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• W2137195835 abstract "The current workflow for clinical Fragile X testing is time consuming and labor intensive. Recently developed PCR-based methods simplify workflow, amplify full mutation alleles, and improve sensitivity for detecting low-level mosaicism. We evaluated the performance characteristics and workflow of two methods using commercially available reagents for determining FMR1 mutation status. We also tested each method's ability to detect mosaicism (range, 100% to 1% for males; 50% to 1% for females). One method used reagents from Asuragen (AmplideX FMR1 PCR, research use only). The second method used analyte specific reagents from Abbott Molecular, including FMR1 Primer 1 (for repeat sizing) and FMR1 Primer 2 (for screening of expanded alleles). Each reaction was evaluated for accuracy, precision, correlation with previous results, and workflow. Both methods performed equally well in accuracy and precision studies using NIST standards and previously characterized Coriell samples. Both methods showed 100% concordance with results from a previous consensus study and for previously analyzed patient samples. The Asuragen reagents were able to detect full mutation mosaicism down to 5% and premutation mosaicism to 1%. The Abbott Molecular Primer 2 reagents were able to detect both full mutation and premutation mosaicism down to 25%. Both PCR-based methods for the determination of FMR1 mutation status performed well, with expected results in their final diagnoses, and differed significantly only in their workflow. The current workflow for clinical Fragile X testing is time consuming and labor intensive. Recently developed PCR-based methods simplify workflow, amplify full mutation alleles, and improve sensitivity for detecting low-level mosaicism. We evaluated the performance characteristics and workflow of two methods using commercially available reagents for determining FMR1 mutation status. We also tested each method's ability to detect mosaicism (range, 100% to 1% for males; 50% to 1% for females). One method used reagents from Asuragen (AmplideX FMR1 PCR, research use only). The second method used analyte specific reagents from Abbott Molecular, including FMR1 Primer 1 (for repeat sizing) and FMR1 Primer 2 (for screening of expanded alleles). Each reaction was evaluated for accuracy, precision, correlation with previous results, and workflow. Both methods performed equally well in accuracy and precision studies using NIST standards and previously characterized Coriell samples. Both methods showed 100% concordance with results from a previous consensus study and for previously analyzed patient samples. The Asuragen reagents were able to detect full mutation mosaicism down to 5% and premutation mosaicism to 1%. The Abbott Molecular Primer 2 reagents were able to detect both full mutation and premutation mosaicism down to 25%. Both PCR-based methods for the determination of FMR1 mutation status performed well, with expected results in their final diagnoses, and differed significantly only in their workflow. Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and is caused by an expansion of the CGG repeat region in the 5′ untranslated region of the FMR1 gene on chromosome Xq27.3. Expansion of the repeats to full mutation range results in hypermethylation of the FMR1 promoter and prevents the production of FMR1 mRNA and protein. Other loss-of-function mutations (ie, point mutations, deletions) can also cause FXS.1Hammond L.S. Macias M.M. Tarleton J.C. Pai G.S. Fragile X syndrome and deletions in FMR1: new case and review of literature.Am J Med Genet. 2011; 72: 430-434Crossref Scopus (86) Google Scholar, 2Gronskov K. Brondum-Nielsen K. Dedic A. Hjalgrim H. A nonsense mutation in FMR1 causing fragile X syndrome.Eur J Hum Genet. 