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Original Article

Acta Cardiol Sin 2005;21:37-45

Genetics

Denaturing High-performance Liquid Chromatography Screening for KvLQT1 Gene Variations in Patients with Atrial Fibrillation Ling-Ping Lai,1,2 Chia-Ti Tsai,2 Yi-Ning Su,3 Juey-Jen Hwang,2 Chwen-Fang Lee,1 Kwan-Lih Hsu,2 Fu-Tien Chiang,2 Chuen-Den Tseng,2 Yung-Zu Tseng2 and Jiunn-Lee Lin2

Background: A mutation in the KvLQT1 gene has been reported to be responsible for autosomal dominant hereditary atrial fibrillation (Af). It is not known whether mutations in this gene are also responsible for Af in common clinical practice. Methods: We enrolled 100 consecutive patients with Af, as well as 50 normal individuals without Af. Total cellular DNA was isolated from peripheral leukocytes. Polymerase chain reactions were performed to amplify the translated region of the KvLQT1 gene. Denaturing high-performance liquid chromatography was subsequently used to screen for the presence of heteroduplexes, and DNA sequencing was applied to these heteroduplexes. Results: Among the 100 patients with Af, we identified six single-nucleotide polymorphisms (SNPs), including three intronic SNPs (in introns 1, 12, and 13, respectively), two synonymous SNPs (C435 to T and G1638 to A) and one non-synonymous SNP (C1343 to G with amino acid change P448 to R). Two of the intronic SNPs were present in only one patient with Af (intron 1 and intron 12). The other four SNPs were more common and were also present in normal individuals. The incidences of heterozygocity of the four common SNPs were not significantly different between normal individuals and patients of Af, nor were the incidences significantly different between patients with lone Af and patients with Af associated with organic heart disease. Conclusion: KvLQT1 mutation was not found in 100 patients with Af. Although common SNPs were identified, their incidences were not significantly different among normal individuals, patients with lone Af and patients with organic Af.

Key Words:

Atrial fibrillation · KvLQT1 · Genetics · DHPLC

great deal of morbidity and mortality.3 The pathogenesis of Af is complex. There are many well-known risk factors for this disease, such as hypertension, valvular heart disease, ischemic heart disease, heart failure and thyroid disease. However, it is also observed that there are individual variations in Af susceptibility. For instance, some patients remain in sinus rhythm despite the presence of advanced heart disease, while some patients have Af without any organic heart disease (lone Af). Therefore, genetic factors are likely to play important roles in the pathogenesis of Af. With the advent of modern molecular genetic techniques, possible genetic factors contributing to the

INTRODUCTION Atrial fibrillation (Af) is the most common sustained tachyarrhythmia in human beings.1,2 It is responsible for a

Received: October 18, 2004 Accepted: November 17, 2004 From the 1Institute of Pharmacology, School of Medicine, National Taiwan University, Taipei; 2Department of Internal Medicine, 3 Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan. Address correspondence and reprint requests to: Dr. Jiunn-Lee Lin, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan S. Road. Taipei, Taiwan. Tel: 886-2-2312-3456 ext. 5001; Fax: 886-2-2395-1841; E-mail: [email protected]

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Ling-Ping Lai et al.

and left ventricular ejection fraction and to detect significant valvular disease (defined as moderate-severe or severe valvular regurgitation or stenosis). Left ventricular dysfunction was defined as a left ventricular ejection fraction less than 55%.8 Organic Af was defined as Af associated with organic heart disease, such as left ventricular dysfunction, significant valvular disease, coronary artery disease or clinical evidence of cardiac abnormalities.

occurrence of Af can therefore be explored. Among many research groups, Chen et al. first identified a gene responsible for Af.4 They reported that a mis-sense mutation in the KvLQT1 gene was responsible for autosomal dominant hereditary Af in a large family in the northern area of China. This gene encodes for the a-subunit of cardiac IKs channels, which contributes significantly to the repolarization process in atrial tissue.5,6 It is now known that mutations with a loss of function are responsible for congenital long QT syndrome,7 while a mutation with a gain of function is responsible for Af. In common clinical practice, Af is not regarded as a hereditary disease even though some familial aggregation is observed. However, hereditary Af could be taken as sporadic due to late onset and variable penetrance. It is also possible that variations in the KVLQT1 gene with minor functional change result in a “forme fruste” of Af, which becomes overt when other aggravating factors are added. We therefore hypothesized that some of the Af in common clinical practice had KVLQT1 mutation or variation. We used a denaturing high-performance liquid chromatography (DHPLC) protocol to screen for variations in KVLQT1 gene in these patients.

