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The Wellcome Trust Centre for Human Genetics, University of. Oxford, Windmill Road, Headington, Oxford OX3 7BN, UK. P. Szepetowski (И). INSERM U491.
Neurogenetics (1999) 2 : 115–120 DOI 10.007/s100489900066

Q Springer-Verlag 1999

Original article

Electronic identification and chromosomal assignment by radiation hybrid mapping of human expressed sequence tags corresponding to new potassium channel genes Pierre Szepetowski 7 Anthony P. Monaco

Received: February 17, 1999 / Accepted: February 22, 1999 / Published online: April 30, 1999

ABSTRACT Human homologues of 36 Caenorhabditis elegans potassium channels were identified by expressed sequence tag (EST) database searching. This approach was combined with radiation hybrid mapping to localize new potassium channel genes in the human genome. In addition, several ESTs whose location was already known were also identified as cDNAs encoding additional potassium channels. The identification and mapping of all these genes will make them useful tools for mutation detection in neurological as well as other human diseases. Key words Potassium channel genes 7 Human expressed sequence tags 7 Radiation hybrid mapping

INTRODUCTION Ion channels play critical roles in a broad range of physiological processes, including neuronal, muscular, cardiac, endocrine, and many other functions. Consequently, ion channels of many different types have been implicated in several human genetic disorders [1–3]. In particular, potassium channel genes are responsible for a large variety of human diseases, including benign neonatal familial convulsions [4–6], episodic ataxia with myokimia [7], the cardiac long-QT syndrome [8], familial persistent hyperinsulinemic hypo-

P. Szepetowski 7 A.P. Monaco The Wellcome Trust Centre for Human Genetics, University of Oxford, Windmill Road, Headington, Oxford OX3 7BN, UK P. Szepetowski (Y) INSERM U491 Faculté de Médecine de la Timone 27, bd Jean Moulin, 13358 Marseille Cedex 5, France Fax: 33-491-804-319 e-mail: [email protected] or [email protected]

glycemia of infancy [9], and the renal Bartter syndrome [10]. The systematic identification and mapping in the human genome of potassium channel genes is essential to link them with many other human diseases. In addition to human potassium channel genes that are already known and mapped (Table 1), it is highly likely that other as yet undiscovered homologous genes do exist in the human genome. Cross-species comparison is a powerful approach to identify new genes and elucidate their function [11–13]. While previous studies have been based either on the screening of cDNA or genomic libraries, or on polymerase chain reaction (PCR) with degenerate primers, the electronic search of expressed sequence tag (EST) databases from different model organisms has been successfully employed in recent years. For example, human cDNAs have been selected on the basis of their homologies to various Drosophila mutant genes [14, 15]. Two main advantages of such approaches are their low cost and their rapidity. Among model organisms, Caenorhabditis elegans represents a powerful source of information [16], especially since determination of its genomic sequence is soon to be achieved [17, 18]. Genes encoding potassium channels have been identified within the genome of C. elegans [19]. We have used the 36 C. elegans potassium channel genes (as indicated at the Sanger Centre database) as tools to identify homologous human transcribed sequences in the EST division (dbEST) of Genbank [20], and have employed electronic and radiation hybrid mapping to localize these clones in the human genome.

MATERIALS AND METHODS Bioinformatics C. elegans potassium channel genes were retrieved from the Sanger Centre database (http://www.sanger.ac.uk/Projects/C Elegans/wormpep fetch.shtml). Human homologous ESTs (KESTs: K channel ESTs) were identified by screening the dbEST data-

Known gene Unigene

Know location

KCNN1

hsu69184-r35526 aa286692-aa315333 hsc2zg032-hsc2zh042-r45192 aa115317-aa380379-aa429995aa470390-aa507024-aa758177 aa349999-h10765-h12995h19104-n57588-r22676 aa195883-hsu55984