2011; 19: 489-491Crossref PubMed Scopus (39) Google Scholar, 3Coffee B. Ikeda M. Budimirovic D.B. Hjelm L.N. Kaufmann W.E. Warren S.T. Mosaic FMR1 deletion causes fragile X syndrome and can lead to molecular misdiagnosis.Am J Med Genet A. 2008; 146A: 1358-1367Crossref PubMed Scopus (79) Google Scholar Prevalence of Fragile X is estimated to be 1 in 4000 males and 1 in 5000 to 8000 females. Indications for testing the repeat region of the FMR1 gene include family history of FXS or undiagnosed intellectual disability; individuals with intellectual disability, developmental delay, or autism; women with fertility problems with elevated follicle-stimulating hormone levels; and men and women with intention tremor and cerebellar ataxia.4Sherman S. Pletcher B.A. Driscoll D.A. Fragile X syndrome: diagnostic and carrier testing.Genet Med. 2005; 7: 584-587Crossref PubMed Scopus (225) Google Scholar Current guidelines define normal alleles as 6 to 44 repeats, intermediate/gray-zone alleles as 45 to 54 alleles, premutation alleles as 55 to 200 repeats, and full mutation alleles as >200 repeats. The categories signify the likelihood of expansion from one generation to the next.5Spector E.B. Kronquist K. Fragile X Molecular Working Group 2005 for the Quality Assurance Committee of the American College of Medical GeneticsTechnical Standards and Guidelines for Fragile X Testing: A Revision to the Disease-Specific Supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics 2006 Edition.http://www.acmg.net/Pages/ACMG_Activities/stds-2002/fx.htmGoogle Scholar Premutation alleles are unstable at meiosis and have an increased risk of expansion to full mutation in the next generation.6Nolin S.L. Brown W.T. Glicksman A. Houck G.E. Gargano A.D. Sullivan A. Biancalana V. Bröndum-Nielsen K. Hjalgrim H. Holinski-Feder E. Kooy F. Longshore J. Macpherson J. Mandel J.-L. Matthijs G. Rousseau F. Steinbach P. Väisänen M.-L. von Koskull H. Sherman S.L. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles.Am J Med Genet. 2003; 72: 454-464Scopus (318) Google Scholar The risk of expansion is dependent on the size of the premutation. The smallest known premutation allele that expanded to a full mutation in one generation was 56 repeats.7Fernandez-Carvajal I. Lopez Posadas B. Pan R. Raske C. Hagerman P.J. Tassone F. Expansion of an FMR1 grey-zone allele to a full mutation in two generations.J Mol Diagn. 2009; 11: 306-310Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar In the same family, a large intermediate allele (52 repeats) expanded to full mutation within two generations.7Fernandez-Carvajal I. Lopez Posadas B. Pan R. Raske C. Hagerman P.J. Tassone F. Expansion of an FMR1 grey-zone allele to a full mutation in two generations.J Mol Diagn. 2009; 11: 306-310Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar Premutation alleles are associated with Fragile X–associated tremor and ataxia syndrome and premature ovarian insufficiency, but also with autism, attention deficit/hyperactivity disorder, and learning disabilities.8Farzin F. Perry H. Hess D. Loesch D. Cohen J. Bacalman S. Gane L. Tassone F. Hagerman P. Hagerman R. Autism spectrum disorders and attention-deficit/hyperactivity disorder in boys with the fragile X premutation.Dev and Behav Peds. 2006; 27: S137-S144Crossref PubMed Scopus (278) Google Scholar Full mutation alleles are associated with autism, intellectual disability, and dysmorphic features.9Rousseau F. Labelle Y. Bussieres J. Lindsay C. The fragile X mental retardation syndrome 20 years after the FMR1 gene discovery: an expanding universe of knowledge.Clin Biochem Rev. 2011; 32: 135-162PubMed Google Scholar FXS is an X-linked dominant disorder, and symptoms are usually milder in affected females. Among individuals with a Fragile X full mutation, many are found with mosaicism for different-sized repeats in different cells. Size mosaicism is commonly seen as a smear in the full mutation range on the Southern blot. However, size mosaicism with premutation and full mutation alleles has also been reported, as well as methylation mosaicism.10Nolin S.L. Glicksman A. Houck Jr., G.E. Brown W.T. Dobkin C.S. Mosaicism in fragile X affected males.Am J Med Genet. 