Development of a Genomic DNA Reference Material Panel for Myotonic Dystrophy Type 1 (DM1) Genetic Testing
2013; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2013.03.008
ISSN1943-7811
AutoresLisa V. Kalman, Jack Tarleton, Monica Hitch, Madhuri Hegde, Nick L. Hjelm, Elizabeth Berry‐Kravis, Lili Zhou, James E. Hilbert, Elizabeth Luebbe, Richard T. Moxley, Lorraine H. Toji,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoMyotonic dystrophy type 1 (DM1) is caused by expansion of a CTG triplet repeat in the 3′ untranslated region of the DMPK gene that encodes a serine-threonine kinase. Patients with larger repeats tend to have a more severe phenotype. Clinical laboratories require reference and quality control materials for DM1 diagnostic and carrier genetic testing. Well-characterized reference materials are not available. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community, the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members, and the Coriell Cell Repositories, has established and characterized cell lines from patients with DM1 to create a reference material panel. The CTG repeats in genomic DNA samples from 10 DM1 cell lines were characterized in three clinical genetic testing laboratories using PCR and Southern blot analysis. DMPK alleles in the samples cover four of five DM1 clinical categories: normal (5 to 34 repeats), mild (50 to 100 repeats), classical (101 to 1000 repeats), and congenital (>1000 repeats). We did not identify or establish Coriell cell lines in the premutation range (35 to 49 repeats). These samples are publicly available for quality control, proficiency testing, test development, and research and should help improve the accuracy of DM1 testing. Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG triplet repeat in the 3′ untranslated region of the DMPK gene that encodes a serine-threonine kinase. Patients with larger repeats tend to have a more severe phenotype. Clinical laboratories require reference and quality control materials for DM1 diagnostic and carrier genetic testing. Well-characterized reference materials are not available. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community, the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members, and the Coriell Cell Repositories, has established and characterized cell lines from patients with DM1 to create a reference material panel. The CTG repeats in genomic DNA samples from 10 DM1 cell lines were characterized in three clinical genetic testing laboratories using PCR and Southern blot analysis. DMPK alleles in the samples cover four of five DM1 clinical categories: normal (5 to 34 repeats), mild (50 to 100 repeats), classical (101 to 1000 repeats), and congenital (>1000 repeats). We did not identify or establish Coriell cell lines in the premutation range (35 to 49 repeats). These samples are publicly available for quality control, proficiency testing, test development, and research and should help improve the accuracy of DM1 testing. 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 planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The authors have no relevant financial relationship to disclose except for Madhuri Hegde, who received honoraria as scientific advisor to Genome Quest, RainDance, Tessarar, and Oxford Genetic Technologies. These relationships were deemed not to be relevant to the educational activity. 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 planning committee members and staff have no relevant financial relationships with commercial interests to disclose. The authors have no relevant financial relationship to disclose except for Madhuri Hegde, who received honoraria as scientific advisor to Genome Quest, RainDance, Tessarar, and Oxford Genetic Technologies. These relationships were deemed not to be relevant to the educational activity. Myotonic dystrophy type 1 (DM1) (Steinert disease), the most common form of adult muscular dystrophy, is a dominantly inherited, multisystem disorder that typically affects skeletal, smooth, and cardiac muscle, the eyes, the brain, and endocrine function.1Harper P.S. Myotonic Dystrophy. Saunders Co, London, W.B2001Google Scholar Although penetrance is approximately 100% by age 50 years, there is variable expressivity, and mild cases may be misdiagnosed or undiagnosed.2Prior T.W. American College of Medical Genetics (ACMG) Laboratory Quality Assurance CommitteeTechnical standards and guidelines for myotonic dystrophy type I testing.Genet Med. 2009; 11: 552-555Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 