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• W2000327615 abstract "Mutations in the GJB2 gene are the most common cause of congenital hearing loss in many populations. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry–based minisequencing assay, TheraTyper-GJB2, for the detection of c.35delG, c.167delT, and c.235delC mutations in the GJB2 gene. This assay was evaluated for analytic performance, including detection limit, interference, cross-reactivity, and precision, using GJB2 reference standards prepared by site-directed mutagenesis of a molecular clone. The detection limit was as low as 0.040 ng of human genomic DNA per PCR. No cross-reactivity with bacteria and viruses and no negative effects of increased levels of various potential interfering substances was observed. A precision test involving repetitive analysis of 2400 replicates showed 99.9% agreement (2397 of 2,400) with 99.8% (95% CI, 99.7%–99.8%) sensitivity and 100.0% (95% CI, 99.3%–100.0%) specificity. TheraTyper-GJB2 and direct sequencing assays showed 100% concordance for detecting mutations in 1,113 clinical specimens. Overall, TheraTyper-GJB2 showed comparable performance for detecting GJB2 mutations in reference and clinical samples with that of direct sequencing, and easier interpretation of results for analysis of a large quantity of samples. Therefore, the TheraTyper-GJB2 assay will be practically useful for the diagnosis of GJB2 mutations associated with congenital hearing loss with faster, cheaper, more reliable, and high-throughput capability. Mutations in the GJB2 gene are the most common cause of congenital hearing loss in many populations. This study describes the development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry–based minisequencing assay, TheraTyper-GJB2, for the detection of c.35delG, c.167delT, and c.235delC mutations in the GJB2 gene. This assay was evaluated for analytic performance, including detection limit, interference, cross-reactivity, and precision, using GJB2 reference standards prepared by site-directed mutagenesis of a molecular clone. The detection limit was as low as 0.040 ng of human genomic DNA per PCR. No cross-reactivity with bacteria and viruses and no negative effects of increased levels of various potential interfering substances was observed. A precision test involving repetitive analysis of 2400 replicates showed 99.9% agreement (2397 of 2,400) with 99.8% (95% CI, 99.7%–99.8%) sensitivity and 100.0% (95% CI, 99.3%–100.0%) specificity. TheraTyper-GJB2 and direct sequencing assays showed 100% concordance for detecting mutations in 1,113 clinical specimens. Overall, TheraTyper-GJB2 showed comparable performance for detecting GJB2 mutations in reference and clinical samples with that of direct sequencing, and easier interpretation of results for analysis of a large quantity of samples. Therefore, the TheraTyper-GJB2 assay will be practically useful for the diagnosis of GJB2 mutations associated with congenital hearing loss with faster, cheaper, more reliable, and high-throughput capability. Congenital hearing loss (HL) is one of the most common sensory impairments in humans, occurring in 1 per 1,000 infants.1Marazita M.L. Ploughman L.M. Rawlings B. Remington E. Arnos K.S. Nance W.E. Genetic epidemiological studies of early-onset deafness in the U.S. school-age population.Am J Med Genet. 1993; 46: 486-491Crossref PubMed Scopus (393) Google Scholar, 2Rennels M. Pickering L.K. Sensorineural hearing loss in children.Lancet. 2005; 365: 2085-2086Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar The onset of HL in early childhood can have serious effects on language acquisition.3Petit C. Levilliers J. Hardelin J.P. Molecular genetics of hearing loss.Annu Rev Genet. 