1994; 15: 509-512Crossref Scopus (125) Google Scholar Although phenotypic variability may reflect the degree of mosaicism for unmethylated alleles, the role of mosaicism in the clinical presentation of the patient is not clear.10Nolin S.L. Glicksman A. Houck Jr., G.E. Brown W.T. Dobkin C.S. Mosaicism in fragile X affected males.Am J Med Genet. 1994; 15: 509-512Crossref Scopus (125) Google Scholar, 11Rousseau F. Heitz D. Tarleton J. Macpherson J. Malmgren H. Dahl N. Barnicoat A. Mathew C. Mornet E. Tajada I. Maddalena A. Spiegel R. Schinzel A. Marcos J.A.G. Schorderet D.F. Schaap T. Maccioni L. Russo S. Jacobs P.A. Schwartz C. Mandel J.L. A multicenter study on genotype-phenotype correlations in the fragile X syndrome, using direct diagnosis with probe StB12.3: the first 2,253 cases.Am J Med Genet. 1994; 55: 225-237Google Scholar, 12Orrico A. Galli L. Dotti M.T. Plewnia K. Censini S. Federico A. Mosaicism for full mutation and normal-sized allele of the FMR1 gene: a new case.Am J Med Genet. 1998; 78: 341-344Crossref PubMed Scopus (26) Google Scholar, 13Schmucker B. Seidel J. Mosaicism for a full mutation and a normal size allele in two fragile X males.Am J Med Genet. 1999; 84: 221-225Crossref PubMed Scopus (29) Google Scholar In a few cases, a mosaic pattern has been reported in patients in whom a full mutation allele coexists with a normal-sized allele.12Orrico A. Galli L. Dotti M.T. Plewnia K. Censini S. Federico A. Mosaicism for full mutation and normal-sized allele of the FMR1 gene: a new case.Am J Med Genet. 1998; 78: 341-344Crossref PubMed Scopus (26) Google Scholar, 13Schmucker B. Seidel J. Mosaicism for a full mutation and a normal size allele in two fragile X males.Am J Med Genet. 1999; 84: 221-225Crossref PubMed Scopus (29) Google Scholar The overall incidence of mosaicism is difficult to estimate because the ability to detect mosaicism may be an inherent limitation of current methodologies, and may vary from one laboratory to another, but has been reported to range from 12% to 41% for full mutation–premutation mosaicism.10Nolin S.L. Glicksman A. Houck Jr., G.E. Brown W.T. Dobkin C.S. Mosaicism in fragile X affected males.Am J Med Genet. 1994; 15: 509-512Crossref Scopus (125) Google Scholar, 11Rousseau F. Heitz D. Tarleton J. Macpherson J. Malmgren H. Dahl N. Barnicoat A. Mathew C. Mornet E. Tajada I. Maddalena A. Spiegel R. Schinzel A. Marcos J.A.G. Schorderet D.F. Schaap T. Maccioni L. Russo S. Jacobs P.A. Schwartz C. Mandel J.L. A multicenter study on genotype-phenotype correlations in the fragile X syndrome, using direct diagnosis with probe StB12.3: the first 2,253 cases.Am J Med Genet. 1994; 55: 225-237Google Scholar The current workflow in many diagnostic laboratories includes Southern blot analysis for determining mutation status (normal, premutation, full mutation) and methylation status of the FMR1 promoter, along with a PCR-based assay for determining repeat number in the normal, intermediate, and low premutation range (typically <110 repeats). Large premutation and full mutation alleles generally cannot be detected by PCR alone, which makes interpreting certain sample types (homozygous normal females and mosaic specimens) difficult without results from Southern blot analysis. Conversely, Southern blot analysis does not accurately size alleles in the normal, intermediate, and low premutation range, and may be limited by the hybridization conditions in its ability to detect mosaicism.10Nolin S.L. Glicksman A. Houck Jr., G.E. Brown W.T. Dobkin C.S. Mosaicism in fragile X affected males.Am J Med Genet. 1994; 15: 509-512Crossref Scopus (125) Google Scholar Therefore, PCR amplification combined with Southern blot analysis has been necessary for accurate CGG repeat detection and sizing. However, this type of testing is time consuming, labor intensive, and not amenable to high-throughput testing. Recently developed triplet-primed PCR-based methods have been designed to simplify workflow, detect full mutation alleles, and improve sensitivity for detecting low-level mosaicism.14Filipovic-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, 15Chen L. Hadd A.G. 