3Bird T.D. Myotonic Dystrophy Type 1.in: GeneReviews [Internet]. Copyright University of Washington, Seattle19932012http://www.ncbi.nlm.nih.gov/books/NBK1499Google Scholar DM1 results from an unstable CTG triplet expansion in the 3′ untranslated region of the DMPK gene (encodes a serine-threonine kinase) located on chromosome 19q13.3.2Prior T.W. American College of Medical Genetics (ACMG) Laboratory Quality Assurance CommitteeTechnical standards and guidelines for myotonic dystrophy type I testing.Genet Med. 2009; 11: 552-555Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 4Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member.Cell. 1992; 68: 799-808Abstract Full Text PDF PubMed Scopus (2027) Google Scholar, 5Fu Y.H. Pizzuti A. Fenwick Jr., R.G. King J. Rajnarayan S. Dunne P.W. Dubel J. Nasser G.A. Ashizawa T. de Jong P. Wieringa B. Korneluk R. Perryman M.B. Epstein H.F. Caskey C.T. An unstable triplet repeat in a gene related to myotonic muscular dystrophy.Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1258) Google Scholar, 6Mahadevan M. Tsilfidis C. Sabourin L. Shutler G. Amemiya C. Jansen G. Neville C. Narang M. Barceló J. O'Hoy K. Leblond S. Earle-Macdonald J. de Jong P.J. Wieringa B. Korneluk R.G. Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene.Science. 1992; 255: 1253-1255Crossref PubMed Scopus (1404) Google Scholar Individuals not affected by DM1 have 5 to 34 CTG triplet repeats in leukocyte DNA. Patients with DMPK alleles in leukocyte DNA with 35 to 49 CTG repeats (premutations) do not have symptoms, but their children have an increased risk of inheriting larger CTG repeats and of having symptoms.2Prior T.W. American College of Medical Genetics (ACMG) Laboratory Quality Assurance CommitteeTechnical standards and guidelines for myotonic dystrophy type I testing.Genet Med. 2009; 11: 552-555Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar DMPK alleles with CTG repeat expansions >49 lead to a wide spectrum of symptoms that characterize the DM1 phenotype. Alleles >49 CTG repeats are unstable and may expand in length during meiosis, causing offspring to inherit CTG repeats that are longer than those in the parent. These children often display anticipation defined as an earlier age at onset of a more severe phenotype than the affected parent.3Bird T.D. Myotonic Dystrophy Type 1.in: GeneReviews [Internet]. Copyright University of Washington, Seattle19932012http://www.ncbi.nlm.nih.gov/books/NBK1499Google Scholar In general, patients with larger CTG repeat expansions in circulating leukocyte DNA tend to have more severe clinical phenotypes.7Harley H.G. Brook J.D. Rundle S.A. Crow S. Reardon W. Buckler A.J. Harper P.S. Housman D.E. Shaw D.J. Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy.Nature. 1992; 355: 545-546Crossref PubMed Scopus (645) Google Scholar, 8Tsilfidis C. MacKenzie A.E. Mettler G. Barceló J. Korneluk R.G. Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophy.Nat Genet. 1992; 1: 192-195Crossref PubMed Scopus (301) Google Scholar, 9Hunter A. Tsilfidis C. Mettler G. Jacob P. Mahadevan M. Surh L. Korneluk R. The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy.J Med Genet. 1992; 29: 774-779Crossref PubMed Scopus (196) Google Scholar, 10Moxley R.T. Meola G. The Myotonic Dystrophies. The Molecular and Genetic Basis of Neurologic and Psychiatric Disease.in: Rosenberg R.N. DiMauro S. Paulson H.L. Ptacek L. Nestler E.J. Wolters Kluwer, Philadelphia2008: 532-541Google Scholar However, most patients display somatic tissue mosaicism in skeletal muscle, heart, and brain, which can complicate prediction of the phenotype severity.10Moxley R.T. Meola G. The Myotonic Dystrophies. The Molecular and Genetic Basis of Neurologic and Psychiatric Disease.in: Rosenberg R.N. DiMauro S. Paulson H.L. Ptacek L. Nestler E.J. Wolters Kluwer, Philadelphia2008: 532-541Google Scholar, 11Ashizawa T. Dubel J.R. Harati Y. Somatic instability of CTG repeat in myotonic dystrophy.Neurology. 1993; 43: 2674-2678Crossref PubMed Google Scholar, 12Anvret M. Ahlberg G. Grandell U. Hedberg B. Johnson K. Edström L. Larger expansions of the CTG repeat in muscle compared to lymphocytes from patients with myotonic dystrophy.Hum Mol Genet. 1993; 2: 1397-1400Crossref PubMed Scopus (174) Google Scholar, 13Thornton C.A. Johnson K. Moxley III, R.T. Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes.Ann Neurol. 