HUMAC

Hs26662

Hs13982 Hs22834 Hs95673

18q12

13q14

12q13 14q22 2q37 16p13

8q21 14q23

8q24 2p24

KCNQ3 KCNS3

Hs22675 Hs27043

1q21 19q13 11p15 20q13

KCNN3 KCNN4 KCNQ1 KCNQ2

KCNS1

1q42-q43

KCNK1

Step 3: already mapped (28 KESTs) h19264-hsu69192-r19352-r34920 hsc0bf081-hsc0bf091-hsu69185

Other KESTs (47):

Step 2: unmapped (15 KESTs) h18119-h18164-h18261-h20365h43697-h49525-h49759-h51390h51391-h85454-h85737-h99478 aa594089-hsc2vh041-m62043

aa001030-aa021294-aa056966aa257164-aa307980-aa308453hsc3ah031-n78391-t74333 aa490752-aa767647 aa122017-t24528 aa177094-w93500 aa091374-aa812513-h23701h23702-h51419-h51461-t03719t15380 aa001392-h08544-r36327-t178692 aa627564

Step 1: already mapped (63 KESTs) aa297822-h22867 AF033382 2p25 K channel aa193556-aa297745 AF033383 Hs122761 20q13 K Channel aa076910-aa247558-aa297037HERG 7q35-q36 h17705-r55596 aa018118-aa018120-aa018214KCNA1 12p13 aa057369 aa220960 KCNA5 12p13 aa166676-aa349883-2106218 KCNB1 20q13 h19662 KCNC1 11p15 aa400462-aa400737-aa442709KCNC4 1p21 aa7818-aa860738 t94029 KCNJ15 21q22 aa247334 KCNJ3 2q24 h23948-h29824-h29912-h41402KCNJ4 22q13 m78731 aa40299-aa400268-aa401506KCNJ8 12p11 w05428 h45962 KCNJ9 1q21-q23

Confirmed K channel genes (78 KESTs)

KEST

Table 1

WI-5815CHLC.GATA27E03/3.36 cR from WI-5815

D20S169CHLC.GATA47F05/3.36 cR from D20S169 ND

AFMA323ZE5-D1S2635/ 1.61 cR from AFM323ZE5

RH mapping (interval/ location)

14q23

20q12-q13

1q21

CCL

DRES61

DRES59 DRES62control

Control

aagatgatgaccacacatgg

ccggtcgcctcttctgtgtt

ggagtagctgagaggaagat

Comment Forward primer

gtgcatgcagtcaactgcat

agttccaccctcagtcacc

acttgtcactgccctgtttc

Reverse primer

5.5e-07 (R186.5) 6.1e-05 5.1e-13 5.7e-29 6.2e-32

2.4e-07 (R03E9.4) 6.1e-07 (M02A10.2) 2.3e-16 (R03E9.4) 7.8e-14 (M02A10.2) 1.1e-41 (M02A10.2) 1.1e-06 (K04G11.5) 8.3e-33 (M02A10.2) 1.1e-08 (K04G11.5)

2 3 6 6 5 3 2 3 3

4

1

1 2 1 1

8 1

5

7

1 2

2 2 7 5

19

3

6.8e-58 (C30D11.1)

4

0.024 (F14F11.1) 5.7e-05 (ZK1321.2) 8.8e-11 (C25B8.1) 0.04 (C32C4.1)

0.01 (ZK1321.2)

0.037 (C40C9.1)

(C03F11.1) (T02E1.8) (C25B8.1) (C25B8.1)

7.0e-05 (R07A4.1)

3.4e-07 (C52B11.2)

0.0048 (C30D11.1) 2.5e-05 (C52B11.2) 3.2e-13 (C52B11.2) 1.9e-43 (C52B11.2)

1.5e-35 (C32C4.1) 1.9e-38 (C30D11.1)

1.8e-30 (T02E1.8)

8.4e-13 (ZK1321.2)

1.0e-18 (C25B8.1) 0.00013 (ZK1321.2)

3.3e-06 3.8e-08 4.1e-47 1.3e-30

1.1e-10 (C40C9.1)

0.032 (F14F11.1)