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, 16Hantash F.M. Goos D.M. Tsao D. Quan F. Buller-Burckle A. Peng M. Jarvis M. Sun W. Strom C.M. Qualitative assessment of FMR1 (CGG)n triplet repeat status in normal, intermediate, premutation, full mutation, and mosaic carriers in both sexes: implications for fragile X syndrome carrier and newborn screening.Genet Med. 2010; 12: 162-173Crossref PubMed Google Scholar, 17Lyon 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, 18Tassone 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 In this paper, we describe the evaluation of two methods using commercially available reagents labeled for research use only (RUO) and analyte specific reagents (ASR) for determining FMR1 mutation status in our laboratory. The National Institute of Standards and Technology (NIST) Fragile X Human DNA Triplet Repeat Standard (SRM2399; NIST A-I) and previously NIST-sequenced DNA samples from five Coriell FX cell lines (NA07174, CD00014, NA06892, NA06906, and NA06891; Coriell Institute for Medical Research, Camden, NJ) were used to assess accuracy of the two methods (Table 1). Twenty-two other control DNA samples from Coriell were used in this study, including 16 samples that had been previously used in a consortium study of nine clinical laboratories (Table 2, Table 3).19Wilson J.A. Pratt V.M. Phansalkar A. Muralidharan K. Highsmith Jr., W.E. Beck J.C. Bridgeman S. Courtney E.M. Epp L. Ferreira-Gonzalez A. Hjelm N.L. Holtegaard L.M. Jama M.A. Jakupciak J.P. Johnson M.A. Labrousse P. Lyon E. Prior T.W. Richards C.S. Richie K.L. Roa B.B. Rohlfs E.M. Sellers T. Sherman S.L. Siegrist K.A. Silverman L.M. Wiszniewska J. Kalman L.V. Fragile Xperts Working Group of the Association for Molecular Pathology Clinical PracticeConsensus characterization of 16 FMR1 reference materials: a consortium study.J Mol Diagn. 2008; 10: 2-12Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar According to the company website, Coriell genomic DNA is purified from fresh blood or immortalized lymphocytes with the Gentra Autopure method using the Qiagen Autopure instrument according to manufacturer's instructions (Qiagen, Valencia, CA).Table 1Accuracy TestingExpectedAsuragenAbbott 2 screeningAbbott 1 sizingNIST-A202019(−1)21(+1)NIST-B30303031(+1)NIST-C41414042(+1)NIST-D51515152(+1)NIST-E60606060NIST-F73737373NIST-G88/89, 9388, 9387, ND(−1, ND)87(−1, ND)NIST-H9697(+1)9696NIST-I118121(+3)120(+2)119(+1)NA07174303031(+1)31(+1)CD0001456565656NA06892939392(−1)92(−1)NA0690696101(+5)99(+3)100(+4)NA06891118120(+2)exp(ND)120(+2)ND, not detected. Open table in a new tab Table 2Consensus Sample AnalysisSample IDSexGenotypeConsensus lengthAsuragenAbbott 2 screeningAbbott 1 sizingNA20230MINT53545454NA20232MINT46464646NA20234FINT31, 4631, 4632, 4631, 46NA20235FINT29, 4529, 4529, 4530, 45NA20236FINT31, 5331, 5431, 5431, 54NA07538FNOR29, 29293030NA20238FNOR29, 3030, 312930, 31NA20243FNOR29, 4129, 4129, 4129, 41NA20244MNOR41414141NA20231MPRE76787877NA20233MPRE117119119118NA20237MPRE100–104⁎No consensus was reached.100, 13710099, 135NA20240FPRE30, 8031, 8230, 8131, 81NA20241FPRE29, 93–110⁎No consensus was reached.30, 9129, exp29, 90NA20242FPRE30, 7330, 7431, 7430, 73NA20239FPRE/FULL20, 183–193⁎No consensus was reached.21, 20021, exp21, 202F, female; M, male; exp, expansion present; FULL, full mutation; INT, gray-zone/intermediate; NOR, normal alleles; PRE, premutation. No consensus was reached. Open table in a new tab Table 3Patient and Additional Coriell DNA Sample AnalysisExpectedAsuragenAbbott 2 screeningAbbott 1 sizingSexGenotypeA1A2GenotypeA1A2A3GenotypeA1A2SexGenotypeA1A2Smear on