Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3

Science

Volumn 280, Issue 5368, 1998, Pages 1447-1451

  Wang, Aihui ^a   Liang, Yong ^a   Fridell, Robert A ^a   Probst, Frank J ^a   Wilcox, Edward R ^a   Touchman, Jeffrey W ^a   Morton, Cynthia C ^a   Morell, Robert J ^a   Noben Trauth, Konrad ^a   Camper, Sally A ^a   Friedman, Thomas B ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

MYOSIN;

ALLELE; ARTICLE; CHROMOSOME 17P; DNA SEQUENCE; GENE LOCUS; GENE MAPPING; GENE MUTATION; HEARING IMPAIRMENT; HUMAN; MOLECULAR CLONING; PRIORITY JOURNAL;

AMINO ACID SEQUENCE; ANIMALS; BRAIN; CHROMOSOME MAPPING; CHROMOSOMES, HUMAN, PAIR 17; COCHLEA; COSMIDS; DEAFNESS; EXONS; FEMALE; GENE EXPRESSION; GENES, RECESSIVE; HUMANS; MALE; MICE; MOLECULAR SEQUENCE DATA; MUTATION; MYOSINS; PEDIGREE; POINT MUTATION; SEQUENCE ALIGNMENT; SEQUENCE ANALYSIS, DNA;

EID: 0032577293     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5368.1447     Document Type: Article

References (52)

