HD) method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome

HD) method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome

Clinica Chimica Acta 280 (1999) 113–125 A single strand conformation polymorphism / heteroduplex (SSCP/ HD) method for detection of mutations in 15 e...

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Clinica Chimica Acta 280 (1999) 113–125

A single strand conformation polymorphism / heteroduplex (SSCP/ HD) method for detection of mutations in 15 exons of the KVLQT1 gene, associated with long QT syndrome Lars Allan Larsen a , Paal Skytt Andersen a , Jørgen K. Kanters b ,c , d a a, Joes Ramsøe Jacobsen , Jens Vuust , Michael Christiansen * a

Department of Clinical Biochemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark b Department of Medicine, Elsinore Hospital, DK-3000 Elsinore, Denmark c Department of Medical Physiology, University of Copenhagen, DK-2200 Copenhagen, Denmark d Department of Paediatrics G, State University Hospital, Rigshospitalet, DK-2100 Copenhagen, Denmark Received 25 August 1998; received in revised form 9 October 1998; accepted 24 October 1998

Abstract Congenital long QT syndrome (LQTS) is characterised by prolongation of the QT interval on ECG and cardiac arrhythmias, syncopes and sudden death. A rapid and reliable genetic diagnosis of the disease may be of great importance for diagnosis and treatment of LQTS. Mutations in the KVLQT1 gene, encoding a potassium-channel subunit of importance for the depolarisation of cardiac myocytes, is believed to be associated with 50% of all LQTS cases. Our data confirms that KvLQT1 isoform 1 is encoded by 16 exons, and not 15, as reported previously. We have used genomic DNA sequences to design intronic PCR primers for amplification of 15 exons of KVLQT1 and optimised a non-radioactive single stranded conformation polymorphism / heteroduplex (SSCP/ HD) method for detection of mutations in KVLQT1. The sensitivity of the method was 100% when it was tested on 15 in vitro constructed mutants. By multiplexing the PCR amplification of KVLQT1, it is possible to cover all 15 exons in four PCR reactions.  1999 Elsevier Science B.V. All rights reserved. Keywords: Long QT syndrome; KVLQT1; Single stranded conformation polymorphism analysis; Heteroduplex analysis; Mutation detection

*Corresponding author. Tel.: 1 45-32-683657; fax: 1 45-32-683878; e-mail: [email protected] 0009-8981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00177-6

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1. Introduction Inherited long QT syndrome (LQTS) is a cardiac disorder characterised by prolongation of the QT interval on electrocardiograms. The clinical presentation is syncopes and sudden death, resulting from repolarisation-related cardiac arrhythmias such as ‘torsade de pointes’ and ventricular fibrillation (reviewed in Ref. [1]). The major criteria for clinical diagnosis of LQTS is QT prolongation. However, approximately 5% of individuals with LQTS have a QT interval in the normal range [2]. As sudden death may be the presenting symptom in these individuals, and b-adrenergic blockade confers a significantly improved prognosis on affected individuals, genetic diagnosis of LQTS may be of importance in the individual patient. This is particularly important for children in LQTS families where ECG is often inconclusive. In addition, assignment of LQTS to specific genes may have implications for prognosis and choice of treatment [3,4]. LQTS is found in a predominantly autosomal dominant form, Romano–Ward syndrome and a predominantly autosomal recessive form, Jervell and LangeNielsen syndrome, the latter associated with congenital deafness. Four genes involved in LQTS have been identified. Two genes encode cardiac potassium-channel subunits, KVLQT1 (11p15.5) [5] and HERG (7q35-37) [6], one gene encodes a cardiac sodium-channel subunit, SCN5 A (3p21-24) [7] and the fourth gene, KCNE1 (21q22.1-2), encodes Isk or MinK, the regulator of the I ks and I kr cardiac potassium currents [8,9]. A fifth gene, involved in LQTS, was mapped at 4q25-27 in one family [10], but has not been characterised. Mutations in KVLQT1 are believed to be associated with 50% of all cases of LQTS [5,11]. The KVLQT1 gene was isolated by positional cloning [5] and sequencing of a partial KVLQT1 cDNA revealed that the gene encodes a protein with predicted structural characteristics of a potassium channel subunit with six membrane spanning domains, S1–S6, and a pore forming domain [5]. Further characterisation of the gene revealed 14 KVLQT1 exons spanning more than 350 kbp of the 11p15.5 region [12]. Transcription analysis and functional studies of the KVLQT1 gene product have shown that the gene is transcribed in at least six different 59 splice variants, isoforms 0–5, which involves splicing of 1–3 additional exons to the 59 end of exon 1 [12–15]. However, only isoform 0 and 1 seem to be translated into functional potassium channel subunits in the human heart [15]. Very recently, the genomic structure of the 16 exons of KVLQT1 encoding isoform 1 was described [16]. So far, more than 30 different mutations in KVLQT1 have been found to be associated with Romano–Ward syndrome [5,15,17–20] while four different KVLQT1 mutations have been associated with Jervell and Lange-Nielsen syndrome [21–23].