gelVCU13FNOR3131NOR3131NOR3031XXNOR3031NoVCU15FNOR2230NOR2330NOR2331XXNOR2330NoVCU16FNOR2931NOR2931NOR3031XXNOR2931NoVCU17FNOR3043NOR3044NOR3044XXNOR3044NoVCU18FNOR3030NOR3030NOR31XXNOR3030NoVCU19FNOR3043NOR3143NOR3143XXNOR3143NoVCU20FNOR3135NOR3135NOR3235XXNOR3236NoVCU14MNOR18NOR20NOR21XYNOR21NoVCU21MNOR18NOR20NOR21XYNOR21NoVCU22MNOR24NOR24NOR25XYNOR25NoVCU23MNOR31NOR31NOR32XYNOR31NoVCU24MNOR20NOR20NOR21XYNOR21NoVCU25MNOR29NOR30NOR29XYNOR30NoVCU26MNOR41NOR41NOR41XYNOR41NoVCU27M (XXY)NOR2929NOR3131NOR31XYNOR3031NoVCU28MNOR20NOR21NOR21XYNOR21NoNA13664FINT2849INT3052INT3152XXINT3052NoVCU30MINT50INT51INT51XYINT51NoVCU31MINT48INT49INT49XYINT49NoVCU42MINT52INT53INT53XYINT53NoVCU32FPRE3190PRE2991PRE2990XXPRE2990NoVCU33FPRE1980PRE2484PRE2483XXPRE2483NoVCU35FPRE3179PRE2479PRE2478XXPRE2478NoVCU36FPRE34121PRE26123EXP27XXPRE26120NoVCU37FPRE34112PRE31115EXP31XXPRE31113NoVCU38FPRE3082PRE3085PRE3084XXPRE3084NoVCU39FPRE4263PRE4266PRE4266XXPRE4265NoVCU29MPRE56PRE56PRE56XYPRE56YesVCU34MPRE84PRE85PRE83XYPRE83NoVCU40MPRE88PRE93PRE92XYPRE92NoVCU41MPRE130PRE138EXPXYPRE136NoVCU43FFULL35FULLFULLm33162>200EXP34XXFULL33YesVCU44FFULL32FULLFULL31>200EXP31XXFULL30YesVCU45FFULL30FULLFULL30>200EXP29XXFULL29YesVCU46FFULL24FULLFULL23>200EXP24XXFULL24YesVCU48FFULL30FULLFULL30>200EXP30XXFULL30YesNA05847FFULL21650FULL20>200EXP21XXFULL21YesNA07537FFULL29FULLFULL29>200EXP30XXFULL29YesVCU47MFULLFULLFULL>200EXPXYFULLYesVCU49MFULLFULLFULL>200EXPXYFULLYesVCU50MFULLFULLFULL>200EXPXYFULLYesVCU51MFULLFULLFULL>200>200EXPXYFULLYesVCU52MFULLFULLFULL>200EXPXYFULLYesNA04025MFULL645FULL>200EXPXYFULLYesNA07862MFULL501–550FULL>200EXPXYFULLYesNA09237MFULL931–940FULL>200EXPXYFULLYesF, female; M, male; EXP, expansion present; FULL, full mutation; FULLm, full mutation mosaic; INT, grey-zone/intermediate; NOR, normal; PRE, premutation. Open table in a new tab ND, not detected. F, female; M, male; exp, expansion present; FULL, full mutation; INT, gray-zone/intermediate; NOR, normal alleles; PRE, premutation. F, female; M, male; EXP, expansion present; FULL, full mutation; FULLm, full mutation mosaic; INT, grey-zone/intermediate; NOR, normal; PRE, premutation. A total of 40 residual patient samples previously tested for Fragile X were also analyzed. These samples were de-identified and given a new number (VCU##). The DNA had been extracted from whole blood with an organic extraction method using phenol:chloroform and isopropanol precipitation, and was tested using both Southern blot analysis and a laboratory-developed PCR-based test.20Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar The de-identified residual patient samples included 9 male samples with repeats in the normal range, 7 normal female, 3 gray-zone/intermediate male, 4 premutation male, 7 premutation female, 5 full mutation male, and 5 full mutation female samples (Table 3). Six Coriell DNA samples (NA20232, NA06892, NA04025, NA20234, NA06903, and NA05847) representing gray-zone/intermediate, premutation, and full mutation alleles for both males and females were used to ascertain precision. Each sample was run in duplicate on three separate days (six data points per sample). Overall, 22 normal, 10 gray-zone/intermediate, 27 premutation, and 17 full mutation samples were tested. Artificial mosaic samples were prepared by diluting DNA with expanded alleles with DNA containing normal-sized alleles. Premutation male DNA with 118 repeats (NA06891) was diluted with normal male DNA with 30 repeats (NA07174) to prepare samples with 100%, 90%, 75%, 50%, 25%, 10%, 5%, and 1% of the expanded allele. The same dilutions were made with full mutation male DNA with 645 repeats (NA04025). Premutation female DNA with 30/100 repeats (NA20242) was diluted with normal female DNA with 29/30 repeats (NA20238) to prepare samples with 50%, 25%, 10%, 5%, and 1% of the expanded allele. The same dilutions were made with full mutation female DNA with 29/>200 repeats (NA07537). Samples were PCR-amplified using AmplideX FMR1 PCR reagents (RUO) by preparing a master mix with 11.45 μL of GC-rich AMP buffer, 0.5 μL of FAM-labeled FMR1 forward and reverse primers, 0.5 μL of FMR1 CGG primers, 0.5 μL of diluent, and 0.05 μL of GC-rich polymerase mix from Asuragen (Austin, TX).15Chen L. Hadd A.G. 