Polymerase chain reaction (PCR) amplification Peripheral blood samples were collected, and genomic DNA was extracted using a modified proteinase K method (Qiagen GmbH). PCR was performed in thin-walled PCR tubes in a total volume of 25 mL containing 100 ng of genomic DNA, 0.12 mM of each primer, 100 mM dNTPs, 0.5 units of AmpliTaq Goldenzyme (PE Applied Biosystems, Foster City, CA, USA), and 2.5 mL of GeneAmp 10x buffer II (10 mM tris-HCl, pH = 8.3, 50 mM KCl), in 2 mM MgCl2 as provided by the manufacturer. Amplification was performed in a multiblock system thermocycler (ThermoHybaid, Ashford, UK). PCR amplifications were performed with an initial denaturation step at 95 °C for ten minutes, followed by 35 cycles consisting of denaturation at 94 °C for 30 seconds, annealing for 30 seconds and elongation at 72 °C for 1 minute. The annealing temperature was 55 °C for most reactions, except 58 °C for exons 1-1, 10, 14, 15, 16 and 63 °C for exon 8. A touch-down protocol from 70 °C to 58 °C was used for exons 1-2, 4 and 5. A final extension step was applied at 72 °C for 10 minutes after 35 cycles. The primer sequences for the PCR reactions are listed in Table 1. The primers were designed to cover the entire coding region of KvLQT1.

METHODS Study patients We included 100 consecutive patients with Af who visited our hospital and were willing to give consent to the study. There were 58 men and 42 women with a mean age of 73.1 years at diagnosis (range 47 to 94). All patients were Chinese. We also included 27 men and 23 women (100 alleles, mean age 71.6 years) without Af as the reference population. The age and sex were not significantly different between the two groups. The Ethical Committee of the Institutional Reviewing Board approved this study, and informed consent was obtained.

DHPLC analysis The DHPLC system used for detecting heteroduplexes was a Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc., San Jose, CA, USA). DHPLC was carried out on automated HPLC instrumentation equipped with a DNASep column (Transgenomic, Inc., San Jose, CA, USA). DHPLC-grade acetonitrile (9017-03, JT Baker, Phillipsburg, NJ, USA) and triethylammonium acetate (TEAA, Transgenomic, Inc., Crewe, UK) were used for the mobile phases. The mobile phases were comprised of 0.05% acetonitrile in 0.1

Clinical assessment The presence of Af was determined by review of patients’ histories, serial ECG and/or ambulatory ECG monitoring. Patients with palpitations without ECG documentation were excluded from both patient and control groups. Transthoracic echocardiography was performed to measure the left atrial and left ventricular dimensions Acta Cardiol Sin 2005;21:37-45

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KvLQT1 and Afib

files were identified by visual inspection of the chromatograms on the basis of the appearance of additional earlier eluting peaks. Corresponding homozygous profiles showed only one peak.