0.0013 (C52B11.2)

0.044 (C30D11.1) 0.0055 (R07A4.1) 3.0e-12 (C52B11.2) 1.7e-09 (C52B11.2)

0.016 (C40C9.1) 2.0e-17 (C30D11.1)

0.048 (R07A4.1)

0.05 (R186.5)

8.7e-09 (C25B8.1) 0.003 (R07A4.1)

0.01 (C03F11.1) 0.05 (C03F11.1) 0.019 (ZK1321.2) 0.026 (ZK1321.2)

0.027 (M04B2.5)

8.3e-13 (M02A10.2) 0.00044 (K04G10.5)

(R186.5) (F14F11.1) (R186.5) (F14F11.1)

4.3e-09 (ZK1321.2)

8

0.033 (F22B7.7)

5.9e-08 (C32C4.1)

0.011 (R186.5)

WSEV

5

NOSM BSEV

116

10q21-q22 5q34 8q22 Hs.107245 1p13-q12 Hs.121498 8q21

21q22 11q24 17q21 11q24 21q22 1q22-q23 11p15 17p11 21q22 17p11 1q41 2p23

KCNE1 KCNJ1 KCNJ2 KCNJ5 KCNJ6 KCNJ10 KCNJ11 KCNJ12 KCNJ14 KCNJN1 KCNK2 KCNK3

KCNMA1 KCNMB1 KCNS2

3q26 12p13 1p36 1p13 11p14 11q14 12p13 19q13 1p13 19q13 19q13 Xp11

KCNA1B KCNA2 KCNA2B KCNA3 KCNA4 KCNA4L KCNA6 KCNA7 KCNA10 KCNC2 KCNC3 KCND1

Appendix: other mapped potassium channel genes

hsc0sc112

h96170

h49142

Hs27212

Hs13285

h11972-hsc3be091-t74525

h49065-h51137

Hs122648

Hs21323

Hs11364Hs42448

Hs118695

aa777982

aa604914

aa533124

aa504857-w25800

aa325048 aa464375

aa243775-aa604625-h97186

Step 4: unmapped (19 KESTs) aa148718

D2S174-WI-4431/1.82 cR from D2S174

WI-5587-WI-5717/21.82 cR from WI-5587

GCT10F11-NIB1054/ 12.33 cR from GCT10F11 CHCL.GATA12H10.101 4-D2S331/12.33 cR from CHLC.GATA12H10.101 4 ND WI-5105CHLC.GATA87F04/ P0.00 cR from WI-5105 WI-7903-WI-9028/8.01 cR from WI-7903 WI-4142-WI-4822/4.71 cR from WI-4142 Tel-NIB1364/P14.39 cR from NIB1364 WI-1409-D11S913/5.87 cR from WI-1409 AFM200VC7-WI6801/ 1.71 cR from AFM200VC7 WI-3706CHLC.GATA26D02/3.15 cR from WI-3706 WI-3706CHLC.GATA26D02/3.0t cR from WI-3706 WI-6442-Tel/31.20 cR from WI-6442 D2S354-D2S111/1.31 cR from D2S354

2p23

Xp11

2q24

17q25

12q13

12q13

8q24

11q13

Control

Control

acccagcagtccctaaagct

gactgatggaaactacatct

caagctggtgattccggct

gggcaagtccagtgcagacg

gtttgggggtacatatcccc

tccttcgaggaggagctggcc

cttctcccaaggaccttgtc

gctaagcagaagtagacgg

atcaccaccgtgggctatgg

ccggtcgcctcttctgtgtt

6q21 1p36

gccatgaccacacagtgtcg

atctctacaaagaaagcgtg

cagtcctgtttcagagatct

gctgggcatgttattcgcat

19q13

1q41

2q33-q37

20q13

accattctgcagactgacag

ttcttctccctcttgggtga

tcgctttcaccaaatccca

ttggctgccaggagctgtcg

tgggagctcaacctaacgac

tcctcgatgcccagcctctt

aggtctatgatggtttggcac

ccattgcctgctgcctgtgc

cagcagcatggtggtcggca

agttccacccctcagtcacc

actagacttgaggctgtggg

taaggatgtgttggcttggg

gacaagactgtgtccctgaatt

cattcaattgcagacgcct

1

1

6

5

7

5

8

17

3

1 20

2

8

3.9e-30 (C30D11.1) 0.0025 (K06B4.12)

