 repeats in base pairs and are listed in Table 2. True homozygous normal samples were distinguished from expanded alleles by the absence of a stuttering pattern denoting an expanded allele. Coriell sample NA20045 had no expanded allele beyond the 15 CAG allele, as illustrated by the flat baseline on the electropherogram (Figure 3A); therefore, it was determined to be a true homozygous HD sample. The same flat baseline in the electropherogram was noted for all 11 homozygous ARUP laboratory HD samples.Table 2Assay Precision: Means ± SD of Five Coriell Samples Used for Within- and Between-Run ComparisonsSampleMeans ± SD CAG repeat size, bpWithin-run validation (n=5 replicates)Between-run validation (n=5 runs)NA20207 Allelle 1996.86 ± 0.0596.72 ± 0.1 Allele 21102.88 ± 0.04102.72 ± 0.1NA20249 Allele 22105.9 ± 0105.72 ± 0.1 Allele 39155.92 ± 0.04155.7 ± 0.1NA20252 Allele 22105.86 ± 0.05105.7 ± 0.1 Allele 65231.36 ± 0.2230.9 ± 0.2NA20253 Allele 22105.86 ± 0.1105.68 ± 0.1 Allele 101334.2 ± 0.1333.6 ± 0.4MGL-14 Allele 1996.88 ± 0.0496.74 ± 0.1 AlleleExpandedExpanded Open table in a new tab For assay interpretation, the prominent peak(s) are used for CAG allele calls and continuous stutter peaks for determining the existence of expanded alleles in apparently homozygous samples. These stuttering peaks are also important in distinguishing heterozygous samples that differ by one or two CAG repeats. Figure 3B illustrates the pattern generated by a 19/21 heterozygote. Note the less prominent peak in the superimposed image between the 19 and 21 allele peaks. The stutter peak pattern generated by Coriell sample NA20253 displays a typical pattern generated by an expanded allele. The prominent peak is located in the center of the stutter pattern and corresponds to the correct allele size of 101 repeats (Figure 4A). In contrast, the stutter peak pattern generated by Coriell sample NA20252 reveals that a unique pattern of the prominent peak skewed one CAG repeat to the right of the center (figure not shown). This is possibly due to mosaicism in lymphocytes commonly found in expanded HD alleles.25Margolis R.L. Ross C.A. Diagnosis of Huntington disease.Clin Chem. 2003; 49: 1726-1732Crossref PubMed Scopus (82) Google Scholar Sequencing data by Kalman and colleagues14Kalman L. Johnson M.A. Beck J. Berry-Kravis E. Buller A. Casey B. Feldman G.L. Handsfield J. Jakupciak J.P. Maragh S. Matteson K. Muralidharan K. Richie K.L. Rohlfs E.M. Schaefer F. Sellers T. Spector E. Richards C.S. Development of genomic reference materials for Huntington disease genetic testing.Genet Med. 2007; 9: 719-723Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar confirm the correct size of this allele to be 65 repeats, the center peak, although not the most prominent. The CAP proficiency sample MGL-14 (Figure 4B) had an expanded allele of >200 CAG repeats, which was detected by the chimeric PCR assay. However, the size could not be detected because the primers failed to amplify the full-length product. Instead, only a stuttering pattern, which faded beyond ≥100 repeats, was seen. Therefore, we set 101 CAG repeats as the upper limit for accurately sized expanded alleles. Thus, samples with expanded alleles >101 CAG repeats will require Southern blot analysis for estimated sizing. Five challenging clinical samples were selected that had required sequencing subsequent to being run with the prior clinical assay (including amplification of the CCG+CAG and the CCG alleles separately). The prior clinical assay was confounded because of the presence of variants (single nucleotide change or CAA deletion) (Figure 1) in the region immediately 3′ of the CAG repeats. With the standard assay, these samples initially appeared homozygous and were reflexed to the CCG/CAG assays, which use primers external to the first-tier assay. The samples revealed two alleles in the normal range with the CCG/CAG assay and were sequenced to identify the reason for the apparent allele dropout. The chimeric assay was able to accurately size both alleles in all samples without additional testing. Short tandem repeat loci are analyzed by PCR, followed by capillary electrophoresis.29Jeffreys A.J. Wilson V. Thein S.L. Individual-specific 'fingerprints' of human DNA.Nature. 1985; 316: 76-79Crossref PubMed Scopus (1512) Google Scholar, 30Smith L.M. Sanders J.Z. Kaiser R.J. Hughes P. Dodd C. Connell C.R. Heiner C. Kent S.B. Hood L.E. Fluorescence detection in automated DNA sequence analysis.Nature. 1986; 321: 674-679Crossref PubMed Scopus (1270) Google Scholar The PCR amplifies the sequence as defined by the flanking fluorescent dye–labeled primers, and the products are then resolved on a capillary electrophoresis instrument. The variable length of each allele is due to variation in the number of CAG repeats contained within the fragment. When a chimeric primer is introduced into a repetitive region, such as those found in the HTT gene, the PCR effect is stuttering of the trinucleotide repeat. This method provides the amplicon size, although it does not provide full sequence information. The generation of allelic bins greatly assists in correct allele genotyping. This assay also eliminates the need for CAG and CAG+CCG analysis because true homozygous and apparent homozygous samples can be easily distinguished based on the presence or absence of an expanded stuttering pattern.10Tassone F. Pan R. Amiri K. Taylor A.K. Hagerman P.J. A rapid polymerase chain reaction-based screening method for identification of all expanded alleles of the fragile X (FMR1) gene in newborn and high-risk populations.J Mol Diagn. 2008; 10: 43-49Abstract Full Text Full Text PDF PubMed Scopus (128
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