A potassium channel mutation in neonatal human epilepsy

Science

Volumn 279, Issue 5349, 1998, Pages 403-406

  Biervert, Christian ^a   Schroeder, Björn C ^b   Kubisch, Christian ^b   Berkovic, Samuel F ^c   Propping, Peter ^a   Jentsch, Thomas J ^b   Steinlein, Ortrud K ^a  

^a UNIVERSITY OF BONN   (Germany)

^b UNIVERSITY OF HAMBURG   (Germany)

^c UNIVERSITY OF MELBOURNE   (Australia)

Author keywords

[No Author keywords available]

Indexed keywords

POTASSIUM CHANNEL;

AMINO ACID SEQUENCE; ARTICLE; CHROMOSOME 20Q; CHROMOSOME 8Q; DEPOLARIZATION; EPILEPSY; GENE LOCUS; MUTATION; PRIORITY JOURNAL; REPOLARIZATION; XENOPUS LAEVIS;

ACTION POTENTIALS; AMINO ACID SEQUENCE; ANIMALS; BRAIN; CHROMOSOME MAPPING; CHROMOSOMES, HUMAN, PAIR 20; CLONING, MOLECULAR; EPILEPSY; FEMALE; FRAMESHIFT MUTATION; HUMANS; INFANT, NEWBORN; KCNQ2 POTASSIUM CHANNEL; MALE; MOLECULAR SEQUENCE DATA; MUTAGENESIS, INSERTIONAL; OOCYTES; OPEN READING FRAMES; PEDIGREE; POTASSIUM; POTASSIUM CHANNELS; POTASSIUM CHANNELS, VOLTAGE-GATED; XENOPUS LAEVIS;

EID: 0032536030     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5349.403     Document Type: Article

References (36)