0.02 (R02E9.4)

0.033 (F22B7.7)

6.4e-11 (C25B8.1)

0.015 (C32C4.1)

0.001 (R07A4.1)

0.037 (M04B2.5)

0.015 (F29F11.4)

0.0069 (F29F11.4)

3.8e-23 (C30D11.1)

3.8e-23 (C30D11.1)

1.1e-14 (C30.D11.1) 1.1e-14 (C30D11.1)

2.0e-27 (R186.5)

1.2e-27 (F14F11.1)

4.2e-18 (C30D11.1)

5.4e-05 (C40C9.1)

1.4e-05 (C40C9.1)

4.8e-10 (R04F11.4)

5.0e-07 (M02A10.2) 0.0011 (K04G11.5)

3.9e-30 (C30D11.1) 5.4e-14 (C40C9.1)

6.6e-07 (R03E9.4)

1.4e-08 (F14F11.1)

117

118 base (http://www.ncbi.nlm.nih.gov/dbEST/) with each C. elegans amino acid sequence, using the TBLASTN program ([21]; http:/ /www.ncbi.nlm.nih.gov/cgi-bin/BLAST/). ESTs displaying E values ~0.05 were selected. Identification of KESTs corresponding to previously known genes was performed by screening all nonredundant GenBankcEMBLcDDBJcPDB sequences. Overlapping KESTs were detected with the UniGene database at NCBI (http://www.ncbi.nlm.nih.gov/Schuler/UniGene/) and the dbEST database, using the BLASTN program. Locations of known genes and ESTs were retrieved in various databases, including the Genome Data Base (GDB: http://gdbwww.gdb.org/), Medline (http://www.ncbi.nlm.nih.gov/PubMed/), Genbank (http://www2.ncbi.nlm.nih.gov/genbank/query form.html), Unigene, GeneMap 98 (http://www.ncbi.nlm.nih.gov/genemap98/), and the Whitehead Institute/MIT server (http://carbon.wi.mit.edu:8000/cgi-bin/contig/phys map).

Radiation hybrid mapping Primers were generated from each uni-KEST clone. Whenever possible, primers were designed from the 3’ ends, which in most cases should correspond to the 3’ untranslated regions. This minimized the chances of high homology to corresponding hamster and human genes, as well as the possibility that the sequence cannot be amplified because of the presence of a large intron in between the two primers. Primers were systematically submitted to a BLASTN search, in order to eliminate those conserved in other potassium channel genes or KESTs sequences. The Genebridge 4 whole-genome radiation hybrid panel (purchased from Research Genetics) was screened with selected KESTs. All PCRs were performed using standard conditions. Briefly, 25 ng of DNA for each of the 93 hybrid clones, plus human and hamster genomic DNA controls, were used for PCR amplification in 96 well microtiter plates. Each PCR was performed in a 15-ml volume, with 25 ng of each primer, 1–3 mM MgCl2, 200 mM of each nucleotide, and 0.2 units of Taq polymerase. PCR results were submitted to the Radiation Hybrid Mapping Server at the Whitehead Institute (http://carbon.wi.mit.edu:8000/cgi-bin/contig/ rhmapper.pl). A cut-off of 15 was chosen for LOD scores.