M TEAA (eluent A) and 25% acetonitrile in 0.1 M TEAA (eluent B). For heteroduplex detection, crude PCR products, subjected to an additional three-minute, 95 °C denaturing step, followed by gradual reannealing from 95 °C to 65 °C over a period of 30 minutes prior to analysis, were eluted at a flow rate of 0.9 mL/min. The start- and end-points of the gradient by mixing eluents A and B and the temperature required for successful resolution of heteroduplex molecules were adjusted by using an algorithm provided by the WAVEmaker system control software, version 4.1.42 (Transgenomic, Inc., San Jose, CA, USA). Eight microliters of PCR product was injected for analysis in each running. Individual analytical gradient conditions for DHPLC running are described in Table 1 and are expressed as a percentage of eluent B. The flow rate was 0.9 mL/min, and the ultraviolet detector was set to 260 nm. For amplicons of exons 6, 10, 11 and 14, two melting domains were present as predicted according to the base sequence. Two DHPLC conditions were applied to these amplicons for better detection sensitivity. The DHPLC conditions for the amplicons are shown in Table 1. Heterozygous pro-

Sequence analysis Amplicons were purified by solid-phase extraction and were bidirectionally sequenced with the PE Biosystems Taq DyeDeoxy terminator cycle sequencing kit (PE Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Sequencing reactions were separated on a PE Biosystems 373A/3100 sequencer. The sequencing results were compared with KvLQT1 mRNA and the gene sequence in GenBank (accession numbers NM000218 and AJ006345, respectively). The adenosine of the start codon (ATG) was numbered as the first nucleotide when expressing genetic variations. Statistical analysis The incidences of heterozygosity were compared using a chi-square test. A p value less than 0.05 was

Table 1. Base sequence of the primer pairs, as well as the DHPLC conditions Tm

B%

Exon

Forward primer

Backward primer

Size (bp)

68 °C 65.5 °C 64 °C 64.5 °C 65 °C 65 °C 62 °C 64 °C 64 °C 64 °C 64 °C 58 °C 61 °C 62 °C 64 °C 63 °C 64.5 °C 63 °C 58 °C 66 °C 65.5 °C

57 53 48 53 48 47 52 51 50 50 54 58 49 50 48 51 54 44 53 48 54

1-1 1-2 2 3 4 5 6 6 7 8 9 10 10 11 11 12 13 14 14 15 16

caggccctcctcgttatg cgccgcgcccccagttgc atgggcagaggccgtgatgctgac gttcaaacaggttgcagggtctga ctcttccctggggccctggc tcagccccacaccatctccttc tcctggagcccgacactgtgtgt tcctggagcccgacactgtgtgt tggctgaccactgtccctct gctggcagtggcctgtgtgga tggctcagcaggtgacagc gcctggcagacgatgtcca gcctggcagacgatgtcca ctgtccccacactttctcct ctgtccccacactttctcct tggccactcacaatctcct cactgcctgcactttgagcc ccagggccaggtgtgactg ccagggccaggtgtgactg ggccctgatttgggtgtttta caccactgactctctcgtct

gacgctcgaggaagttgtag cagagctcccccacaccag atccagccatgccctcagatgc cttcctggtctggaaacctgg tgcgggggagcttgtggcacag ctgggcccctaccctaaccc tgtcctgcccactcctcagcct tgtcctgcccactcctcagcct ccccaggaccccagctgtccaa aacagtgaccaaaatgacagtgac gacacaggctgtaccaagccaa caactgcctgaggggttct caactgcctgaggggttct tgagctccagtcccctccag tgagctccagtcccctccag gccttgacaccctccacta gtgaggagaagggggtggtt tgggcccagagtaactgaca tgggcccagagtaactgaca gcaggagcttcacgttcaca ccatcccccagccccatc

364 224 165 256 170 154 238 238 195 191 280 216 216 195 195 222 304 119 119 186 297

B% = percentage of eluent B; bp = base pairs; DHPLC = denaturing high-performance liquid chromatography; Tm = melting temperature. 39



tures for these heteroduplex patterns. Further DNA sequencing reaction identified six SNPs in these five heteroduplexes (two in amplicons for exon 13).

considered significant.