1. S. F. Berkovic and I. E. Scheffer, Brain Dev. 19, 13 (1997).
2. G. M. Ronen, T. O. Rosales, M. Connolly, V. E. Anderson, M. Leppert, Neurology 43, 1355 (1993).
3. M. Leppert et al., Nature 337, 647 (1989).
4. T. B. Lewis, R. J. Leach, K. Ward, P. O'Connell, S. G. Ryan, Am. J. Hum. Genet. 53, 670 (1993).
5. O. Steinlein et al., Genomics 22, 493 (1994).
6. A human fetal brain cDNA library (Clontech) was screened with the insert of cosmid C19-2 (5), and a single clone was isolated (insert size: 3.4 kb). It was extended in seven consecutive steps with gene-specific primers, using the 5′ RACE kit (Life Technologies) with adult human brain cDNA. Sequences were validated by sequencing independent RACE clones. The resulting composite sequence differed at the 3′ end from HNSPC, a previously reported partial cDNA (8). This may be due to an unspliced intron at the 3′ end of the HNSPC clone. Using an appropriate 5′ primer and a 3′ primer after the stop codon, we amplified a full-length cDNA from a human fetal brain cDNA library (Clontech) and subcloned it into the expression vector PTLN (28). The cDNA sequence of KCNQ2 has been deposited in the European Molecular Biology Laboratory-Gen-Bank database (accession number Y15065).
7. Q. Wang et al., Nature Genet. 12, 17 (1996).
8. M. Yokoyama, Y. Nishi, J. Yoshii, K. Okubo, K. Matsubara, DNA Res. 3, 311 (1996). The partial cDNA was called HNSPC (for human neuron-specific potassium channel).
9. KVLQT1 will be renamed to KCNQ1 by the HUGO/ GDB Nomenclature Committee.
10. B. C. Schroeder, C. Kubisch, T. J. Jentsch, unpublished results.
11. L. Y. Jan and Y. N. Jan, Nature 371, 119 (1994).
12. L. J. Ptacek, Neuromuscular Disord. 7, 250 (1997).
13. We obtained partial genomic structure of the KCNQ2 gene by amplifying human genomic DNA with gene-specific primers and sequencing the resulting polymerase chain reaction (PCR) fragments with an ABI 377 DNA sequencer. We were guided by the partial genomic structure of KVLQT1 (29), which is largely conserved in KCNQ2 (Fig. 1B). We then designed intronic primers to amplify single exons from genomic DNA. The primers used to amplify the mutant exon in the BFNC pedigree are 5′-GCAGACAAGAGGGGCAAGTCCAGC-3′ (forward) and 5′-CTGCACTCCCACCATGGGCCACAG-3′ (reverse).
14. Using intronic primers, single exons were amplified from genomic DNA by PCR. These were either sequenced directly with an ABI 377 sequencer or were first checked by single-strand conformational analysis and heteroduplex analysis as described (30).
15. S. F. Berkovic et al., Arch. Neurol. 51, 1125 (1994).
16. 2, and 5 mM HEPES (pH 7.4). For ion substitution experiments, NaCl and KCl were partially substituted by equivalent amounts of KCl (for NaCl), RbCl, or CsCl. Expression studies were performed with the variant lacking the additional exon (Fig. 1B). KCNQ2 that had this additional exon gave similar currents.
17. B. Barhanin et al., Nature 384, 78 (1996).
18. M. C. Sanguinetti et al., ibid., p. 80.
19. B. Wollnik et al., Hum. Mol. Genet. 6, 1943 (1997).
20. T. Takumi, H. Ohkubo, S. Nakanishi, Science 242, 1042 (1988).
21. I. Splawski, M. Tristan-Firouzi, M. H. Lehmann, M. C. Sanguinetti, M. T. Keating, Nature Genet. 17, 338 (1997); C. Rubie et al., ibid., p. 267.
22. I. Splawski, M. Tristan-Firouzi, M. H. Lehmann, M. C. Sanguinetti, M. T. Keating, Nature Genet. 17, 338 (1997); C. Rubie et al., ibid., p. 267.
23. B. C. Schroeder, C. Kubisch, T. J. Jentsch, unpublished observations.
24. C. Chouabe et al., EMBO J. 16, 5472 (1997).
25. N. Neyroud et al., Nature Genet. 15, 186 (1997).
26. D. L. Browne et al., ibid. 8, 136 (1994); E. R. P. Brunt and T. W. van Weerden, Brain 113, 1361 (1990).
27. D. L. Browne et al., ibid. 8, 136 (1994); E. R. P. Brunt and T. W. van Weerden, Brain 113, 1361 (1990).
28. O. Steinlein et al., Nature Genet. 11, 201 (1995).
29. C. F. Fletcher et al., Cell 87, 607 (1996); D. L. Burgess, J. M. Jones, M. H. Meisler, J. L. Noebels, ibid. 88, 385 (1997).
30. C. F. Fletcher et al., Cell 87, 607 (1996); D. L. Burgess, J. M. Jones, M. H. Meisler, J. L. Noebels, ibid. 88, 385 (1997).
31. C. Lorenz, M. Pusch, T. J. Jentsch, Proc. Natl. Acad. Sci. U.SA. 93, 13362 (1996).
32. M. P. Lee, R. J. Hu, L. A. Johnson, A. P. Feinberg, Nature Genet. 15, 181 (1997).
33. O. Steinlein et al., Hum. Mol. Genet. 6, 943 (1997).
34. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
35. 32P-labeled 430-bp fragment of KCNQ2 (encoding amino acids 144 through 288), using protocols provided by the manufacturer, and the membranes were exposed to photographic films (Kodak XR).
36. We thank J. Stoodt and P. Hausmann for excellent technical assistance, G. A. Nicholson and M. L. Kennerson for the original mapping of the pedigree to 20q13 (15), and M. M. Nöthen for the controls. Supported by grants from the Deutsche Forschungsgemeinschaft to O.K.S. (Ste769/1-1 ; SFB 400/85) and to T.J.J. (Je164/3-1), the Fonds der Chemischen Industrie to T.J.J., and the National Health and Medical Research Council to S.F.B.