European Journal of Human Genetics (2002) 10, 36 ± 43 ã 2002 Nature Publishing Group All rights reserved 1018-4813/02 $25.00 www.nature.com/ejhg ARTICLE Characterisation of the human voltage-gated potassium channel gene, KCNA7,acandidategenefor inherited cardiac disorders, and its exclusion as cause of progressive familial heart block I (PFHBI) Soraya Bardien-Kruger1,4, Heike Wulff2, Zainu Arieff1,5, Paul Brink3, K George Chandy2 and Valerie Corfield*,1 1University of Stellenbosch/Medical Research Council Centre for Molecular and Cellular Biology, Department of Medical Physiology and Biochemistry, University of Stellenbosch Medical School, Tygerberg, South Africa; 2Department of Physiology and Biophysics, Medical School, University of California Irvine, Irvine, USA; 3Department of Internal Medicine, University of Stellenbosch Medical School, Tygerberg, South Africa Mutations in genes encoding cardiac ion channels and their subunits are responsible for several genetic cardiac disorders. We characterised the human gene KCNA7, encoding the voltage-gated potassium channel Kv1.7 and compared its coding sequence with that of the mouse orthologue, kcna7. Both genes are encoded by two exons separated by a conserved intron, unlike all the other Kv1-family genes that contain intronless coding regions. KCNA7 and kcna7 encode proteins of 456 amino acid residues that share 495% sequence identity, and the mouse channel has biophysical and pharmacological properties closely resembling the ultra- rapidly activating delayed rectifier (IKur) in cardiac tissue. Using reverse transcriptase-PCR, KCNA7 mRNA was detected in adult human heart. We determined that KCNA7 resides on chromosome 19q13.3 in a region that also contains the progressive familial heart block I (PFHBI) locus. Direct sequencing of KCNA7's coding sequence in PFHB1-affected individuals revealed no pathogenic sequence changes, but two single nucleotide polymorphisms detected in exon 2 result in amino acid substitutions. These results provide evidence for the exclusion of this candidate as the PFHB1-causative gene, although mutations in regulatory and non-coding regions cannot be excluded. As ion channel-encoding genes have been implicated in a growing number of genetic conditions, the data presented may facilitate further analysis of the role of KCNA7 and its product in the heart. European Journal of Human Genetics (2002) 10, 36 ± 43. DOI: 10.1038/sj/ejhg/5200739 Keywords: KCNA7; Kv1.7; kcna7; potassium channels; PFHB1; inherited cardiac conduction disorders; IKur Introduction Ion channel defects are implicated in a number of inherited cardiac diseases, many of which are characterised by sudden death and associated with conduction abnormalities. The *Correspondence: V Corfield, US/MRC Centre for Molecular and Cellular Long QTsyndromes (LQTS) are associated with mutations in at Biology, Department of Medical Physiology and Biochemistry, PO Box least five genes, four of which encode potassium (K+) channels. 19063, Tygerberg 7505, Republic of South Africa. Tel: +27-21-938-9405; + Fax: +27-21-931-7810; E-mail: [email protected] These K channel genes act as two paired sets, namely KCNQ1/ Current addresses: 4South African National Bioinformatics Institute, KCNE1 and KCNH2/KCNE1, with the products of each pair co- 5 University of Western Cape, Bellville, South Africa; Department of assembling to form the ion channels responsible for the slowly Biochemistry, University of Western Cape, Bellville, South Africa Received 1 May 2001; revised 18 October 2001; accepted 18 October activating potassium current (IKs) and the rapidly activating 1 2001 delayed rectifier potassium current IKr, respectively. Interest- KCNA7 and cardiac conduction disorder, PFHB1 S Bardien-Kruger et al 37 ingly, the fifth LQTS-causative gene, SCN5A1 has also been regions of KCNA7 for the presence of triplet repeat motifs implicated in three distinct cardiac diseases: Brugada syn- which might be pathologically expanded in PFHB1-affected drome, in which patients have marked abnormalities in family members displaying genetic anticipation. ventricular conduction,1 progressive cardiac conduction defect (PCCD);2 and an isolated cardiac conduction disease.3 Progressive familial heart block type I (PFHBI) is an Materials and methods autosomal dominantly inherited cardiac conduction dis- All procedures used were reviewed and approved by the order, which has been mapped to chromosome 19q13.3 in a University of Stellenbosch's Ethics Committee (reference 86/ large South African Afrikaner family.4 Clinically, PFHBI is 085). The clinical details and pedigrees of the Afrikaner families characterised by right bundle branch block, left anterior in which PFHBI segregates were described previously.4,5 hemiblock, or complete heart block with broad QRS complexes.5 Evidence of genetic anticipation in this disorder, Characterisation of KCNA7 with an increase in severity of the disease's presentation in Determining coding sequence A BLAST search of the succeeding generations, has been reported.5 A second cardiac