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Nearly all mutations have been found in and between the membrane spanning domains S2–S6, and the pore forming region. However, in most of these studies, the mutation analysis has been limited to exon 2–7, in which the first mutation study was performed [5]. We have used genomic DNA sequences spanning the KVLQT1 gene to design intronic PCR primers for SSCP analysis of KVLQT1 in order to improve the genetic analysis of LQTS.

2. Materials and methods

2.1. DNA samples from LQTS patients Blood samples were collected from LQTS patients and family members identified from routine molecular-genetic workup on patients with symptomatic LQTS admitted to hospitals in Copenhagen. All mutations were associated with prolonged QT c and classical clinical signs of LQTS. Clinical details will be presented elsewhere. Genomic DNA was extracted from whole blood or filter paper blood spots using a QIAamp kit (Qiagen, Germany).

2.2. Construction of point mutations Point mutations in exon 6 of the KVLQT1 gene were constructed by PCR using degenerate oligo nucleotides or by taq polymerase induced errors, provoked by raising the dNTP concentration to 400 mmol / l and the MgCl 2 concentration to 1.5 mmol / l. The degenerate oligonucleotides were designed with single base-substitutions at the desired points (i.e. the primer: 59GGCTGACCA CTGTCCCTCTCCCTGC AGCTGCAGGTCACAGV CACCACC-39 was used to generate mutations in codon 310). The sequence of the other degenerate primers will be available by request to L.A. Larsen ([email protected]). The PCR fragments were cloned using the pCR-script vector (Stratagene, La Jolla, CA). A number of mutated clones were identified by DNA sequencing.

2.3. PCR amplification PCR was performed in 200 ml thin-walled PCR tubes in a total reaction volume of 50 ml, containing 1–2 ml DNA template, 5 ml of 10 3 reaction buffer (100 mmol / l Tris–HCl, pH 8.85, 250 mmol / l KCl, 50 mmol / l (NH 4 ) 2 SO 4 ), 1–4 mmol / l MgSO 4 (optimised for each primer pair, see Table 1), 200 mmol / l dNTP and 1 Unit of pwo DNA polymerase (Boehringer Mannheim, Germany). Previously published amplification primers were used, supplemented with additional intronic primers (Table 1). The additional primers were designed from published genomic DNA sequences (Genbank accession numbers:

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Table 1 Intronic primers for amplification of KVLQT1 exons Nucleotide sequence

Amplified a region

Optimal MgSO 4 b concentration (mmol/l) (single exons)

Optimal MgSO 4 b concentration (mmol/l) (multiplex PCR)

Fragment size (bp)