1994; 35: 104-107Crossref PubMed Scopus (239) Google Scholar Patients with 50 to 100 repeats are mildly affected, usually with cataracts and/or mild myotonia, which develop later in adulthood. Those with the classic phenotype (Steinert's disease) have 101 to 1000 CTG repeats in leukocyte DNA and have classic DM1 symptoms, including cataracts, grip myotonia, and distal weakness. The age at onset for the classical group is approximately late teens to 30 years. Patients with congenital or childhood myotonic dystrophy typically present between birth and 10 years of age and have more severe manifestations. These patients typically have >1000 CTG repeats in circulating blood cells. Molecular genetic testing for DM1 relies primarily on measurement of the number of CTG repeats in the DMPK gene isolated from circulating leukocyte DNA. PCR can be used to measure repeat length in DMPK alleles up to approximately 100 CTG repeats. Southern blot analysis is used to detect larger expansions. There are currently no US Food and Drug Administration-approved or cleared molecular genetic tests for DM1. All clinical testing is currently performed using tests developed in the individual laboratory. Clinical laboratories use characterized reference materials for a variety of quality assurance purposes, including test development, test validation, quality control, and for alternative assessment. Proficiency testing programs often distribute characterized genomic DNA reference materials to their participants. The use of reference materials is also mandated by regulatory requirements and professional guidelines for clinical laboratories.14International Organization for StandardizationISO 15189 Medical Laboratories: Particular Requirements for Quality and Competence. International Organization for Standardization, Geneva2007Google Scholar, 15Centers for Medicare and Medicaid Services, US Department of Health and Human Services: Part 493-Laboratory Requirements: Clinical Laboratory Improvement Amendments of 1988. 42 CFR §493.1443-1495Google Scholar, 16Chen B. Gagnon M. Shahangian S. Anderson N.L. Howerton D.A. Boone D.J. Good laboratory practices for molecular genetic testing for heritable diseases and conditions.MMWR Morb Mortal Wkly Rep. 2009; 58: 1-29PubMed Google Scholar, 17Association for Molecular Pathology statement: recommendations for in-house development and operation of molecular diagnostic tests.Am J Clin Pathol. 1999; 111: 449-463PubMed Google Scholar, 18Chen B. O'Connell C.D. Boone D.J. Amos J.A. Beck J.C. Chan M.M. et al.Developing a sustainable process to provide quality control materials for genetic testing.Genet Med. 2005; 7: 534-549Crossref PubMed Scopus (43) Google Scholar, 19CLSI. Molecular Methods for Clinical Genetics and Oncology Testing; Approved Guideline-Third Edition CLSI document MM01-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2012Google Scholar, 20Clinical and Laboratory Standards InstituteVerification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline, MM17-A. Clinical and Laboratory Standards Institute, Wayne, PA2008Google Scholar [American College of Medical Genetics: Standards and Guidelines for Clinical Genetics Laboratories, http://www.acmg.net/AM/Template.cfm?Section=Publications1; Washington State Department of Health, http://www.doh.wa.gov/hsqa/fsl/lqa_home.htm; College of American Pathologists, http://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=accreditation (paid subscription required); and New York State Clinical Laboratory Evaluation Program, http://www.wadsworth.org/clep, all websites last accessed January 11, 2013.] Reference materials should be well characterized, homogeneous, and closely resemble an actual clinical specimen.20Clinical and Laboratory Standards InstituteVerification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline, MM17-A. Clinical and Laboratory Standards Institute, Wayne, PA2008Google Scholar Ideally, the laboratory should use a set of reference materials that contain the range of mutation types expected for the test that will be performed. This will allow development and evaluation of assays to detect a range of variants. Reference materials containing a range of CTG repeat lengths (from <35 to several thousand repeats) should be used for development and evaluation of DM1 genetic tests. There are no commercial or other sources of characterized reference materials for molecular genetic testing of DM1. Laboratories that perform this test typically use characterized genomic DNA from cell lines, such as those available from the National Institute of General Medical Sciences (NIGMS) repository at the Coriell Institute for Medical