2001; 35: 589-646Crossref PubMed Scopus (255) Google Scholar Although more than 50% of cases of HL are hereditary, at the present time the best provision for congenital HL is early diagnosis and continuous management. Because of the high prevalence and clinical impact of this condition, the NIH and the CDC have advocated early detection through genetic diagnosis as the most effective support for the prevention and treatment of hereditary HL. Approximately 70% of congenital cases associated with genetic factors are classified as nonsyndromic. The remaining 30% of cases have 1 of more than 400 forms of syndromic deafness that can be diagnosed by associated clinical findings. The inheritance pattern of nonsyndromic HL is autosomal-recessive transmission (80%), autosomal-dominant transmission (15% to 20%), X-linked (2% to 3%), or mitochondrial (1%).4Van Camp G. Willems P.J. Smith R.J. Nonsyndromic hearing impairment: unparalleled heterogeneity.Am J Hum Genet. 1997; 60: 758-764PubMed Google Scholar Congenital HL is a highly heterogeneous disorder, in which 50 dominant loci corresponding to 24 genes and 79 recessive loci corresponding to 40 genes can cause the same phenotype, especially in nonsyndromic cases.5Lenz D.R. Avraham K.B. Hereditary hearing loss: from human mutation to mechanism.Hear Res. 2011; 281: 3-10Crossref PubMed Scopus (41) Google Scholar Despite this large genetic heterogeneity, mutations in the GJB2 (OMIM 121011) gene are responsible for approximately 50% of cases of severe to profound recessive nonsyndromic HL in many populations.6Kenneson A. Van Naarden Braun K. Boyle C. GJB2 (connexin 26) variants and nonsyndromic sensorineural hearing loss: a HuGE review.Genet Med. 2002; 4: 258-274Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar GJB2 encodes the gap junction protein connexin 26 protein, one of the most important members of the connexin family. Connexin 26 protein is expressed in epithelial and connective tissue cells in the inner ear.7Kammen-Jolly K. Ichiki H. Scholtz A.W. Gsenger M. Kreczy A. Schrott-Fischer A. Connexin 26 in human fetal development of the inner ear.Hear Res. 2001; 160: 15-21Crossref PubMed Scopus (26) Google Scholar This gap junction protein serves as the structural basis for recycling potassium ions back to the endolymph of the cochlear duct after sensory hair cell stimulation.8Wangemann P. K+ cycling and the endocochlear potential.Hear Res. 2002; 165: 1-9Crossref PubMed Scopus (336) Google Scholar The loss of connexin 26 protein would be expected to disrupt potassium ion flow and lead to hearing loss.9Holt J.R. Corey D.P. Ion channel defects in hereditary hearing loss.Neuron. 1999; 22: 217-219Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The most frequent pathogenic mutations, c.35delG and c.167delT, together account for 70% and 40% of GJB2-related HL in Caucasians10Gasparini P. Rabionet R. Barbujani G. Melchionda S. Petersen M. Brondum-Nielsen K. Metspalu A. Oitmaa E. Pisano M. Fortina P. Zelante L. Estivill X. High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG.Eur J Hum Genet. 2000; 8: 19-23Crossref PubMed Scopus (347) Google Scholar and Ashkenazi Jews,11Lerer I. Sagi M. Malamud E. Levi H. Raas-Rothschild A. Abeliovich D. Contribution of connexin 26 mutations to nonsyndromic deafness in Ashkenazi patients and the variable phenotypic effect of the mutation 167delT.Am J Med Genet. 2000; 95: 53-56Crossref PubMed Scopus (74) Google Scholar, 12Morell R.J. Kim H.J. Hood L.J. Goforth L. Friderici K. Fisher R. Van Camp G. Berlin C.I. Oddoux C. Ostrer H. Keats B. Friedman T.B. Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness.N Engl J Med. 1998; 339: 1500-1505Crossref PubMed Scopus (472) Google Scholar, 13Dong J. Katz D.R. Eng C.M. Kornreich R. Desnick R.J. Nonradioactive detection of the common Connexin 26 167delT and 35delG mutations and frequencies among Ashkenazi Jews.Mol Genet Metab. 2001; 73: 160-163Abstract Full Text PDF PubMed Scopus (18) Google Scholar respectively. In contrast, East Asian patients with hereditary HL rarely display these mutations; instead, another single base pair deletion, c.235delC, is present in 43% of Korean patients.14Lee K.Y. Choi S.Y. Bae J.W. Kim S. Chung K.W. Drayna D. Kim U.K. Lee S.H. Molecular analysis of the GJB2, GJB6 and SLC26A4 genes in Korean deafness patients.Int J Pediatr Otorhinolaryngol. 2008; 72: 1301-1309Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Shin J.W. Lee S.C. Lee H.K. Park H.J. Genetic screening of GJB2 and SLC26A4 in Korean cochlear implantees: experience of Soree Ear Clinic.Clin Exp Otorhinolaryngol. 