RESULTS Incidences of SNPs in patients with Af and in the normal population The incidences of heterozygosity of the SNPs in the KVLQT1 gene are shown in Table 2. We identified six SNPs in patients with Af, including three intronic SNPs (introns 1, 12, and 13), two synonymous SNPs (C435 to

PCR amplification for all 18 exons was successfully performed as revealed by agarose gel electrophoresis. Further DHPLC screening identified five different heteroduplex patterns in amplicons for exons 1-2, 2, 10, 12 and 13, respectively. Figure 1 shows the DHPLC pic-

Figure 1. Five heteroduplex elution patterns identified by denaturing high-performance liquid chromatography (DHPLC) in patients with atrial fibrillation. Heteroduplex patterns are characterized by the appearance of early eluting peaks, while homoduplex profile shows only one peak. DNA sequencing results are shown below the DHPLC patterns.

Table 2. Incidences of SNPs in the study patients SNP

a.a. change

Af (n = 100)

Control (n = 50)

p

P448R -

1/100 (1%) 12/100 (12%) 19/100 (19%) 1/100 (1%) 31/100 (31%) 31/100 (31%)

0 11/50 (22%) 14/50 (28%) 0 23/50 (46%) 23/50 (46%)

0.109 0.210 0.07 0.07

C(387+10)A C435T C1343G A(1590+31)T G1638A G(1686+36)A

a.a. = amino acid; Af = atrial fibrillation; SNP = single-nucleotide polymorphism. Acta Cardiol Sin 2005;21:37-45

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KvLQT1 and Afib

T and G1638 to A) and one non-synonymous SNP (C1343 to G). The intronic SNPs in intron 1 [C(387+ 10)A] and intron 12 [A(1590+31)T] were both identified in only one patient with Af, respectively. These two intronic SNPs were not found in normal individuals. The C435T SNP was a synonymous SNP in exon 2. The incidence of heterozygosity was 12% in Af patients and 22% in normal individuals. The incidences were not significantly different between the two groups. The C1343G SNP was a non-synonymous SNP causing an amino acid change from P (proline) to R (arginine) in exon 10. The incidences of heterozygocity were not significantly different between Af patients and control subjects (19% vs 28%, p < 0.21). For the heteroduplex pattern identified in exon 13, further DNA sequencing reaction revealed two tightly linked SNPs in this heteroduplex. All amplicons with G1638A heterozygosity were also heterozygous for another SNP in intron 13 [G(1686+36)A]. The incidence of heterozygocity of the two linked SNPs was not significantly different between Af patients and normal subjects (31% vs 46%, p < 0.07).

groups according to the presence or absence of organic heart disease. There were 67 patients with organic heart disease (organic Af) and 33 patients without (lone Af). The clinical and echocardiographic features are shown in Table 3. Patients with organic Af had significantly larger LA and LV chamber sizes. The left ventricular ejection fraction was significantly lower in patients with organic Af. As for the KvLQT1 gene variations, the two patients with the rare intronic SNP in intron 1 and intron 12 were both in the organic Af group. For the other four common SNPs, the incidences of heterozygosity were not significantly different between patients with organic Af or lone Af.

DISCUSSION Up to now, KVLQT1 is the only known gene responsible for familial Af, although linkage analysis has revealed that other genes are also responsible.9-11 Functional studies of KVLQT1 mutation responsible for Af have demonstrated a gain in function. This supports the hypothesis that IKs channels play important roles in atrial tissue. We therefore performed the present study to answer the question whether mutations or variations in this

Comparison between patients with organic Af and lone Af We further divided the patients with Af into two

Table 3. Clinical characteristics and SNPs in patients with organic Af and lone Af

Age (years) Sex (M/F) LA (mm) LVEF (%) LVEDD (mm) LVESD (mm) HT DM CHO (mg/dL) TG (mg/dL) C(387+10)A C435T C1343G A(1590+31)T G1638A G(1686+36)A

Organic Af (n = 67)

Lone Af (n = 33)

p

74.1 ± 10.3 36/31 50.2 ± 8.5 55.7 ± 14.8 48.7 ± 7.4 33.7 ± 8.2 39/67 (58.2%) 9/67 (13.4%) 175.8 ± 40.1 123.6 ± 105.4 1/67 (1.5%) 7/67 (10.4%) 14/67 (20.9%) 1/67 (1.5%) 19/67 (28.3%) 19/67 (28.3%)