Research (Camden, NJ) or residual patient samples as reference materials. Laboratories and proficiency testing programs have been unable to obtain many of the necessary reference materials because cell lines and patient samples with the complete range of CTG repeat lengths are not readily available. To address this need, the Centers for Disease Control and Prevention's Genetic Testing Reference Material (GeT-RM) Coordination Program collaborated with members of the genetic testing community, the NIGMS repository, and the NIH-funded National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members.21Hilbert J.E. Kissel J.T. Luebbe E.A. Martens W.B. McDermott M.P. Sanders D.B. Tawil R. Thornton C.A. Moxley III, R.T. Registry Scientific Advisory CommitteeIf you build a rare disease registry, will they enroll and will they use it? methods and data from the National Registry of Myotonic Dystrophy (DM) and Facioscapulohumeral Muscular Dystrophy (FSHD).Contemp Clin Trials. 2012; 33: 302-311Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar This report describes the characterization of new and preexisting cell lines in three clinical genetic testing laboratories and the development of a DM1 reference material panel for research and improved clinical testing. DM1 cell lines with a range of repeat lengths were identified in the NIGMS repository at the Coriell Institute for Medical Research. Myotonic dystrophy repeat lengths that were not represented in the NIGMS repository were also identified, and patients with these repeat lengths were recruited and asked to donate whole blood for lymphoblast cell line development. DNA from 13 preexisting cell lines from the NIGMS repository and 12 newly established lines were selected for further characterization in one laboratory to identify candidate reference materials. New DM1 cell lines were established through collaboration with the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members at the University of Rochester Medical Center (Rochester, NY). A protocol and study materials were prepared and approved by the University of Rochester Research Subjects Review Board. Patients with CTG repeats in selected size ranges (30 to 49, 50 to 199, and ≥2000) were recruited by the National Registry through mailed requests. Patients and their families who wished to donate blood for cell line creation returned the signed consent form to the University of Rochester. Consented patients were then mailed a blood collection kit containing instructions for the patient, a letter for the medical care provider who would collect the blood, blood collection tubes labeled with the deidentified research code, and preaddressed and prepaid shipping labels for sending the blood tubes to the Coriell Cell Repositories. When the patient visited his or her physician for a regular visit, blood was collected and sent (deidentified) to Coriell using the return shipping label and materials provided. Coriell does not have access to patients' names or other identifying information. When the blood specimens were received, each was assigned a new number and was prepared for cell line establishment. When the cell line was established, the data manager requested the deidentified clinical information from the patient registry using the corresponding research code. Cell line creation was performed as previously described.22Kalman L. Leonard J. Gerry N. Tarleton J. Bridges C. Gastier-Foster J.M. Pyatt R.E. Stonerock E. Johnson M.A. Richards S. Schrijver I. Ma T. Rangel Miller V. Adadevoh Y. Furlong P. Beiswanger C. Toji L. Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel.J Mol Diagn. 2011; 13: 167-174Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Whole blood samples collected from consenting patients or their families were sent to the Coriell Cell Repositories for Epstein-Barr virus transformation of B lymphocytes, as previously described.23Beck J.C. Beiswanger C.M. John E.M. Satraiano E. West D. Successful transformation of cryopreserved lymphocytes: a resource for epidemiological studies.Cancer Epidemiol Biomarkers Prev. 2001; 10: 551-554PubMed Google Scholar, 24Bernacki S.H. Stankovic A.K. Williams L.O. Beck J.C. Herndon J.E. Snow-Bailey K. Prior T.W. Matteson K.J. Wasserman L.M. Cole E.C. Stenzel T.T. Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.J Mol Diagn. 