A multiplex PCR strategy to screen for known mutations in families with sudden cardiac death burden
Keywords:sudden cardiac death, personalized medicine, point-of-care testing, PCR
Ventricular tachyarrythmia occurring in ischemic heart disease, dilated/hypertrophic cardiomyopathies or rare monogenic mutations of cardiac ion channels or associated proteins belong to the most frequent causes of sudden cardiac death (SCD). In further decades, next generation sequencing and bioinformatic analysis will become the gold standard of SCD risk stratification. At the moment, Sanger-sequencing is still obligatory in genetic diagnosis. A multiplex polymerase chain reaction (PCR) assay detecting eight SCD mutations in one reaction-tube was developed. To test the general validity of the assay, it was used with 12 patients, who had one or two of the eight mutations (LMNA, p.V256V; SCN5A, p.R1583C; RYR2, p.G1885E; MYH7, V606M; DSG2, p.T335A; KCNJ8, p.S422L; MYBPC, p.E441K; TNNT2, A38V). Thereafter, we tested the multiplex assay in a real diagnostic environment within a high risk family of several past SCD cases. This method allows efficient discrimination of multiple mutations by allele-specific PCR with standard PCR conditions. It relies on obtaining a PCR product specific to the mutation or wildtype—using primers that have the 3´end base complementary to the DNA template site, i.e. a specific primer only permits amplification to take place when its 3´terminal nucleotide matches with its target sequence. The PCR products are further analyzed by length, with Tape Station®(Agilent Technologies, Germany), a high-fidelity capillary chromatography test. The novel multiplex PCR assay strategy could be a good additional test used for SCD risk stratification. Advantages of the test are high velocity and ease of implementation, low price and flexibility of application within cardiomyopathy families for screening purposes.
Martens E, Sinner MF, Siebermair J,Raufhake C, Beckmann BM, Veith S, Düvel D, Steinbeck G, Kääb S, (2012) Incidence of sudden cardiac death in Germany: results from an emergency medical service registry in Lower Saxony, Europace. 16 1752–1758, doi: 10.1093/europace/euu153. PMID: 25061228
Chopra N, Knollmann BC., (2011) Genetics of Sudden Cardiac Death Syndromes, Curr Opin Cardiol. 26 196–203. doi:10.1097/HCO.0b013e3283459893. PMID: 21430528
Douglas P. Zipes; Hein J.J. Wellens, Sudden Cardiac Death, (1998) Circulation. 98 2334–2351. doi: 10.1161/01. PMID:9826323
Rajat Deo, MD, MTR; Christine M. Albert, MD, (2012) Sudden Cardiac Death Epidemiology and Genetics of Sudden Cardiac Death, Circulation. 125 620–637. doi:10.1161/CIRCULATIONAHA.111.023838. PMID:22294707
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF., (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS), Nucleic Acids Res. 17 2503–16. doi:10.1093/nar/17.7.2503. PMID:2785681
Germer S, Higuchi R., (1999) Single-tube genotyping without oligonucleotide probes., Genome Res. 9 72–78. doi:10.1101/gr.9.1.72. PMID:9927486
Myakishev MV, Khiripin Y, Hu S, Hamer HD, (2001)High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers, Genome Res 163–169. doi. 10.1101/gr.157901. PMID:11156625
Mohd Nazif Darawi, Chin Ai-Vyrn, Kalavathy Ramasamy, Philip Poi Jun Hua, Tan Maw Pin, Shahrul Bahyah Kamaruzzaman and Abu Bakar Abdul Majeed, (2013) Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms. 2013 14:27., BMC Med. Genet. 14. doi:10.1186/1471-2350-14-27. PMID:23419238
Hezard N, Cornillet P, Droulle C, Gillot L, Potron G, Nguyen P., (1997) Factor V Leiden: detection in whole blood by ASA PCR using an additional mismatch in antepenultimate position., Thromb Res. 88 59–66. doi:http://dx.doi.org/10.1016/S0049-3848(97)00191-6. PMID:9336874
Mirasena S, Shimbhu D, Sanguansermsri M, Sanguansermsri T, Detection of beta-thalassemia mutations using a multiplex amplification refractory mutation system assay., Hemoglobin. (n.d.) 403–409. doi:10.1080/03630260701798391. PMID:1865489
Chen CH, (2016) Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood, PLoS One. 11. doi:http://dx.doi.org/10.1371/journal.pone.0153593. PMID:27078154
Sapkota BR, Ranjit C, Neupane KD, Macdonald M, (2008) Development and evaluation of a novel multipleprimer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of mycobacterium leprae, J Med Microbiol. 57 179–184. doi:10.1099/jmm.0.47534-0. PMID:18201983
Teh LK, Lee WL, Amir J, Salleh MZ, Ismail R, (2007)Single step PCR for detection of allelic variation of MDR1 gene (P-glycoprotein) among three ethnic groups in Malaysia, J J Clin Pharm Ther. 32 313–319. doi:10.1111/j.1365-2710.2007.00822.x. PMID:17489883
Yaku H, Yukimasa T, Nakano S, Sugimoto N, Oka H., Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem., Electrophoresis. 29 (2008) 4130–4140. doi:10.1002/elps.200800097. PMID:18991264
Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ., (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies., Nucleic Acids Res. 18 999–1005. PMID: 2179874
Little S., (2001) Amplification-refractory mutation system (ARMS) analysis of point mutations., Curr Protoc Hum Genet. 9.8.1-9.8.12. doi:10.1002/0471142905.hg0908s07. PMID:18428319
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