Primer concentration (mmol/l) in multiplex PCR (tube no.)c

2F 2R 3F 3R 4F 4R 5F 5R 6F 6R 7F 7R 8F 8R 9F 9R 10F 10R 11F 11R 12F 12R 13F 13R 14F 14R 15F 15R 16AF 16AR 16BF 16BR

59 CGTCATGCTGACTGCCGTG 39 59 CCAGGGAGATGCCAGCTTC 39 59 CATGGCTGGGTTCAAACAGGT 39 59 GGAAACCTGGGCGTGACCTC 39 59 CTCTTCCCTGGGGCCCTGGC 39 59 TGCGGGGGAGCTTGTGGCACA G 39 59 CCCTCAGCCCCACACCATC 39 59 TCTGCTCCCTCCGTGCTGTC 39 59 TCCTGGAGCCCGAACTGTGTGT 39 59 TGTCCTGCCCACTCCTCAGCCT 39 59 AGGCTGACCACTGTCCCTCT 39 59 CCCCAGGACCCCAGCTGTCCAA 39 59 AGCCTCCTGTCCATTCCTTCC 39 59 AACAGTGACCAAAATGACAGTGAC 39 59 GGGAACAGGGAGGGGGAGC 39 59 TGGTGGCAGGTGGGCTACTC 39 59 ATGTCCAGGAACCGCTAATCTG 39 59 GGCTCCCAAAAAAGGCAGTG 39 59 ACGTGCTGTCCCCACACTTTC 39 59 CCAGCCCTTCACGCACATG 39 59 TGAGGGGATGACCAGCACAGG 39 59 GGGCAGGAAGGCTCAGCACAG 39 59 CCCGAGGGCAGACACTGTCACTG 39 59 CCCTCCCCGCTGCCGTTTG 39 59 GGTGTGAACTGGTGTCTGTGTCC 39 59 AGGTGCGGGAGAGTCCATTG 39 59 CGGCCCACCCCAGCACTTG 39 59 CCCCAGGACGCTAACCAGAACCAC 39 59 CTTCCCACCACTGACTCTCTCG 39 59 CGTAGGTGGGCAGGGTGTTG 39 59 CTCCGTCGACCCTGAGCTCT 39 59 CATGGGGTATTGGGGCCTCT 39

Exon 2 (1)

1

1.5

186

2.4 (tube 3)

Exon 3 (2)

1.5

2.0

254

2 (tube 4)

Exon 4 (3)

1.5

1.5

170

2 (tube 3)

Exon 5 (4)

1

1.5

186

2.4 (tube 2)

Exon 6 (5)

2

3

238

2 (tube 1)

Exon 7 (6)

1.5

1.5

194

2 (tube 2)

Exon 8 (7)

4

3

166

2 (tube 1)

Exon 9 (8)

1.5

1.5

233

2 (tube 2)

Exon 10 (9)

1.5

1.5

246

2.4 (tube 2)

Exon 11 (10)

1

2.0

179

2.8 (tube 4)

Exon 12 (11)

1

1.5

163

1.6 (tube 3)

Exon 13 (11)

1

3

213

2,4 (tube 1)

Exon 14 (12)

1.5

2.0

140

2 (tube 4)

Exon 15 (13)

1

3

158

2.4 (tube 1)

Exon 16 (14)

1

1.5

234

2.8 (tube 3)

Exon 16 (14)

1.5

2.0

232

2 (tube 4)

a

[5] [5]

[5] [5] [5] [5] [5]

Exon numbering according to Ref. [16], numbers in brackets refer to the numbering according to Ref. [12]. Under the conditions descibed in Section 2. c Primer pairs are combined in four tubes as indicated by the tube no. b

Reference

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No.

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AC000377, AC00375, U90095 and HSAC001228) or from genomic sequences, obtained by sequencing of PCR products spanning intron sequences. Thermal cycling was performed in a PTC200 DNA engine (MJ Research, USA), with the temperature profile: 948C for 4 min followed by 34 cycles of 948C for 20 s, 678C for 20 s, 728C for 40 s, and ending with 7 min extension at 728C. Multiplex PCR was performed with the following modifications: 1.25 U pwo polymerase was used and the annealing and extension times were expanded to 1 min. Only 32 cycles were performed. PCR products were purified with spin columns according to the manufacturers instructions (Boehringer Mannheim, Germany).