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14. Myo15 and MYO15 gene symbols were approved by the HUGO/GDB Nomenclature Committee.
15. Human participation was approved by the Institutional Review Boards of National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communicative Disorders (OH97-DC-N006 and OH93-DC-016), Brigham and Women's Hospital (87-02294), Udayana University, Denpasar Bali (1997-1), and the All India Institute of Medical Genetics (S-8066-01).
16. Twenty-seven primer pairs were synthesized based on the sequence of predicted mouse Myo15 exons. Only four primer pairs from exons 11, 14, 16, and 18 amplified human genomic DNA.
17. The 5′ and 3′ RACE reactions were performed with human fetal brain and placenta Marathon-Ready cDNAs (7402-1 and 7411-1; Clontech Laboratories).
18. 32P-labeled MYO15 cDNA fragment from exon 11 in Hybrisol I (Oncor). Filters were washed in 0.015 M NaCl containing 0.0015 M sodium citrate and 0.5% SDS at 55°C for 1 hour and autoradiographed. Northern blot analysis used the same hybridization conditions as above except that final washes were done at 65°C. Random fragments of cosmid 155d02 were subcloned and sequenced; 868 M13 sequence reads on PE/ABI 377s gave an average of 10-fold coverage of this cosmid [R. K. Wilson and E. R. Mardis, Analyzing DNA, B. Birren, E. D. Green, S. Klapholz, R. M. Myers, J. Roskams, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), p. 398]. Contig assembly and editing were done with the Phred/Phrap/Consed suite of programs [B. Ewing, L. Hillier, M. C. Wendl, P. Green, Genome Res. 8, 175 (1998); D. Gordon et al., ibid., p. 195 (available at http://www.genome. washington.edu/UWGC/)].
19. 32P-labeled MYO15 cDNA fragment from exon 11 in Hybrisol I (Oncor). Filters were washed in 0.015 M NaCl containing 0.0015 M sodium citrate and 0.5% SDS at 55°C for 1 hour and autoradiographed. Northern blot analysis used the same hybridization conditions as above except that final washes were done at 65°C. Random fragments of cosmid 155d02 were subcloned and sequenced; 868 M13 sequence reads on PE/ABI 377s gave an average of 10-fold coverage of this cosmid [R. K. Wilson and E. R. Mardis, Analyzing DNA, B. Birren, E. D. Green, S. Klapholz, R. M. Myers, J. Roskams, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), p. 398]. Contig assembly and editing were done with the Phred/Phrap/Consed suite of programs [B. Ewing, L. Hillier, M. C. Wendl, P. Green, Genome Res. 8, 175 (1998); D. Gordon et al., ibid., p. 195 (available at http://www.genome. washington.edu/UWGC/)].
20. 32P-labeled MYO15 cDNA fragment from exon 11 in Hybrisol I (Oncor). Filters were washed in 0.015 M NaCl containing 0.0015 M sodium citrate and 0.5% SDS at 55°C for 1 hour and autoradiographed. Northern blot analysis used the same hybridization conditions as above except that final washes were done at 65°C. Random fragments of cosmid 155d02 were subcloned and sequenced; 868 M13 sequence reads on PE/ABI 377s gave an average of 10-fold coverage of this cosmid [R. K. Wilson and E. R. Mardis, Analyzing DNA, B. Birren, E. D. Green, S. Klapholz, R. M. Myers, J. Roskams, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1997), p. 398]. Contig assembly and editing were done with the Phred/Phrap/Consed suite of programs [B. Ewing, L. Hillier, M. C. Wendl, P. Green, Genome Res. 8, 175 (1998); D. Gordon et al., ibid., p. 195 (available at http://www.genome. washington.edu/UWGC/)].
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23. Human cosmid and mouse BAG sequences were compared [J. Pustell and F. C. Kafatos, ibid. 12, 643 (1984)].
24. MYO15 cDNA fragments were subcloned into pGEM T-Easy (Promega) and sequenced on a PE/ABI 377 using Thermo Sequenase dye terminators (Amersham Life Science).
25. Mouse Myo15 has a predicted mini exon 6 with conserved amino acids and donor and acceptor splice sites. We have not yet identified this mini exon in Myo15 cDNA clones.
26. Abbreviations for 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.
27. A. Wang, data not shown.
28. No MYO15 expressed sequence tags (ESTs) were identified in dbEST and TIGR databases and MYO15 clones were not present among 235.392 I.M.A.G.E. Consortium cDNAs (Genome Systems). The following are the top three P(N) scores obtained by a BLASTX search of GenBank with the MYO15 cDNA sequence: MYO7A (accession number U39226), 1.3e-1B2; Myo7A (accession number U81453), 1.3e-177; C. elegans HUM-6 (accession number U80848), 6.2e-177.
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30. A search of PROSITE reveals another potential ATP-guanosine triphosphate (GTP) binding site in exon 18. Available at http://www.ebi.ac.uk/searches/prosite_input.html.
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35. A gapped BLASTP (15) search of the tail region of MYO15 (exons 23 and 25 to 50) gave statistically significant alignments to both of the two MyTH4 domains and the first talin-like domain of MYO7A (GenBank accession numbers U55208 and U39226). The bit score and E value of MYO15 to the first MyTH4 and talin-like domains of MYO7A are 59.7, 4e-09 and 43.4, 8e-04, respectively. The bit score and E value to the second MyTH4 domain are 48 and 1e-05.
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39. MYO1C, an unconventional myosin that maps to 17p13, was suggested as a DFNB3 candidate [F. Crozet et al., Genomics 40, 332 (1997); T. Hasson et al., ibid. 36, 431 (1996)]. There is 37% amino acid identity between MYO1C and MYO15 in the motor domain.
40. MYO1C, an unconventional myosin that maps to 17p13, was suggested as a DFNB3 candidate [F. Crozet et al., Genomics 40, 332 (1997); T. Hasson et al., ibid. 36, 431 (1996)]. There is 37% amino acid identity between MYO1C and MYO15 in the motor domain.
41. Primer pairs to each MYO15 exon were designed to amplify both the exon and 20 to 100 bp of flanking intronic sequence. Genomic DNA from affected individuals in three DFNB3 families and normal controls were used as a template for PCR amplification. Sequences of these 50 primer pairs and the PCR amplification conditions can be obtained from the authors. PCR products were gel purified (Qiagen) and sequenced with Thermo Sequenase radiolabeled terminators (Amersham Life Science).
42. 2] in a cycle condition of 94°C for 1 min and then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s followed by 72°C for 6 min. PCR products were purified (Qiagen), digested with Xmn I, and separated in a 2% agarose gel.
43. A nonsense mutation may affect mRNA stability and result in degradation of the transcript [L. Maquat, Am. J. Hum. Genet. 59, 279 (1996)].
44. + RNA from 50 human tissues (The Human RNA Master Blot, 7770-1, Clontech Laboratories) was hybridized with a probe from exons 29 to 47 of MYO15 using the same condition as Northern blot analysis (13).
45. Smith-Magenis syndrome (SMS) is due to deletions of 17p11.2 of various sizes, the smallest of which includes MYO15 and perhaps 20 other genes [(6); K-S Chen, L. Potocki, J. R. Lupski, MRDDRes. Rev. 2, 122 (1996)]. MYO15 expression is easily detected in the pituitary gland (data not shown). Haploinsufficiency for MYO15 may explain a portion of the SMS phenotype such as short stature. Moreover, a few SMS patients have sensorineural hearing loss, possibly because of a point mutation in MYO15 in trans to the SMS 17p11.2 deletion.
46. R. A. Fridell, data not shown.
47. K. B. Avraham et al., Nature Genet. 11, 369 (1995); X-Z. Liu et al., ibid. 17, 268 (1997); F. Gibson et al., Nature 374, 62 (1995); D. Weil et al., ibid., p. 60.
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49. K. B. Avraham et al., Nature Genet. 11, 369 (1995); X-Z. Liu et al., ibid. 17, 268 (1997); F. Gibson et al., Nature 374, 62 (1995); D. Weil et al., ibid., p. 60.
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51. + selection over oligo(dT) columns. First-strand cDNA was prepared using an Advantage RT-for-PCR kit (Clontech Laboratories). A portion of the first-strand cDNA (4%) was amplified by PCR with Advantage cDNA polymerase mix (Clontech Laboratories) using human MYO15-specific oligonucleotide primers (forward, 5′-GCATGACCTGCCGGCTAAT-GGG-3′; reverse, 5′-CTCACGGCTTCTGCATGGT-GCTCGGCTGGC-3′). Cycling conditions were 40 s at 94°C; 40 s at 66°C (3 cycles), 60°C (5 cycles), and 55°C (29 cycles); and 45 s at 68°C. PCR products were visualized by ethidium bromide staining after fractionation in a 1% agarose gel. A 688-bp PCR product is expected from amplification of the human MYO15 cDNA. Amplification of human genomic DNA with this primer pair would result in a 2903-bp fragment.
52. We are grateful to the people of Bengkala, Bali, and the two families, from India. We thank J. R. Lupski and K.-S. Chen for providing the human chromosome 17 cosmid library. For technical and computational assistance, we thank N. Dietrich, M. Fergusson, A. Gupta, E. Sorbello, R. Torkzadeh, C. Varner, M. Walker, G. Bouffard, and S. Beckstrom-Sternberg (National Institutes of Health Intramural Sequencing Center). We thank J. T. Hinnant, I. N. Arhya, and S. Winata for assistance in Bali, and T. Barber, S. Sullivan, E. Green, D. Drayna, and J. Battey for helpful comments on this manuscript. Supported by the National Institute on Deafness and Other Communication Disorders (NIDCD) (Z01 DC 00035-01 and Z01 DC 00038-01 to T.B.F. and E.R.W. and R01 DC 03402 to C.C.M.), the National Institute of Child Health and Human Development (R01 HD30428 to S.A.C.) and a National Science Foundation Graduate Research Fellowship to F.J.P. This paper is dedicated to J. B. Snow Jr. on his retirement as the Director of the NIDCD.