RESULTS AND DISCUSSION Electronic screening The dbEST database [20] was screened with 36 amino acid sequences corresponding to the C. elegans potassium channels as indicated at Sanger Centre, using the TBLASTN program [21]. In total, 125 clones (which were called KESTs, for K channel ESTs) displaying significant homologies were selected for further analysis (Table 1). A low-significance cut-off (E value ~0.05) was chosen since it rapidly appeared that several human clones, although displaying very low significant homology, were actual potassium channel genes in human. For example, KEST AA767647, which is the KCNN3 potassium channel gene, was selected with 1 single C. elegans clone (C03F11.1) with statistical significance (E value) at 0.01 only. AA604625 (actually the KCNJ13 potassium channel gene) was only found with 2 C. elegans clones, R03F9.4 and M02A10.2, with E values at 0.02 and 0.0064, respectively. Similarly, AA018214 (KCNA1) matched ZK1321.2 with Ep0.01, and R35526, which is 85% homologous to the rat potassium channel ELK2 at the nucleotide level, was se-

lected by one single clone (C30D11.1), with E at 0.044. Two main steps of subsequent electronic screening were used. First, 63 KESTs, corresponding to 20 potassium channel genes that had been previously identified and mapped, were identified and eliminated by submitting all KESTs to a BLASTN search of the non-redundant sequence databases (Table 1, step 1). In some cases, sequences differed from known human genes because of a few undetermined nucleotides, leading to an incomplete albeit very high homology. Second, ESTs were submitted to a systematic search in the Unigene database at NCBI. Consequently, sets of KESTs (uniKESTs) were found that corresponded to the same cDNA clone, thus decreasing the number of KESTs that were analyzed further. Moreover, KESTs were systematically submitted to a BLASTN search of the dbEST database in order to detect overlapping KESTs that were not contained within the Unigene database. At this stage, 47 KESTs, corresponding to 21 uniKESTs, could be defined that did not correspond to any known potassium channel gene, and were used in subsequent analyses. In addition, 15 other KESTs did correspond to two known but unmapped potassium channel genes (KCNN1 and KCNS1) and were also used for radiation hybrid mapping (Table 1, step 2). Electronic mapping The next stage was to look in various databases for those uni-KESTs that had previously been mapped on the human genome. Eight uni-KESTs whose location was already known could be found (Table 1, step 3), among which three (corresponding to DRES 59, 61, and 62) had been identified in a previous study on the basis of their homology to various Drosophila potassium channel genes [14]. This, in addition to the detection of known potassium channel genes, clearly showed that the strategy used is reliable and reproducible. Although mapping data were already available, this result provided interesting new information, since these cDNAs can now be considered as new putative potassium channel genes. Radiation hybrid mapping Thirteen uni-KESTs (Table 1, step 4) and two known potassium channel genes (KCNN1 and KCNS1) remained unmapped. Radiation hybrid mapping of these clones was performed in order to evaluate their possible role in previously mapped human disorders. The Genebridge 4 panel (Research Genetics), which is composed of 93 human/hamster clones, was screened by PCR. A unique PCR product could be obtained from 14 of the 15 clones. In 1 case (AA325048), no product could be seen when using human DNA from the radiation hybrid panel, even when different sets of primers