2.4. Mutation analysis Single stranded conformation polymorphism and heteroduplex analysis (SSCP/ HD) was performed on PCR products essentially as described [24]. Analysis was performed on 1–2 ml purified PCR product in a precast 10% polyacrylamide gel at 4 and 208C, using a Multiphor apparatus (Pharmacia Biotech, Sweden). Bands were visualised by silver staining. Automated ‘Dye terminator’ cycle sequencing (Perkin Elmer) was performed according to the manufacturers instructions on an ABI373 DNA sequencer.

3. Results

3.1. Confirmation of a corrected genomic structure of KVLQT1 Genomic DNA sequences around 11p15.5 were obtained from Genbank sequences (accession numbers: AC000377, AC00375, U90095 and HSAC001228), and used to design intronic PCR primers for amplification of KVLQT1 exons (Table 1). Each PCR fragment was identified by direct sequencing. Inspection of the genomic DNA sequence around ‘exon 11’ (using the numbering from the first published genomic structure of KVLQT1 [12]) indicated the presence of an insert of 7 kbp in the exon. The insert was bordered by intron–exon splice site consensus sequences, strongly indicating the existence of an intron in ‘exon 11’. Amplification of KVLQT1 DNA with primers bordering ‘exon 11’ was not possible, while amplification of two fragments was achieved with additional primers located in the intron. Sequence analysis of the PCR fragments confirmed that ‘exon 11’ is interrupted by an intron (Fig. 1). Thus, our results confirm the genomic structure determined by Splawski et al. [16], and consequently, we have used their exon numbering in the rest of this article. In Table 1, the numbering by Lee et al. [12] is shown in parentheses.

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Fig. 1. Confirmation of the presence of an intron between exon 12 and 13 of KVLQT1. Part of the published KVLQT1 cDNA and the derived amino acid sequence is shown on top. Below is shown parts of the DNA sequence of two PCR fragments amplified from genomic DNA, with primers flanking exon 12 and 13, respectively. Intronic DNA sequences are shown in lower case.

3.2. SSCP/HD analysis for KVLQT1 mutations The PCR reaction conditions were optimised to run under identical thermocycling conditions. The PCR reactions were performed with pwo DNA polymerase, an enzyme with 39–59 exonuclease activity (proofreading activity), in order to reduce polymerase induced errors. SSCP/ HD analysis was initially performed using DNA from eight unrelated clinically well described LQTS patients and healthy family members. We found three novel KVLQT1 mutations in patients with clinically diagnosed congenital LQTS (Table 2). Clinical characteristics of these mutations will be presented elsewhere (manuscript in preparation). None of these mutations was found in healthy members of the LQTS families. Two previously published polymorphisms in exon 13 and 16 [12] were also detected by the method (Table 2). Examples of SSCP/ HD conformation patterns are shown in Fig. 2A and B. The sensitivity of the SSCP/ HD method was tested on in vitro constructed mutants (Table 2). The in vitro mutants were made, as described in Section 2, by introducing mutations in the PCR fragment covering exon 7 of KVLQT1. The mutated PCR fragments were cloned in pCR-script (Stratagene, La Jolla, CA) and the mutated clones were identified by DNA sequencing. Each clone was

Table 2 KVLQT1 mutations analysed in this study Deduced amino acid change

Effect

DNA region

Protein region

LQTS associated mutations 579 T → G 1141 G → A 1205 C → T

F157C G344G (none) R366W

Missense IVS6 59 DS Missense

exon 2 exon 7 exon 8

S2 S6 S6– b

In vitro constructed mutations IVS5 39 ss 2 8 C → G IVS5 39 ss 2 8 C → A 1031 G → T 1031 G → A 1031 G → T and 1088 A → G 1034 A → T 1034 A → C 1034 A → G 1035 C → T and 1036 A → T 1038 T → G 1038 T → C 1038 T → A 1043 A → T 1043 A → G 1134 T → C and IVS6 59 ss 1 22 T → C Polymorphisms 1638 G /A 2095 C / T