2012; 5: S10-S13Crossref PubMed Scopus (26) Google Scholar, 16Yan D. Park H.J. Ouyang X.M. Pandya A. Doi K. Erdenetungalag R. Du L.L. Matsushiro N. Nance W.E. Griffith A.J. Liu X.Z. Evidence of a founder effect for the 235delC mutation of GJB2 (connexin 26) in east Asians.Hum Genet. 2003; 114: 44-50Crossref PubMed Scopus (96) Google Scholar The location of c.35delG, c.167delT, and c.235delC mutations in the GJB2 were shown and minor allele frequencies were compared among different ethnic groups (Figure 1A). These three mutations have been reported to cause abnormal protein function. One of the most common mutations of the GJB2 gene is c.35delG, the deletion of one guanine residue in a stretch of six between nucleotide positions 30 and 35 that introduces a stop codon at amino acid position 13 in the N-terminus of the protein.17Scott D.A. Kraft M.L. Carmi R. Ramesh A. Elbedour K. Yairi Y. Srisailapathy C.R. Rosengren S.S. Markham A.F. Mueller R.F. Lench N.J. Van Camp G. Smith R.J. Sheffield V.C. Identification of mutations in the connexin 26 gene that cause autosomal recessive nonsyndromic hearing loss.Hum Mutat. 1998; 11: 387-394Crossref PubMed Scopus (210) Google Scholar The c.167delT mutation introduces a premature stop codon in the first extracellular loop at amino acid 56.12Morell R.J. Kim H.J. Hood L.J. Goforth L. Friderici K. Fisher R. Van Camp G. Berlin C.I. Oddoux C. Ostrer H. Keats B. Friedman T.B. Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness.N Engl J Med. 1998; 339: 1500-1505Crossref PubMed Scopus (472) Google Scholar, 18Zelante L. Gasparini P. Estivill X. Melchionda S. D'Agruma L. Govea N. Mila M. Monica M.D. Lutfi J. Shohat M. Mansfield E. Delgrosso K. Rappaport E. Surrey S. Fortina P. Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans.Hum Mol Genet. 1997; 6: 1605-1609Crossref PubMed Scopus (548) Google Scholar The c.235delC mutation of GJB2 causes a loss of targeting activity to the cell membrane and severe deterioration of gap junction activity.19Choung Y.H. Moon S.K. Park H.J. Functional study of GJB2 in hereditary hearing loss.Laryngoscope. 2002; 112: 1667-1671Crossref PubMed Scopus (35) Google Scholar Schematic representations of normal connexin 26 protein and three abnormal connexin 26 proteins produced by three mutations are shown in Figure 1, B and C. Accordingly, these mutations were selected for screening during genetic testing of HL. Currently, genotyping of single-nucleotide polymorphisms or frameshift mutations in GJB2 is performed by allele-specific PCR,20Han B. Zong L. Li Q. Zhang Z. Wang D. Lan L. Zhang J. Zhao Y. Wang Q. Newborn genetic screening for high risk deafness-associated mutations with a new Tetra-primer ARMS PCR kit.Int J Pediatr Otorhinolaryngol. 2013; 77: 1440-1445Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar real-time PCR,21Van Eyken E. Van Camp G. Hendrickx J.J. Demeester K. Vandevelde A. Azza J.B. Van de Heyning P. Van Laer L. A new, easy, and rapid high-throughput detection method for the common GJB2 (CX26), 35delG mutation.Genet Test. 2007; 11: 231-234Crossref PubMed Scopus (3) Google Scholar microarray,22Choi S.Y. Lee K.Y. Kim Y.E. Bae J.W. Oh S.K. Kim S.Y. Hwang S.J. Kim U.K. Lee S.H. Application of allele-specific primer extension-based microarray for simultaneous multi-gene mutation screening in patients with non-syndromic hearing loss.Int J Mol Med. 2010; 25: 315-320PubMed Google Scholar, 23Rodriguez-Paris J. Pique L. Colen T. Roberson J. Gardner P. Schrijver I. Genotyping with a 198 mutation arrayed primer extension array for hereditary hearing loss: assessment of its diagnostic value for medical practice.PLoS One. 2010; 5: e11804Crossref PubMed Scopus (20) Google Scholar or other methods. These screening assays for mutations associated with deafness are useful, but cannot be applied easily to various populations and require optimization for multiplex reactions. TheraTyper-GJB2 targets the GJB2 mutations c.35delG, c.167delT, and c.235delC that are associated with HL in major populations—Caucasians, Ashkenazi Jews, and Asians, respectively. Moreover, previous