Genoscope database (http://www.genoscope.org) revealed conduction disorder, designated isolated cardiac conduction several 19q13.3 human genomic sequences (eg, disorder (ICCD), showing similar clinical features to PFHBI, R0AA003ZF08G1) that aligned with the published mouse has been mapped to the same region of chromosome 19q13.3 kcna7 cDNA sequence (AF032099). Subsequent BLAST in a Lebanese family.6 Although PFHBI and ICCD exhibit searches of GenBank with this inferred human KCNA7 phenotypic similarities and are linked to the same micro- sequence identified significant matches with human BAC satellite markers on chromosome 19q13.3, it is not known if clone BC52309 (synonym: CITB-60B18; GenBank accession they are the same clinical entity. Recent studies from our no. AC008687), mouse genomic contig (AC073711), as well group using a number of microsatellite markers have refined as three mouse placental expressed sequence tags, ESTs the PFHB1 locus to a 4 cM region on 19q13.3 between (AA021711, AI322534, AI324179). The coding region of markers D19S606 and D19S866 (unpublished data). KCNA7 was deduced by aligning the mouse cDNA sequence A plausible candidate gene for PFHBI and ICCD by mapped with the human BAC clone and the three mouse EST position, expression profile and function is the voltage-gated sequences retrieved from the databases. Based on this K+ channel gene7 designated hKv1.7 in the functional determined coding sequence of KCNA7, three overlapping classification and KCNA7 in the HUGO classification (by sets of PCR primers were designed to span exon 1 and four which it will be referred to in the text). This member of a sets were designed to span the larger exon 2 (Table 1). These multigene family8 has been mapped by fluorescent in situ seven primer sets were used in direct DNA sequencing to hybridisation (FISH) to human chromosome 19q13.3, 7 confirm the sequence deduced by the in silico techniques. placing it within the PFHBI locus, where it is flanked by the muscle glycogen synthase gene (GSY1) and histidine-rich Electrophysiology of kcna7 Since the published mouse calcium binding protein (HRC).4 The mouse homologue, cDNA sequence was shown to contain an error (see Results), kcna7, a member of the Shaker-related Kv1-subfamily of K+ the corrected mouse kcna7 sequence was generated by PCR channels, is abundantly expressed in mouse heart,7 strength- from the original published construct and then cloned in- ening the candidacy of the human homologue KCNA7 as the frame downstream to enhanced green fluorescent protein in PFHBI-causative gene. the eGFP-C3 vector. COS-7 cells were transiently transfected Consequently, we determined the complete coding se- with this construct using FuGeneTM 6 (Roche) according to quence of KCNA7, defined its genomic organisation, demon- the manufacturer's protocol and kcna7 currents were strated its presence in human heart, refined its chromosomal recorded 6 ± 12 hours after transfection. COS-7 cells were location relative to the PFHBI locus and screened PFHBI- maintained in Dulbecco's modified Eagle's medium (DMEM) affected family members for mutations. Moreover, we containing 10% heat-inactivated foetal calf serum (Summit conducted an in silico search in the 5' and 3'-untranslated Biotechnology, Fort Collins, CO, USA), 4 mML-glutamine Table 1 Primer sets A ± G spanning the two exons of KCNA7 Exon: Set Forward primer (5' ±3') Reverse primer (5' ±3') PCR product (bp) 1 : A ACA CGT CGG TTC GCG GGT CG AGT AGA GCA CGG CGT CGA AG 248 1 : B CCG CGA GTA TTT CTT CGA CC TCT CGG GAA ACT CGA AAA GC 253 1 : C TGG CAC GCC TGC GCG AGG AC CGC CCA TCT CCT CCC AAA CC 297 2 : D ATG TGT AAA GAG CTG GG AAG TAG GGA AGG ATA GCC AC 303 2 : E GTC CAA GCA AGG CTA TCT TC AGA GGA CCA CAC CGA TGA AG 284 2 : F AAT CTT GGG CCA GAC GCT TC AAT TGG AGA CAA TGA CGG GC 292 2 : G AAG ATA GTG GGC TCT CTG TG TCT AGG GAG GTG TGA GGT CC 296 European Journal of Human Genetics KCNA7 and cardiac conduction disorder, PFHB1 SBardien-Krugeret al 38 a European Journal of Human Genetics KCNA7 and cardiac conduction disorder, PFHB1 S Bardien-Kruger et al 39 + and 1 mM Na pyruvate. All experiments were carried out in PCR was performed using the KCNA7-specific primers in the whole-cell configuration of the patch-clamp technique Table 1 and the products were separated by electrophor- with a holding potential of 780 mV. Currents were recorded esis on a 12% polyacrylamide gel and subsequently silver- in normal Ringer solution (160 mM NaCl, 4.5 mM KCl, 2 mM stained. CaCl2,1mM MgCl2,10mM HEPES, pH 7.4, 290 ± 310 mOsm) with an internal pipette solution containing 134 mM KF, In silico searches Bioinformatic-based in silico mapping 2mM MgCl2,10mM HEPES, 10 mM EGTA (pH 7.2, 290 ± using the EST2GENOME software (http://ftp.sanger.ac.uk/ 310 mOsm). 200-ms depolarising pulses to 40 mV were pub/EMBOSS) was used to position KCNA7 relative to applied every 30 s and Kd-values were determined by fitting microsatellite markers and to other genes of interest on the Hill equation to the reduction of peak current; for chromosome 19q.