2003; 5: 227-230Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar All the samples were placed in culture and were expanded to yield approximately 2 × 108 total viable cells. The culture medium was antibiotic free to increase the likelihood that contamination would be readily detected. The cell suspension was dispensed into forty 1-mL ampules containing 5 × 106 viable cells each. Cultures were cryopreserved in heat-sealed borosilicate glass ampules and were stored in liquid nitrogen (liquid phase). Successful cultures were free of bacterial, fungal, and mycoplasma contamination and were viable after cryopreservation in liquid nitrogen, as evidenced by doubling of the cell number within 4 days of recovery. DNA was prepared as previously described.22Kalman L. Leonard J. Gerry N. Tarleton J. Bridges C. Gastier-Foster J.M. Pyatt R.E. Stonerock E. Johnson M.A. Richards S. Schrijver I. Ma T. Rangel Miller V. Adadevoh Y. Furlong P. Beiswanger C. Toji L. Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel.J Mol Diagn. 2011; 13: 167-174Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Approximately 2 mg of DNA was prepared from cultures of each of the selected cell lines by the Coriell Cell Repositories using the Gentra Autopure system (Qiagen Corp., Valencia, CA) per the manufacturer's instructions or previously described methods.25Miller S.A. Dykes D.D. Polesky H.F. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17628) Google Scholar Three clinical genetics laboratories that offer testing for DM1 volunteered to participate in the study. All three laboratories are located in the United States and are accredited by the College of American Pathologists. Each volunteer laboratory received 20 to 30 μg of DNA from each of the 10 selected DM1 cell lines. The expected repeat length was not revealed to the laboratories. The laboratories genotyped each DNA sample using their standard DM1 assay. The results were collected by the study coordinator (L.K.), who examined the data for discrepancies. The number of CTG repeats was determined using PCR specific for DMPK alleles with less than approximately 100 repeats.4Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member.Cell. 1992; 68: 799-808Abstract Full Text PDF PubMed Scopus (2027) Google Scholar PCR was performed using the following primers: 5′-CTTCCCAGGCCTGCAGTTTGCCCATC-3′ and 5′-GAACGGGGCTCGAAGGGTCCTTGTAGC-3′, with the latter primer being modified by the addition of 6-FAM (6-carboxyfluorescein) to detect the product by capillary electrophoresis. Approximately 200 to 250 ng of genomic DNA was amplified in a total reaction volume of 20 μL. The final concentrations of PCR reagents were as follows: 1× GeneAmp PCR buffer II, 1.2 mmol/L MgCl2, 200 μmol/L deoxynucleotide mix, 10 ng/μL of each primer, and 1.6 U of AmpliTaq Gold polymerase (Applied Biosystems/Life Technologies, Grand Island, NY). Thermocycling conditions in an Applied Biosystems 9700 or 2720 thermal cycler (Applied Biosystems/Life Technologies) were as follows: 95°C × 10 minutes for initial denaturation followed by 25 cycles at 95°C × 30 seconds, 62°C × 30 seconds, and 72°C × 1 minute, with an additional 5 minutes at 72°C after the final temperature cycle. One microliter of the amplified product was mixed with 0.5 μL of GeneScan-500 ROX size standard and 10 μL of Hi-Di formamide (Applied Biosystems/Life Technologies). Immediately before loading onto an Applied Biosystems 3130 or 3130 XL genetic analyzer (Applied Biosystems/Life Technologies), the samples were denatured for 5 minutes at 100°C and then were placed on ice. Southern blot analysis was performed as previously described26Buxton J. Shelbourne P. Davies J. Jones C. Van Tongeren T. Aslanidis C. de Jong P. Jansen G. Anvret M. Riley B. Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy.Nature. 1992; 355: 547-548Crossref PubMed Scopus (557) Google Scholar using the DNA probe p5B1.4 [a gift from Keith Johnson (Charing Cross and Westminster Medical School, London, UK)]. Genomic DNA (5 μg) was separately digested with 50 U each of EcoRI and BamHI (Promega, Madison, WI) and was separated by electrophoresis on a 24-cm-long 1% SeaKem agarose gel (Lonza, Rockland, ME) containing ethidium bromide (a 0.5-μg/mL final concentration) (Sigma-Aldrich, St. Louis, MO) for approximately 18 hours at 40 V. Expanded CTG repeat alleles were estimated after autoradiography by comparing migration with a 1-kilobase (kb) DNA ladder (Invitrogen/Life Technologies) on the agarose gel used for Southern blot analysis. Electrophoretic migration of the autoradiographic signal from expanded CTG repeats was measured from the top of the Southern blot autoradiograph and was visually compared with a image of the ethidium