studies exploiting the restriction fragment mass polymorphism strategy for a multiplex assay for viral mutations did not necessitate repetitive PCR or multiple mass spectrometric analyses.24Han K.H. Hong S.P. Choi S.H. Shin S.K. Cho S.W. Ahn S.H. Hahn J.S. Kim S.O. Comparison of multiplex restriction fragment mass polymorphism and sequencing analyses for detecting entecavir resistance in chronic hepatitis B.Antivir Ther. 2011; 16: 77-87Crossref PubMed Scopus (35) Google Scholar, 25Shim J.H. Suh D.J. Kim K.M. Lim Y.S. Lee H.C. Chung Y.H. Lee Y.S. Efficacy of entecavir in patients with chronic hepatitis B resistant to both lamivudine and adefovir or to lamivudine alone.Hepatology. 2009; 50: 1064-1071Crossref PubMed Scopus (55) Google Scholar On the basis of the prevalence of c.35delG, c.167delT, and c.235delC mutations, we developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry–based minisequencing assay, termed TheraTyper-GJB2, for the detection of these mutations in the GJB2 gene. The TheraTyper-GJB2 assay recently received approval from the Korea Ministry of Food and Drug Safety for in vitro diagnostic use [http://emed.mfds.go.kr/kfda2?cmd=CEBAA05P1&EntpSeq=2013000015&ItemSeq=2013000072&REMOTE_IP=116.123.164.162 (Korean), last accessed April 14, 2014]. To evaluate the performance characteristics of the TheraTyper-GJB2 assay, a series of studies were performed to assess its detection limit, interference, cross-reactivity, and precision. The ability to detect specific mutations also was evaluated using human genomic DNA samples and DNA derived from molecular clones carrying the HL-associated mutations c.35delG, c.167delT, and c.235delC, and the assay performance was compared with that of direct sequencing. This study was performed on 1,113 dried blood spot specimens from unrelated Korean neonates that were collected by the Green Cross Reference Laboratory (Yongin, Korea) between April 2005 and January 2006. The experimental protocol followed the ethical guidelines of the 1975 Declaration of Helsinki and received institutional review board approval. The parents of all neonates provided signed informed consent. Dried blood spot specimens were obtained within 1 hour after birth. The heel of neonates was cleaned with alcohol wipes and pricked using a sterile lancet. The resulting blood drops were applied to a QIAcard FTA One Spot (Qiagen, Chatsworth, CA), soaking through a printed circle that holds 125 μL or more of blood. Samples were labeled, allowed to dry, placed in an envelope, and then sealed in a plastic bag for transport to the laboratory. The dried blood spot specimens were punched out from the QIAcard FTA One Spot using a Harris UNI-CORE Punch (Ted Pella, Inc., Redding, CA). DNA specimens were extracted using the QIAamp Blood Mini assay (Qiagen) according to the manufacturer's instructions and dissolved in 30 μL of distilled water. DNA concentration was quantified using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT). PCR was performed in a total reaction volume of 5 μL using 20 ng of the genomic DNA. Plasmids carrying wild-type and mutated GJB2 sequences were prepared as standards. The c.35delG, c.167delT, and c.235delC mutations were introduced into the GJB2 gene using the Altered Sites II in vitro mutagenesis system (Promega, Madison, WI) and cloned into the PCR-Script Amp SK(+) cloning vector (Stratagene, La Jolla, CA). Prepared reference standards were used to assess the analytic performance of the TheraTyper-GJB2 assay. Moreover, we additionally tested reference materials received from the College of American Pathologists through the Green Cross Reference Laboratory, which is heterozygous for a c.35delG deletion mutation of the GJB2 gene. To examine the performance effects of different potentially interfering substances that might be present during the TheraTyper-GJB2 assay, test DNA samples in phosphate-buffered saline (pH 8.0) were spiked with anticoagulants (warfarin, heparin, EDTA, or sodium citrate), antiplatelet agents (aspirin, ticlopidine), anticoagulant preservatives (acid citrate dextrose, citrate phosphate dextrose, or citrate phosphate dextrose