Forensic Science International: Genetics Supplement Series 3 (2011) e389–e390
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Development of a Long QT-Syndrome mutation detection method M. Nastainczyk a,*, R. Lessig b, D. Husser-Bollmann c, J. Dreßler a, J. Edelmann a a
Institute of Legal Medicine, University of Leipzig, Germany Institute of Legal Medicine, Martin-Luther-University of Halle-Wittenberg, Germany c Heart Centre Leipzig, Department of Internal Medicine/Cardiology, University of Leipzig, Germany b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 August 2011 Accepted 15 September 2011
Sudden cardiac death (SCD) especially in younger adults with no previous symptoms is a challenging problem in forensic diagnostics, occurring with an incidence of about 80,000 per year in Germany. Long QT-Syndrome (LQTS) and other cardiac disorders associated with abnormality of cardiac rhythm triggered by mutations of cardiac ion channels have to consider if no cause of death is detectable during autopsy. A molecular genetic screening can help to ensure. Meanwhile twelve genes are known in which more than 700 different mutations are associated with LQTS. The three major LQTS-susceptibility genes are KCNQ1, KCNH2 and SCN5A. They are responsible for 75% of all congenital LQTS cases. We developed a rapid, sensitive and reasonable method for the simultaneously screening of the most common LQTS mutations, focused on these three genes. Using the SNaPshot1 minisequencing primer extension assay a total of 91 Single-Nucleotide Polymorphisms (SNPs) were examined in six multiplex assays. The data suggest that this technique is applicable, solid, flexible and a good alternative to complete sequencing. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sudden cardiac death Long QT-Syndrome Single-nucleotide polymorphism KCNQ1 KCNH2 SCN5A
1. Introduction Long QT-Syndrome (LQTS) is a cardiac disorders associated with abnormality of cardiac rhythm triggered by mutations of cardiac ion channels. It can be acquired (drug-induced) but primarily it is a congenital disorder caused by Single-Nucleotide Polymorphisms (SNPs) in cardiac ion channel genes. It is characterised by delayed ventricular repolarisation leading to
a prolonged QT interval and can affect a ventricular tachyarrhytmia, usually torsade de point. The arrhythmia results in sudden cardiac death (SCD) especially in younger, apparently healthy individuals. In most cases the clinically disorder of congenital Long QT-Syndrome becomes manifest during childhood. To date more than 700 mutations in 12 different genes have been associated with LQTS. The three major LQTS-susceptibility
Table 1 Position of SNPs within the KCNQ1, KCNH2 and SCN5A gene. Position and nucleotide of the wildtype alleles and variants KCNQ1 assay I
KCNQ1 assay II
KCNQ1 assay III
KCNH2 assay I
KCNH2 assay II
KCNH2 assay III
SCN5A assay I
A551C T560C G565A C939G G940A/T/C A944C/G C959A/T/C C1030T G1032A/C G1515A C1552T/G G1556A
C520T G521A/C C568T G805A C691T G692A G724A G914A/C C772A G941A A964G C965T C1027T C1028T/G
G898A C784G T839A/C G806A C830G/T C905T/A G535A G569A G947A/T G973A C1031T/A C1022A/T A773G G781A/C/T
G1285C G1468A A1478T G1575T G1583C C1609T C1863G C2204T G2362A G2398T G2626T G2660A A2690C
G1235A C1283A/T C1600T G1601T T1655C G1681C/A C1682T G1714T/A/C G1810A G1825A/C G1882A/G G1883T
G1715A C1838T C1841T C1868T C1922T G1745T C1979T G2044T C2086T C2117G C2162T C2173T C2254T G2255A
T635C C647T G674A C1715A/T C1735A G1844A C4826G G4868A/T G4931A G5349A G5350A G5455A
* Corresponding author. E-mail address:
[email protected] (M. Nastainczyk). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.056
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M. Nastainczyk et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e389–e390
genes are KCNQ1, KCNH2 and SCN5A. They are responsible for 75% of all congenital LQTS cases. 2. Material and methods Genomic DNA was isolated from blood by standard methods. More than 80 samples, including samples from cases with autopsy negative results (with possible LQTS), samples from unrelated persons who have given their consent for research purposes and positive as well as negative controls were examined. The control samples have been previously analyzed by direct sequencing, whereby sequencing was stopped when the first mutation was detected. Self designed and previously published exon primers and extension primers were used to amplify the selected candidate genes fragments [1–3]. Using the SNaPshot1 minisequencing primer extension assay a total of 91 Single-Nucleotide Polymorphisms (SNPs) were examined in six multiplex assays (shown in Table 1). 3. Results and discussion After analysis of more than 80 samples with the 6 multiplex reactions, we were able to confirm 2 positive controls with the heterozygote mutation C691T in KCNQ1 gene and the heterozygote mutation C1922T in KCNH2 gene. Furthermore we found a few of samples with a homozygote or heterozygote genetic variant of A2690C in KCNH2 gene, which had previously been reported with a frequency of about 0.24 in a healthy German population [4] and associated with a lower risk of QTc interval prolongation [5]. The remaining samples from unrelated individuals and cases with
autopsy negative results were unremarkable and neither mutation could be detected. We developed a rapid, sensitive and reasonable method for the simultaneously screening of the most common LQTS mutations, focused on the KCNQ1, KCNH2 and SCN5A gene. Using the SNaPshot1 minisequencing primer extension assay a total of 91 SNPs were examined in six multiplex assays with which it is possible to detect 116 mutations. The data suggest that this technique is applicable, solid, flexible and a good alternative to complete sequencing. Further studies with higher numbers of samples are realised in the future. 4. Conflict of interest None. References [1] T. Itoh, T. Tanaka, R. Nagai, T. Kamiya, T. Sawayama, T. Nakayama, H. Tomoike, H. Sakurada, Y. Yazaki, Y. Nakamura, Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome, Hum. Genet. 102 (1998) 435–439. [2] I. Splawski, J. Shen, K.W. Timothy, G.M. Vincent, M.H. Lehmann, M.T. Keating, Genomic structure of three long QT syndrome genes: KVLQT1, HERG, and KCNE1, Genomics 51 (1998) 86–97. [3] J. Edelmann, S. Schumann, M. Nastainczyk, D. Husser-Bollmann, R. Lessig, Long QT syndrome mutation detection by SNaPshot technique, Int. J. Legal Med. (2011), doi:10.1007/s00414-011-0598-x. [4] C.R. Bezzina, A.O. Verkerk, A. Busjahn, et al., A common polymorphism in KCNH2 (HERG) hastens cardiac repolarization, Cardiovasc. Res. 59 (2003) 27–36. [5] L. Gouas, V. Nicaud, M. Berthet, A. Forhan, L. Tiret, B. Balkau, P. Guicheney, the D.E.S.I.R. Study Group, Association of KCNQ1, KCNE1, KCNH2 and SCN5A polymorphisms with QTc interval length in a healthy population, Eur. J. Hum. Genet. 13 (2005) 1213–1222.