IVS6 c IVS6 c exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 exon 7 and IVS7 d S546S (none) Y662Y (none)

Normal variant Normal variant

13 16

L. A. Larsen et al. / Clinica Chimica Acta 280 (1999) 113 – 125

Nucleotide change a

S6– b S6– b

a

Position in the cDNA sequence of isoform 1 (Genbank Accession No. AF000571). Region downstream of S6. c Nucleotide 2 8 from the 39 splice site of IVS6. d Nucleotide 1 22 from the 59 splice site of IVS7. b

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Fig. 2. Examples of SSCP/ HD conformation patterns. (A) Missense mutation, R366W. (B) Normal variant, 2095 C / T. (C) In vitro constructed mutants. AC, abnormal conformer; HD, heteroduplex.

reamplified with the primer pair spanning exon 6, and the PCR product was mixed with the PCR product from a wild-type clone before SSCP/ HD analysis, in order to mimic a heterozygote sample. Fifteen out of 15 in vitro constructed mutants were detected by the method (Table 2 and Fig. 2C).

3.3. Multiplex SSCP/HD analysis A multiplex PCR panel was optimised in order to bring down the number of PCR reactions needed for each analysis. The primer pairs were combined according to size and optimal MgSO 4 concentration. The primer concentrations were adjusted in order to control the competition between small and large exons

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Fig. 3. Examples of multiplex SSCP/ HD conformation patterns. (A) Multiplex analysis of 15 KVLQT1 exons from a normal control individual and a LQTS patient carrying a donor splice site mutation in exon 6 (IVS6 1 1 G → A). (B) Multiplex analysis of four KVLQT1 exons from a normal control individual (lane 5) and a LQTS patient (lane 6) carrying a missense mutation in exon 2 (F157C). Lanes 1–4 show the normal conformation pattern of each exon. The amplified exons are indicated below each lane. AC, abnormal conformer.

(Table 1). The annealing and extension times were prolonged to one minute each, and the enzyme concentration was raised to 1.25 Units per 50-ml reaction. Combining the primer pairs, as shown in Table 1, made it possible to amplify all sixteen KVLQT1 PCR fragments in four PCR tubes. Detection of a donor splice site mutation, (IVS6 1 1 G → A) and a missense mutation (F157C) in two different LQTS patients by multiplex SSCP/ HD is demonstrated in Fig. 3A and B, respectively.

4. Discussion Our results confirm the corrected genomic structure of KVLQT1 [16], and as a consequence we have used the same exon-numbering in this work.