with adenine), or a DNA extraction agent (ethanol; test concentrations of 5% and 10%). To examine the performance effects of different potential cross-reactivity that might be present during the TheraTyper-GJB2 assay by contaminated specimens, bacteria, or virus from infected people, the DNA of Gardnerella vaginalis, Trichomonas vaginalis, herpes simplex virus (HSV) 1, HSV-2, Candida albicans, Haemophilus ducreyi, Mycoplasma genitalium, Mycoplasma hominis, Neisseria gonorrhoeae, Chlamydia trachomatis, Bacteroides fragilis, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Lactobacillus acidophilus, Nesseria meningitides, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae was obtained from ATCC (Manassas, VA). The DNA of hepatitis C virus was obtained from the National Institute for Biological Standards and Control (London, UK). The DNA of hepatitis B virus and HIV was obtained from SeraCare (Milford, MA). The DNA of Ureaplasma urealyticum and Treponema pallidum was synthesized by Integrated DNA Technologies (Coralville, IA). The concentration of microbial and viral DNA was determined by a branched DNA assay (Versant 3.0; Bayer Healthcare LLC Diagnostic Division, Tarrytown, NY), which had a detection limit of 2,000 copies/mL. To test the effect of microbial and viral sequences on the performance of the TheraTyper-GJB2 assay, the GJB2 negative control was spiked with G. vaginalis, T. vaginalis, C. albicans, H. ducreyi, M. genitalium, M. hominis, N. gonorrhoeae, C. trachomatis, B. fragilis, E. cloacae, E. faecalis, E. coli, L. acidophilus, N. meningitides, P. mirabilis, S. aureus, S. epidermidis, S. pneumoniae, U. urealyticum, T. pallidum, hepatitis B virus, hepatitis C virus, HIV, HSV-1, or HSV-2 DNA (all at 50,000 copies/mL). The precision test was conducted by consecutive analysis of 24 replicates with two GJB2 reference standards (wild type and mutant) at two concentrations (30 and 1000 copies/PCR) over 20 days for the determination of intraday and interday reproducibility. The 24 replicates comprised two independent runs with two replicates performed more than 4 hours apart by three operators in two laboratories (GeneMatrix, Inc., Yongin Laboratory, Yongin, Korea, and GeneMatrix, Inc., Seongnam Laboratory, Seongnam, Korea). The strategy of the TheraTyper-GJB2 assay is shown in Figure 2. The first step requires PCR amplification with forward and reverse primers that introduce the FokI restriction site, GGATG. Linker bases were added to restriction core sequences in primers specific for c.35delG, c.167delT, or c.235delC to introduce a size difference in the restriction fragments (Table 1). Genotypic analysis of GJB2 mutations was performed using the TheraTyper-GJB2 assay according to the manufacturer's recommendations (GeneMatrix, Inc., Seongnam, Korea). Briefly, 5 μL of genomic DNA was used for PCR. For genotyping, PCR was performed in an 18 μL reaction mixture containing 20 mmol/L Tris–HCl (pH 8.4), 50 mmol/L KCl, 0.2 mmol/L of each dNTP, 10 pmol of each primer, and 0.4 units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). The amplification conditions included initial denaturation at 94°C for 10 minutes, 40 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds, followed by extension at 72°C for 5 minutes. All primers were modified to insert a new FokI site and eliminate any naturally occurring FokI sites in the PCR products (Table 2). Restriction enzyme digestion of PCR products was performed by mixing the PCR reaction mixture with 10 μL of buffer containing 50 mmol/L potassium acetate, 20 mmol/L Tris–acetate, 10 mmol/L magnesium acetate, 1 mmol/L dithiothreitol, and 1 unit of FokI. The reaction mixture was incubated at 37°C for 2 hours. The resulting digest was desalted by vacuum filtration through a 96-well sample preparation plate containing 5 mg of polymeric solvent (Waters, Milford, MA) per well. The desalted reaction mixtures were resuspended with matrix solution containing 50 mg/mL 3-hydroxy picolinic acid, 0.05 mol/L ammonium citrate, and 30% acetonitrile, and were dotted 2 μL volumes on an AnchorChip plate (Bruker Daltonics, Billerica, MA). Mass spectra