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 Two microliters of DNA (30 ng/μL) were used for each reaction. Samples were amplified with an initial denaturation step of 95°C for 5 minutes, followed by 10 cycles of 97°C for 35 seconds, 62°C for 35 seconds, and 68°C for 4 minutes, and then 20 cycles of 97°C for 35 seconds, 62°C for 35 seconds, and 68°C for 4 minutes (auto + 20 seconds/cycle). The final extension step was 72°C for 10 minutes. Samples were PCR amplified using Abbott Molecular FMR1 Primer 1 reagents by preparing a master mix with 13 μL of High GC PCR buffer, 0.6 μL of Gender Primers (ASR), 0.8 μL of FMR1 primers (ASR), 1.2 μL of TR PCR Enzyme mix, and 1.4 μL of nuclease-free water from Abbott Molecular (Abbott Park, IL).19Wilson J.A. Pratt V.M. Phansalkar A. Muralidharan K. Highsmith Jr., W.E. Beck J.C. Bridgeman S. Courtney E.M. Epp L. Ferreira-Gonzalez A. Hjelm N.L. Holtegaard L.M. Jama M.A. Jakupciak J.P. Johnson M.A. Labrousse P. Lyon E. Prior T.W. Richards C.S. Richie K.L. Roa B.B. Rohlfs E.M. Sellers T. Sherman S.L. Siegrist K.A. Silverman L.M. Wiszniewska J. Kalman L.V. Fragile Xperts Working Group of the Association for Molecular Pathology Clinical PracticeConsensus characterization of 16 FMR1 reference materials: a consortium study.J Mol Diagn. 2008; 10: 2-12Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Three microliters of DNA (10 ng/μL) were used for each reaction. The reagents were thawed on ice, and the PCR reactions were set up on ice. Samples were amplified with 15 cycles of 98.5°C for 10 seconds, 58°C for 1 minute, and 75°C for 6 minutes, followed by 15 cycles of 98.5°C for 10 seconds (auto + 0.1°C/cycle), 56°C for 1 minute, and 75°C for 6 minutes with a final hold at 4°C. Abbott Molecular FMR1 Primer 1 PCR products were evaluated using agarose gel electrophoresis. One microliter of loading dye was added to 5 μL of PCR product. The samples were run on a 1.5% agarose gel with Tris/borate/EDTA buffer with 150 V for 60 minutes. Gels were stained with ethidium bromide and visualized with UV light. Before capillary electrophoresis analysis, the Abbott Molecular FMR1 Primer 1 PCR products were cleaned by adding 3 μL of Clean Up Enzyme (Abbott Molecular) and 2 μL of PCR product to a 96-well plate. The samples were incubated at 75°C for 10 minutes, followed by a 4°C hold. Samples were PCR amplified using Abbott Molecular FMR1 Primer 2 reagents by preparing a master mix with 13 μL of High GC PCR buffer, 0.8 μL of FMR1 Primers 2 (ASR), 1.2 μL of TR PCR Enzyme mix, and 2 μL of nuclease-free water from Abbott Molecular.17Lyon 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 Three microliters of DNA (10 ng/μL) were used for each reaction. The reagents were thawed on ice, and PCR reactions were set up on ice. Samples were amplified with 50 cycles of 98.5°C for 30 seconds, 53°C for 30 seconds, and 75°C for 1 minute, with a final hold at 4°C. The Asuragen AmplideX FMR1 PCR products were analyzed on an ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA) using POP-7 polymer (Applied Biosystems) with a 50-cm capillary. Samples were prepared for analysis by mixing 2 μL of PCR product with 2 μL of ROX 1000 Size Standard (Asuragen) and 11 μL of Hi-Di Formamide (Applied Biosystems). These samples were heat denatured at 95°C for 2 minutes and cooled to 4°C. The run conditions included injection voltage/time of 2.5 kV/15 seconds and run voltage/time of 15 kV/4200 seconds; all other settings were default for the POP-7/50-cm capillary. The Abbott Molecular FMR1 Primer 1 PCR products were analyzed on an ABI 3130xl Genetic Analyzer using POP-6 polymer (Applied Biosystems) with a 50-cm capillary. Samples were prepared for analysis by adding 3 μL of ROX 1000 Size Standard and 17 μL of Hi-Di Formamide to the 5 μL of cleaned-up