bromide-stained agarose gel (before blotting) that contained a fluorescent ruler overlaying the 1-kb DNA ladder. The fluorescent ruler measures migration from the top of the agarose gel. The autoradiographic signal measurement from the top of the Southern blot filter corresponds to the agarose gel migration distance as measured by the ruler on the image. When the autoradiographic signal from expanded repeats was diffuse, a frequent occurrence during repeat expansion, the midpoint of the signal was used. CTG repeats were estimated by correlating the measurement after autoradiography with the same distance on the agarose gel and then comparing this measurement with electrophoretic migration of the known 1-kb DNA ladder fragments to obtain an estimate of the size of the expanded repeat allele. The DM1 gene region containing the expanded CTG repeat was PCR amplified with primer pair 5′-AACGGGGCTCGAAGGGTCCT-3′ (forward) and 5′-GCCGAAAGAAAGAAATGGTCTGT-3′ (reverse) in a GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA). The PCRs were performed using 0.5 U of Taq polymerase (Fisher Scientific, Waltham, MA) in a total volume of 25 μL containing 50 ng of genomic DNA, 0.8 μmol/L primers, 1 mmol/L MgCl2, and 0.2 mmol/L dNTPs, 0.1 μCi of 32[P]dCTP (PerkinElmer/NEN Life Sciences, Boston, MA), and 5% dimethyl sulfoxide. The amplification was performed with an initial denaturation at 94°C × 5 minutes, followed by 30 cycles of denaturation at 94°C × 30 seconds, annealing at 60°C × 30 seconds, and extension at 72°C × 30 seconds. The final extension was at 72°C for 10 minutes. The PCR products were separated by electrophoresis on a 6% denaturing polyacrylamide gel and were visualized by autoradiography. Allele repeat number was determined by comparison with standard fragments of known size for which repeat number had been previously determined by DNA sequencing analysis. Southern blot analysis was also performed using 10 μg of DNA digested with 10 U of SacI restriction enzyme (New England Biolabs, Ipswich, MA) incubated overnight at 37°C. DNA was precipitated with 3 mol/L sodium acetate and resuspended in 45 μL of hydration solution (Qiagen Inc.). All digested DNAs were applied to a 14-cm 1% agarose gel with 1× Tris-acetate-EDTA buffer (pH 8.3). Electrophoresis was performed for 3.5 hours at 94 V. The Bioline HyperLadder I DNA ladder with 14 bands ranging from 200 to 10,000 bp (Bioline, Taunton, MA) was used as the size marker. The DNA was transferred to a positively charged Millipore S4056 nylon membrane (Millipore, Billerica, MA) using the Probe Tech transfer system (Oncor Inc., Gaithersburg, MD) according to the manufacturer's directions. The probe pM10M627Pizzuti A. Friedman D.L. Caskey T.C. The myotonic dystrophy gene.Arch Neurol. 1993; 50: 1173-1179Crossref PubMed Scopus (34) Google Scholar was radiolabeled with α-32[P]dCTP, incubated overnight with the membrane at 42°C, washed with 2× standard saline citrate (SSC)/0.1% SDS for 30 minutes twice and once at 65°C with 0.5× SSC/0.1% SDS for 30 minutes, and, finally, autoradiographed for 2 to 5 days using Fuji medical X-ray film (Fisher Scientific). In the first analysis, bands representing expanded alleles were estimated by comparison of migration distances of bands and smears representing expanded alleles with migration distances of bands in the Bioline HyperLadder I DNA ladder run on the same gel. In the second analysis, band size was recalculated from the original autoradiogram. Distance of migration of bands on the ladder was measured from the origin and was plotted on semilog paper. Distance of migration of the midpoint of the band or smear representing the expanded allele in the samples was measured and size of the expansion was calculated for the measured distance using the log scale from the size standards. PCR was performed using approximately 100 ng of genomic DNA amplified in a total volume of 15 μL using the primers 5′-CTTCCCAGGCCTGCAGTTTGCCCATC-3′ (forward, 5′ end labeled with 6-Fam) and 5′-GAACGGGGCTCGAAGGGTCCTTGTAGC-3′ (reverse). The final concentration of PCR components was 0.333 μmol/L primers, 1× Q-Solution (Qiagen Inc.), 2% dimethyl sulfoxide (Fisher, Houston, TX), dNTP (200 μmol/L dATP, CTP, and dTTP and 50 μmol/L dGTP; Roche Applied Science, Indianapolis, IN), 167 μmol/L 7-deaza dGTP (Roche Applied Science), 1× ABI buffer without MgCl2, 2 mmol/L MgCl2, and 1.25U of AmpliTaq Gold (Applied Biosystems/Life Technologies). The PCR conditions were as follows: 94°C ×
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