were acquired on a linear matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Biflex IV; Bruker Daltonics) workstation in a positive-ion, delayed-extraction mode. Genotypic analysis by the TheraTyper-GJB2 assay was compared with direct sequencing analysis performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).Table 1Sequence of Primers in This StudyNameSequencePolarity35delG forward5′-GTCCGTCCCTTAAGGTGGTTGGATGGCAGACGATCCTGGGG-3′Sense35delG reverse5′-AACATCCCGGCCCAGCAGGTGGATGTGGAGTGTTTGTTCAC-3′Antisense235delC forward5′-CAATGCCGTGGAACCTGAAAGGATGATCCGGCTATGGGC-3′Sense235delC reverse5′-GACGACGCCTCTCCTTTCCTGGATGACGAAGATCAGCTGC-3′Antisense167delT forward5′-GAGGTTCTGGGCGCGACTTTGGATGTTGTCTGCAACACC-3′Sense167delT reverse5′-TTAAAGAGCGCTCCAAAGCCGGATGTTCTTGCAGCCTGGCTG-3′AntisenseA five-nucleotide sequence (GGATG) embedded in the primer to introduce a FokI site in amplicon is indicated in bold. Open table in a new tab Table 2Expected Masses of Oligonucleotides Resulting from Restriction Enzyme Digestion of PCR ProductsNucleotide variantVariationCalculated fragments∗Letters in bold indicate target site of deletion (-).Calculated molecular mass (Da)UpperLowerUpperLowerc.35delGWT(G)5′-CCTGGGGGGTGTG-3′5′-TGTTCACACCCCC-3′4142.63910.6Deletion5′-CCTGGGGG-TGTG-3′5′-TGTTCACA-CCCC-3′3813.43621.4c.167delTWT(T)5′-ACACCCTGCAGC-3′5′-CCTGGCTGCAGG-3′3655.43742.4Deletion5′-ACACCC-GCAGC-3′5′-CCTGGCTGC-GG-3′3351.23429.2c.235delCWT(C)5′-TGGGCCCTGC-3′5′-AGCTGCAGGG-3′3100.03173.0Deletion5′-TGGGCC-TGC-3′5′-AGCTGCA-GG-3′2810.82843.8WT, wild type.∗ Letters in bold indicate target site of deletion (-). Open table in a new tab A five-nucleotide sequence (GGATG) embedded in the primer to introduce a FokI site in amplicon is indicated in bold. WT, wild type. CIs for two categoric variables were computed using the Cochran–Mantel–Haenszel χ2 test, and a limit of detection test was performed by probit analysis to compute the analytic sensitivity of the TheraTyper-GJB2 assay using the SPSS statistical package (version 13; SPSS, Inc., Chicago, IL). The TheraTyper-GJB2 assay is based on mass spectrometric analysis of small DNA fragments including mutation sites.26Hong S.P. Ji S.I. Rhee H. Shin S.K. Hwang S.Y. Lee S.H. Lee S.D. Oh H.B. Yoo W. Kim S.O. A simple and accurate SNP scoring strategy based on typeIIS restriction endonuclease cleavage and matrix-assisted laser desorption/ionization mass spectrometry.BMC Genomics. 2008; 9: 276Crossref PubMed Scopus (5) Google Scholar, 27Hong S.P. Shin S.K. Lee E.H. Kim E.O. Ji S.I. Chung H.J. Park S.N. Yoo W. Folk W.R. Kim S.O. High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry.Nat Protoc. 2008; 3: 1476-1484Crossref PubMed Scopus (37) Google Scholar, 28Lee H.P. Cho W. Bae J.M. Shin J.Y. Shin S.K. Hwang S.Y. Min K.T. Kim S.N. Lee S.J. Kim S.O. Yoo W.D. Hong S.P. Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.J Clin Virol. 2013; 57: 130-135Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 29Lee J.H. Hachiya A. Shin S.K. Lee J. Gatanaga H. Oka S. Kirby K.A. Ong Y.T. Sarafianos S.G. Folk W.R. Yoo W. Hong S.P. Kim S.O. Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients.Clin Microbiol Infect. 2013; 19: E263-E270Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar The fragments released by enzymatic digestion consist of oligomers of different sizes ranging from 9 to 13 nt for c.35delG, c.167delT, and c.235delC mutations, allowing facile identification of sequence variations in multiple codons in a single mass spectrum by mass spectrometric analysis (Table 2). Results of the TheraTyper-GJB2 assay showed distinct peaks relevant to each allele, with mass values for each diagnostic fragment exactly as predicted (Table 2). Representative test results of the TheraTyper-GJB2 assay and direct sequencing are shown in Figure 3. The multiplex TheraTyper-GJB2 assay showed six distinct mass peaks for the three mutations, the genotype of each fragment in the multiplex assay was exactly the same as that yielded by direct sequencing of wild-type (Figure 3A) and mutant (Figure 3B) reference standards. GJB2 reference standards prepared from molecular clones of wild-type and