71.4 ± 10.2 22/11 41.3 ± 5.9 65.6 ± 7.6 48.0 ± 4.9 30.2 ± 5.0 17/33 (51.5%) 6/33 (18.2%) 188.5 ± 36.1 131.5 ± 70.0 0 5/33 (15.2%) 5/33 (15.2%) 0 12/33 (36.4%) 12/33 (36.4%)

0.747 0.155 0.066 0.000* 0.031* 0.006* 0.349 0.344 0.832 0.614 0.353 0.345 0.416 0.416

* p < 0.05; Af = atrial fibrillation; CHO = serum cholesterol level, DM = diabetes mellitus; HT = hypertension; LA = left atrial dimension, LVEDD = left ventricular end-diastolic dimension; LVEF = left ventricular ejection fraction; LVESD = left ventricular end-systolic dimension; SNP = single-nucleotide polymorphism; TG = serum triglyceride level.

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in the White and Hispanic populations. In the present study, C1343G had an incidence of heterozygosity of 19% to 28%. The incidence is similar to that in the Japanese population. The incidences were not significantly different between normal individuals and patients with Af, nor were the incidences significantly different between patients with lone Af and patients with organic Af. The SNP in intron 13 is tightly linked to G1638A in exon 13. All patients with G1638 heterozygosity were also heterozygous for intron 13 SNP. The SNP in intron 13 was not located at the donor or recipient site for splicing. It is unlikely that this SNP has functional significance. In summary, none of the six SNPs were likely to contribute to the pathogenesis of Af.

gene are present in Af in common clinical practice. In a consecutive series of 100 patients with Af, we did not find any mutations. We therefore conclude that mutations in the KVLQT1 gene are rare in patients with Af, if not totally absent.

DHPLC screening In the present study, we used the DHPLC technique to screen for genetic variations in the KvLQT1 gene. Traditionally, PCR and DNA sequencing strategies are used for mutation detection. However, these procedures are time and effort consuming. Therefore, a reliable mutation screening method greatly enhances the efficacy of identifying mutations. Single-strand conformation polymorphism analysis was a commonly used method for mutation screening before DHPLC was made available. However, it has a maximal sensitivity around 80%. 12 In the present study, we used the DHPLC method, which has demonstrated a sensitivity of nearly 100% in previous reports.13-18 This method is based on the difference between the melting points of homoduplex and heteroduplex PCR products. This application is simple, rapid and economical. We successfully identified six SNPs in the KvLQT1 gene, and two of them had never been reported before. We demonstrated that DHPLC is a useful tool in screening for variations in the KvLQT1 gene.

Role of IKs potassium channels in Af I Ks potassium channels are responsible for the slow component of delayed rectifier potassium current and participate in the cardiac repolarization process. During Af, the depolarization rate is fast, and the diastolic period shortens. The I Ks channels in an open state tend to accumulate because of their slow opening and closing kinetics. Therefore, IKs channels become a very important determinant of an effective refractory period and affect the vulnerability to Af. There are currently two genes known to encode the cardiac I Ks channels. The KVLQT1 gene encodes the a-subunit of the cardiac I Ks potassium channel, while KCNE1 encodes the b-subunit. 5-6 A mutation of KvLQT1 with gain of function has been reported to be responsible for familial Af. An association study has demonstrated the association between KCNE1 gene G38S polymorphism and Af. 26-27 These reports demonstrated the importance of I Ks in the pathogenesis of Af. In the present study, mutations in KvLQT1 were not identified. However, we still cannot exclude the possible role of KvLQT1 in common Af . First, the presence of homozygous mutations cannot be detected by DHPLC screening, although this kind of condition is extremely rare. Second, somatic mutation of the KvLQT1 gene or epigenetic modification of the gene in the atrial tissue cannot be detected by screening methods using genomic DNA as the material. Finally, transcriptional regulation, post-transcriptional and post-translational modification of the KvLQT1 gene was not addressed in the present study.