PCR product. The samples were heat denatured at 93°C for 30 seconds followed by a 25°C hold. Each sample was injected and analyzed with two different run settings, targeting small and large fragments. For small fragments, the run conditions included injection voltage/time of 10.0 kV/1 second and run voltage/time of 15 kV/6000 seconds. For large fragments, the run conditions included injection voltage/time of 8.0 kV/22 seconds and run voltage/time of 15 kV/6800 seconds; all other settings were default for the POP-6/50-cm capillary. The Abbott Molecular FMR1 Primer 2 PCR products were analyzed on an ABI 3130xl Genetic Analyzer using POP-6 polymer with a 50-cm capillary. Samples were prepared for analysis by mixing 2 μL of PCR product with 2 μL of ROX 1000 Size Standard and 20 μL of Hi-Di Formamide. These samples were heat denatured at 95°C for 2 minutes, followed by a 25°C hold. The run conditions included injection voltage/time of 8.0 kV/8 seconds and run voltage/time of 15 kV/5000 seconds; all other settings were default for the POP-6/50-cm capillary. Capillary electrophoresis data were analyzed on GeneMapper 4.0 software (Applied Biosystems). Asuragen provided panels for the software as well as a macro to analyze the data. We developed panels for displaying and analyzing the Abbott Molecular data. Figure 1 shows a full mutation female sample (NA05847) amplified with the Asuragen AmplideX FMR1 reagents (Figure 1A) and the two Abbott Molecular FMR1 Primer sets (Figure 1, B–D). Samples tested with the Asuragen reagents were analyzed with GeneMapper 4.0 and an Excel-based macro, which calculated repeat sizes and assigned a genotype (Figure 1A and Table 3). The formula used by the macro (2.946x + 230.2) was generated using a process control on the 3130xl instrument at the beginning of the validation.14Filipovic-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 Using the Abbott Molecular reagents, a sample is first analyzed with the FMR1 Primer 2 reaction (Figure 1B), which is CGG primed and gives a ladder motif in the presence of an expanded allele (premutation or full mutation). Repeat lengths can be calculated for normal, intermediate, and some premutation alleles [formula: repeat number = (peak size − 134)/3]. The Abbott Molecular FMR1 Primer 1 reaction can be used to amplify and size normal, intermediate, and some premutation alleles [formula: repeat number = (peak size − 193)/3]. This reaction will also reveal sex (Y chromosome, 170 bp; X chromosome, 203 bp) (Figure 1, C and D). The PCR products are analyzed with both agarose gel electrophoresis and capillary electrophoresis. Unlike capillary electrophoresis, agarose gel electrophoresis will usually detect an expanded allele as a distinct band or a smear on the gel (Figure 1D, gel). Any sample with an expansion should be reflexed to Southern blot analysis. To ascertain accuracy, standards with certifiable repeat numbers were tested with all three reactions. The NIST Fragile X Human DNA Triplet Repeat Standard and DNA from previously NIST-sequenced Coriell samples have been previously used to validate various platforms.19Wilson J.A. Pratt V.M. Phansalkar A. Muralidharan K. Highsmith Jr., W.E. Beck J.C. Bridgeman S. Courtney E.M. Epp L. Ferreira-Gonzalez A. Hjelm N.L. Holtegaard L.M. Jama M.A. Jakupciak J.P. Johnson M.A. Labrousse P. Lyon E. Prior T.W. Richards C.S. Richie K.L. Roa B.B. Rohlfs E.M. Sellers T. Sherman S.L. Siegrist K.A. Silverman L.M. Wiszniewska J. Kalman L.V. Fragile Xperts Working Group of the Association for Molecular Pathology Clinical PracticeConsensus characterization of 16 FMR1 reference materials: a consortium study.J Mol Diagn. 2008; 10: 2-12Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Table 1 shows results for all three reactions. Even the Abbott Molecular Primer 2 reaction, which is designed for detecting expanded alleles, not for sizing repeats, performed equally w" @default.
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