mutant GJB2 were diluted serially in phosphate-buffered saline (pH 8.0) to assess the lower limit of detection of the TheraTyper-GJB2 assay. Probit analysis of wild-type and mutant GJB2 reference standards showed detection limits of 0.017 (95% CI, 0.009–0.064) and 0.040 (95% CI, 0.019–0.174) ng DNA per PCR, respectively (Table 3). The limit of detection tested by GJB2 assay from the College of American Pathologists was similar to the results of GJB2 reference standards prepared by site-directed mutagenesis. The detection limits were confirmed using a dilution series of human genomic DNA, and found to be in agreement with the earlier-described calculated values (data not shown).Table 3Limit of Detections of the TheraTyper-GJB2 KitGenotypeCopies/PCRCalculated DNA (ng/PCR)Detected/testedObserved positive hit ratesLOD, copies/PCR(95% CI)LOD, ng/PCR (95% CI)Wild type711023.7560/60100%11.81 (5.63–51.56)0.040 (0.019–0.174)7112.3860/60100%71.100.2460/60100%7.110.02454/6090%0.710.002441/6068%Mutant type (35delG+167delT+235delC)529017.6660/60100%4.92 (2.62–18.76)0.017 (0.009–0.064)5291.7760/60100%52.900.1860/60100%5.290.01857/6095%0.530.001839/6065%Defined dilutions of GJB2 reference standards of the wild and mutant types were diluted serially in phosphate-buffered saline (pH 8.0), and analytic sensitivities were calculated by probit analysis at a 95% detection level. The calculated sensitivity was expressed as copies/mL.LOD, limit of detection. Open table in a new tab Defined dilutions of GJB2 reference standards of the wild and mutant types were diluted serially in phosphate-buffered saline (pH 8.0), and analytic sensitivities were calculated by probit analysis at a 95% detection level. The calculated sensitivity was expressed as copies/mL. LOD, limit of detection. To determine the effect of different interfering substances on assay performance, GJB2 reference standards in phosphate-buffered saline (pH 8.0) at a titer of 30 copies/PCR were spiked with warfarin, heparin, EDTA, sodium citrate, aspirin, ticlopidine, acid citrate dextrose, citrate phosphate dextrose, citrate phosphate dextrose with adenine, or ethanol as described in Materials and Methods. For each sample, TheraTyper-GJB2 assay data from samples spiked with the various interfering substances were compared with those from nonspiked samples. Each chemical was tested in triplicate. None of the samples spiked with a potentially interfering substance had a false-positive test result. None of the interfering substances had a negative effect, with the exception of heparin and 20% ethanol (Table 4).Table 4Effects of Interfering Substances on the TheraTyper-GJB2 AssayInterfering substancesWild typeMutant type∗The mutant types included 35delG, 167delT, and 235delC.ConcentrationConcordance (%)†The concordance was tested three times.ConcentrationConcordance (%)†The concordance was tested three times.Anticoagulants Warfarin5% or 10%1005% or 10%100 Heparin5% or 10%05% or 10%0 EDTA5% or 10%1005% or 10%100 Sodium citrate5% or 10%1005% or 10%100Antiplatelet agents Aspirin5% or 10%1005% or 10%100 Ticlopidine5% or 10%1005% or 10%100Preservatives Acid citrate dextrose5% or 10%1005% or 10%100 Citrate phosphate dextrose5% or 10%1005% or 10%100 Citrate phosphate dextrose with adenine5% or 10%1005% or 10%100Extraction reagents Ethanol5% or 10%1005% or 10%100 Ethanol>20%0>20%0∗ The mutant types included 35delG, 167delT, and 235delC.† The concordance was tested three times. Open table in a new tab To determine the effect of clinically relevant viruses or microbes on assay performance, the GJB2 negative control was spiked with DNA from G. vaginalis, T. vaginalis, C. albicans, H. ducreyi, M. genitalium, M. hominis, N. gonorrhoeae, C. trachomatis, B. fragilis, E. cloacae, E. faecalis, E. coli, L. acidophilus, N. meningitides, P. mirabilis, S. aureus, S. epidermidis, S. pneumoniae, U. urealyticum, T. pallidum, hepatitis B virus, hepatitis C virus, HIV, HSV-1, or HSV-2 as described in Materials and Methods. Each pathogen was tested in triplicate. None o" @default.
• W2000327615 created "2016-06-24" @default.
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