Characteristics of SNPs identified in the present study There were six SNPs identified in the present study. Two of the intronic SNPs (intron 1 and intron 12) were novel. The incidences of both SNPs were low, and they were observed in only one patient with Af. The positions of these intronic SNPs were not at the donor site or recipient site for RNA splicing. The possibility that they are functionally significant is low. The other four SNPs were more common and have been reported before. 19-25 The C435T and G1638A are synonymous SNPs. Since there was no amino acid change, it is also unlikely that they were functionally important. C1343G is the only non-synonymous SPN. It is associated with a change of amino acid from P to R at position 448. Functional studies are still lacking. This SNP has been reported in the Japanese population with an allele frequency of 20-30%, while its incidence is extremely low Acta Cardiol Sin 2005;21:37-45

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KvLQT1 and Afib

11. Ellinor PT, Shin JT, Moore RK, et al. Locus for atrial fibrillation maps to chromosome 6q14-16. Circulation 2003;107:2880-3. 12. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: a review. Hum Mutat 2001;17:439-74. 13. Eng C, Brody LC, Wagner TMU, et al. Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1. J Med Genet 2001;38: 824-33. 14. Bennett RR, den Dunnen J, O’Brien KF, et al. Detection of mutations in the dystrophin gene via automated dHPLC screening and direct sequencing. BMC Genet 2001;2:17. 15. Han S, Cooper DN, Upadhyaya M. Evaluation of denaturing high-performance liquid chromatography (dHPLC) for the mutational analysis of the neurofibromatosis type 1 (NF1) gene. Hum Genet 2001;109:487-97. 16. Taliani MR, Roberts SC, Dukek BA, et al. Sensitivity and specificity of denaturing high-pressure liquid chromatography for unknown protein C gene mutations. Genet Test 2001;5: 39-44. 17. Matyas G, DePaepe A, Halliday D, et al. Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene. Hum Mutat 2002;19: 443-56. 18. Colosimo A, Guida V, DeLuca A, et al. Reliability of dHPLC in mutational screening of beta-globin (HBB) alleles. Hum Mutat 2002;19:287-95. 19. Jongbloed R, Marcelis C, Velter C, et al. DHPLC analysis of potassium ion channel genes in congenital long QT syndrome. Hum Mutat 2002;20:382-91. 20. Ackerman MJ, Tester DJ, Jones GS, et al. Ethnic differences in cardiac potassium channel variants: implications for genetic susceptibility to sudden cardiac death and genetic testing for congenital long QT syndrome. Mayo Clin Proc 2003;78:147987. 21. Iwasa H, Itoh T, Nagai R, et al. Twenty single-nucleotide polymorphisms (SNPs) and their allelic frequencies in four genes that are responsible for familial long QT syndrome in the Japanese population. J Hum Genet 2000;45:182-3. 12. Splawski I, Shen J, Timothy KW, et al. Spectrum of mutations in long-QT syndrome genes: KVLQT1, HERG, SCN5A, KCNE1, and KCNE2. Circulation 2000;102:1178-85. 23. Lee MP, Hu RJ, Johnson LA, et al. Human KVLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements. Nat Genet 1997;15: 181-5. 24. Yang P, Kanki H, Drolet B, et al. Allelic variants in long-QT disease genes in patients with drug-associated torsades de pointes. Circulation 2002;105:1943-8. 25. Itoh T, Tanaka T, Nagai R, et al. Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome. Hum Genet 1998;103:290-4. 26. Lai LP, Deng CL, Moss AJ, et al. Polymorphism of the gene

Limitations The comparisons of genotypes between Af and control groups were done in 100 patients with Af and 50 control subjects. Because the number of control subjects was small, a b-error in statistical analysis could not be excluded.

CONCLUSION Although mutations in the KVLQT1 gene have been demonstrated in patients with autosomal dominant Af, the molecular genetic basis of common Af is unknown. We have now completely analyzed the translated region of the KvLQT1 gene in 100 patients with Af, and we found no mutations in the KVLQT1 gene. Further molecular genetic studies are needed to unravel the molecular basis for this common type of Af.