119

were designed. Since a clear PCR product was obtained with human DNAs from healthy individuals, the failure of the PCR was probably due to mutation of this EST in this particular cell line, or to rearranged or incomplete panel. For KCNN1, strong homology with hamster DNA impeded successful mapping. Positive results of PCR analyses were submitted to the Radiation Hybrid Mapping Server at the Whitehead Institute. In total, 12 uni-KESTs and 1 known potassium channel gene (KCNS1) were successfully mapped (Table 1). Of course not all KESTs identified may encode true potassium channels. However, 78 of the 125 KESTs were known potassium channel genes, while none corresponded to another type of gene. Moreover, confirmation that several KESTs were true potassium channel genes has been obtained during the course of this study: three KESTs (AA243775, AA604625, and H97186) correspond to the potassium channel gene KCNJ13 that has been identified very recently [22], while 12 KESTs are actually the KCNS1 gene [23]. In addition, a new gene, TASK-2, has just been published [24], and corresponds to KEST AA533124. In all three cases, locations are now known and are consistent with our radiation hybrid mapping data. To ensure that our mapping data were reliable, several control experiments were performed by mapping genes whose location had already been determined, and in each case our results confirmed previous data (Table 1). Among all potassium channel genes that were known and mapped, 22 could be detected (Table 1, steps 1c2), while 29 were not (Table 1, appendix), among which 5 had no sequence available in databases at the time the analysis was performed. This could indicate that many putative KESTs were not identified. There are several possible explanations for this: not all potassium channel genes are identified within the genome of C. elegans; some families of potassium channel genes probably have appeared in more-recent species; ESTs that are specific to one given gene are too divergent from the corresponding C. elegans sequence, or correspond to untranslated regions; ESTs have been added to databases after we performed the analysis. To further address this issue, we systematically searched ESTs in the dbEST database with the nucleotide sequences of the 24 known human potassium channel genes that had not been detected with the C. elegans potassium channels, using the BLASTN program. Interestingly, most of these genes (KCNA2,3,4,6,10, KCNC3, KCNE1, KCNJ1,2,5,6,11,12, KCNJN1, and Hs121498) did not have any corresponding EST in the dbEST database. It is thus not surprising that they could not be detected. In the end, 9 (approximately 18%) of the 51 previously known and mapped potassium channel genes (KCNA1B, KCNA2B, KCND1, KCNJ10, KCNK3, KCNMA1, KCNMB1, KCNN3, and Hs.107245) could not be identified, although corresponding ESTs did exist. With respect to the 21 new putative potassium channel genes that were identified (Table 1, steps 3c4), we should have missed about 4

(18%) putative potassium channel genes contained as ESTs in dbEST. All KESTs that were mapped in this study obviously represent new candidate genes for diseases located in the corresponding genomic regions. Ion channel genes are good candidates for the epilepsies [25]: 6p21 (KEST AA533124) is where linkage for juvenile myoclonic epilepsy had been found; another clone (KESTs H11972, HSC3BE091, and T74525) maps at 8q24, where one susceptibility gene for idiopathic generalized epilepsies is located; region 19q13 (KESTs AA504857 and W25800) contains one gene for familial infantile convulsions. Region 2q33-q37, which hosts KESTs AA243647, AA604625, and H97186 (all corresponding to KCNJ13) contains a gene for familial paroxysmal dyskinesia [26, 27]. 20q13 (KEST AA148718 and KCNS1) hosts a gene contributing to body fat and insulin [28]. Our results thus provide important tools to investigate the putative role of potassium channel genes in a large variety of human diseases.

Concluding remarks Tentative efforts to exhaustively map genes on the human genome are in progress. A large and increasing number of known potassium channel genes have already been identified and mapped in human (Table 1). For example, when screening the Genome Data Base with “channel” as a key word, a large number of potassium channel genes can be directly identified. In most cases, mapping information is directly available. When ambiguous or absent, mapping data can also be retrieved in other databases. Deloukas et al. [29] recently have mapped about 30,000 human genes, among which some also encode potassium channels (see GeneMap 98, at NCBI). The fact that many other new putative potassium channel genes could be identified and mapped in the present study emphasizes the need for more-specific studies devoted to particular classes of genes. The strategy used should apply to any other gene family, provided that it exists in a model organism whose genome is being sequenced. Acknowledgements This work was funded by a CEC Marie Curie fellowship to P.S. and the Wellcome Trust. A.P.M. is a Wellcome Trust Principal Research Fellow.

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Note added in proof The genome sequence of C. elegans is essentially complete (The C. elegans Sequencing Consortium, Science 1998, vol.282:20122018). As expected, it appears that there are more than 36 potassium channel genes within the genome of C. elegans (Bargmann, Science 1998, vol.282:2028-2033), a fact that was discussed in the present article. Although the use of most of these additional C. elegans genes would probably lead to detection of the same human KESTs that were identified in this study (for example, about 50 predicted C. elegans potassium channel genes belong to one single family – the two pore/TWIK channels), it will be useful to perform the same kind of analysis with these channels when sequences are available.