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The location of the previously reported KvLQT1 mutations between the transmembrane domains S2 and S6 suggests a functional significance of the pore region and these domains [25]. This is particularly evident with respect to the pore region and the central part of the membrane spanning region S6, since these domains seem to constitute a ‘hot spot’ for mutations in KvLQT1. Mutations in this part of the protein may interfere directly with the pore function. However, the preferred method for detection of point mutations in KVLQT1 [5] is limited to analysis of exon 3–8. This region covers only 30% and 37%, respectively, of the sequences coding for KvLQT1 isoforms 1 and 0, assumed to be the functionally translated isoforms in the heart [13,14]. Thus, this method may miss a number of LQTS associated mutations. We have developed a method for non-radioactive SSCP/ HD analysis of 15 exons in KVLQT1, using the intronic primers listed in Table 1. These 15 exons code for the ‘core’ protein of KVLQT1, and cover 79 and 94% of KvLQT1 isoforms 0 and 1, respectively. Mutations in exon 1a and 1d of isoform 0 and 1, respectively, will not be detected by this method, but primer sequences for amplification of exon 1d was described recently [16]. We believe that use of the method presented here will improve the genetic diagnosis of LQTS and subsequently lead to detection of new mutations of significance for the study of the function of KvLQT1. SSCP analysis is often the method of choice for detection of unknown point mutations in genomic DNA, due to the simplicity of the method. The major disadvantage of the method is a varying sensitivity, found to range from 67–97% [26] when analyzing DNA fragments ranging from 100–450 bp. As a rule, the sensitivity is inversely proportional to the fragment size, but factors such as gel composition, temperature and the base composition of the fragment may have a great influence on the sensitivity and should be optimised for each assay. The assay sensitivity was 100% when tested on 15 in vitro constructed mutants. We can not exclude that the assay would miss some mutations if the study was expanded to a larger number of mutated templates and more exons were included in the study. However, exon 7 was used as a model system, because it codes for the important pore region of KvLQT1, and seems to be a mutational ‘hot spot’. Furthermore, the method’s ability to distinguish between all three possible base substitutions in the same position indicate a high sensitivity. The high sensitivity demonstrated by the assay is most likely due to the fact that all of the PCR products are relatively short (140–254 bp) and all fragments were analysed at two temperatures. But heteroduplex formation during renaturation of some of the DNA before the fragments migrate into the gel, as seen in Fig. 2A, may also have a positive effect on the sensitivity. This renaturation combines two different mutation detection techniques (i.e. SSCP analysis and heteroduplex analysis) in one assay. Since these two techniques are based on

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very different mechanisms, we would expect them to work together in a complementary way, resulting in very high sensitivity, as described previously [27]. The method was used to analyse for KVLQT1 mutations in eight Danish families with inherited LQTS. The analysis revealed three novel mutations (Table 2), confirming the importance of KVLQT1 in LQTS. One of these mutations was located in exon 2, a part of the gene, where mutations have not previously been described. LQTS may be treated efficiently by either b-blockade alone, or in combination with insertion of pacemaker or perhaps gene-specific pharmacological treatment. Since the syndrome may have sudden cardiac death as the first clinical presentation, it may be of importance to diagnose asymptomatic cases, particularly as they may become symptomatic upon treatment with certain drugs [28]. The recent finding that 12 / 24 children dying from sudden infant death syndrome had prolonged QT [29] further stresses the need for efficient molecular diagnosis of LQTS. A further step would be to perform neonatal screening using blood samples already collected for PKU and TSH screening in new-borns. Attempts to perform screening among schoolchildren based on ECG have not been successful due to the inefficiency of ECG as a marker for LQTS, particularly in children [30]. Genetic screening for LQTS will require knowledge on the efficiency of available prophylaxis as well as the prognostic significance and frequency of mutations and will necessitate the use of efficient mutation screening procedures such as the multiplex SSCP/ HD method presented here. Multiplex SSCP/ HD was optimised for amplification of all sixteen KVLQT1 fragments in four PCR reactions (Fig. 3). One disadvantage with the multiplex SSCP/ HD assay is the theoretical possibility of a decrease in sensitivity. Since the number of bands in each lane increases dramatically, it is possible that normal conformers may mask an abnormal conformer, resulting in false negative results. Therefore, for diagnosing LQTS in a small number of patients, the single exon SSCP/ HD method is preferred, while the multiplex SSCP/ HD method may be preferred for large sample materials, such as in population screening, where a low cost is mandatory. The sensitivity of our multiplex SSCP/ HD assay remains to be examined on a large material of LQTS patients, but so far all the LQTS associated mutations and the normal variant listed in Table 2 were detectable in the multiplexed setup.

Acknowledgements We would like to thank Jette Rasmussen and Mads Dahm Johansen for excellent technical assistance. The work was supported by the Danish Heart Foundation (grant no. 97-2-4-22-22525), The Novo Nordisk Foundation, Kong

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Christian den Tiendes Fond and the Research Center for Medical Biotechnology under the Danish biotechnological recearch and development programme.

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