REFERENCES 1. Kannel WB, Abbott RD, Savage DD, et al. Epidemiologic features of atrial fibrillation. N Engl J Med 1982;306:1018-22. 2. Onundarson PT, Thorgeirsson G, Jonmundsson E, et al. Chronic atrial fibrillation: epidemiologic features and 14-year follow-up: a case control study. Eur Heart J 1987;8:521-7. 3. Alpert JS, Petersen P, Godtfredsen J. Atrial fibrillation: natural history, complications and management. Ann Rev Med 1988;39: 41-52. 4. Chen YH, Xu SJ, Bendahhou S, et al. KCNQ1 gain-of-function mutation in familial atrial fibrillation. Science 2003;299:251-4. 5. Sanguinetti MC, Curran ME, Zou A, et al. Coassembly of KvLQT1 and mink (IsK) proteins to form cardiac Iks potassium channel. Nature 1996;384:80-3. 6. Barhanin J, Lesage F, Guillemare E, et al. KvLQT1 and Isk (minK) proteins associate to form the Isk cardiac potassium current. Nature 1996;384:78-80. 7. Wang Q, Curran ME, Splawski I, et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nat Genet 1996;12:17-23. 8. Kennedy JW, Baxley WA, Figley MM, et al. Quantitative angiocardiography: the normal left ventricle in man. Circulation 1966;34:272-8. 9. Brugada R, Tapscott T, Czernuszewicz GZ, et al. Identification of a genetic locus for familial atrial fibrillation. New Engl J Med 1997;336:905-11. 10. Shah G, Brugada R, Gonzalez O, et al. The cloning, genomic organization and tissue expression profile of the human DLG5 gene. Genomics 2002;3:6. 43



encoding a human minimal potassium ion channel (minK). Gene 1994;151:339-40. 27. Lai LP, Su MJ, Yeh HM, et al. Association of the human minK


gene 38G allele with atrial fibrillation: evidence of possible genetic control on the pathogenesis of atrial fibrillation. Am Heart J 2002;144:485-90.

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Original Article

Acta Cardiol Sin 2005;21:37−45

以變性高效能液態層析法偵測心房顫動病患 KvLQT1 基因之變異賴凌平 1

蔡佳醍 2 蘇怡寧 3 黃瑞仁 2 李純芳 1 許寬立 2 江福田 2 曾春典 2 曾淵如 2 林俊立 2 台北市台大醫學院藥理學科１台北市台大醫院內科部2 基因醫學部3

背景 KvLQT1 基因的突變可以造成遺傳性的心房顫動，然而，一般非遺傳性的心房顫動是否也和 KvLQT1 基因有關，則目前仍不明白。方法本研究納入連續 100 名心房顫動患者以及 50 名沒有心房顫動的對照組，我們自週邊血液抽取白血球的 DNA，並以聚合酶連鎖反應的方式放大其 KvLQT1 基因。我們接著以變性高效能液態層析法偵測其中的異結合子，並以 DNA 定序的方法決定其 DNA 變異。結果在這 100 名心房顫動患者中，我們發現了 6 個單一核苷酸多形性，其中三個在 intron 中 (intron1, 12, 13) 兩個沒有胺基酸變化 (C435T 及 G1638A) 一個有胺基酸變化 (C1343G, P448R)。其中 intron 1 及 12 的多形性只在一個病患中發現，至於其他 4 個多形性則較為常見而且也存在於正常人中，這四個多形性的發生率在病人組及正常人組之間並沒有顯著差異，若將心房顫動病患分成原發性心房顫動及因器實性心臟病所引起的心房顫動兩組，則兩組之間的多形性機率並沒有顯著差異。結論在 100 名心房顫動患者中，我們並沒有發現任何 KvLQT1 基因的突變。我們雖然在病患中找到了一些單一核苷酸多形性，然而這些多形性的發生率在正常人及病患之間並無顯著差異，而在原發性心房顫動及器實性心房顫動患者間，也沒有顯著差異。關鍵詞：心房顫動、KvLQT1、遺傳學、變性高效能液態層析法。

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