Nonsyndromic Deafness DFNA1 Associated with Mutation of a Human Homolog of the Drosophila Gene diaphanous Eric D. Lynch,* Ming K. Lee, Jan E. Morrow, Piri L. Welcsh, Pedro E. Leo ´ n, Mary-Claire King The gene responsible for autosomal dominant, fully penetrant, nonsyndromic sensori- neural progressive hearing loss in a large Costa Rican kindred was previously localized to chromosome 5q31 and named DFNA1. Deafness in the family is associated with a protein-truncating mutation in a human homolog of the Drosophila gene diaphanous. The truncation is caused by a single nucleotide substitution in a splice donor, leading to a four– base pair insertion in messenger RNA and a frameshift. The diaphanous protein is a profilin ligand and target of Rho that regulates polymerization of actin, the major component of the cytoskeleton of hair cells of the inner ear. DFNA1 defines the autosomal dominant, fully penetrant, sensorineural progressive hearing loss of kindred M of Costa Rica (OMIM 124900) (Fig. 1) (1, 2). In this kindred, low-frequency deafness begins at about 10 years of age and progresses by age 30 to profound, bilateral deafness involving all frequencies. The ratio of affected to un- affected children of deaf parents is 1:1; males and females are equally likely to be affected. Deafness in kindred M is a senso- rineural cochleosaccular dysplasia specific to the membranous structures of the inner ear. Speech development before onset, in- telligence, life expectancy, and fertility are normal. Hearing loss in this kindred has been traced eight generations to a common ancestor, born in 1713 in Cartago, Costa Rica. As with other nonsyndromic forms of deafness, identification of the gene respon- sible is important for the understanding of human hearing because the wild-type prod- uct of the gene is likely to be critical to development and maintenance of hearing. DFNA1 in kindred M was mapped to a region of 1 centimorgan on chromosome 5q31 by linkage analysis, then a complete 800-kb bacterial artificial chromosome (BAC) contig was constructed of the linked region (3). In order to identify all genes in the linked region, we sequenced BACs composing the contig after shotgun sub- cloning each into an M13 vector (4, 5). We developed the computer program SeqHelp to organize sequences from the chromato- grams, to call bases and align sequences using the computer programs PHRED and PHRAP, and to apply existing, publicly available, software to evaluate the novel genomic sequences (6). SeqHelp displayed putative coding regions, CpG islands, re- peat sequences, and matches to known genes and expressed sequence tags (ESTs) from all databases in an interactive format for further analysis. A previously unidentified human gene homologous to the Drosophila gene dia- phanous (GenBank U11288) and to the mouse gene encoding p140mDia (GenBank U96963) was revealed by the genomic se- quence of BACs 293C24, 45M22, and 249H5 (Fig. 2) (7). Given that the mouse and human predicted amino acid sequences are 97% identical for the regions identified from BACs, we estimated the sizes of gaps from the mouse sequence, constructed prim- ers from the human coding sequence, and used these to amplify intervening exons from human cDNA and to carry out 5'RACE on polyadenylated [poly(A) + ] RNA from lym- phoblastoid lines (8). A total of 3511 base pairs (bp) of coding sequence have been identified; about 250 bp remain to be deter- mined. The human diaphanous gene compris- es at least 18 exons with 3800 bp of coding sequence and a 3' untranslated region (UTR) of 918 or 1891 bp (9). To screen the DFNA1 gene for mutation in the M family, we designed primers to amplify exons and flanking splice junc- tions from genomic DNA of affected and unaffected members of the M family and from control individuals. Each product was screened for single-strand conforma- tion polymorphisms (SSCPs). Variant bands were gel-purified, reamplified, and se- quenced (10). A guanine-to-thymine substitution in the splice donor of the penultimate exon of human DFNA1 was observed in affected members of the M kindred (Fig. 3E). The guanine-to-thymine substitution at this site disrupts the canonical splice donor sequence E. D. Lynch, M. K. Lee, J. E. Morrow, P. L. Welcsh, M.-C. King, Departments of Medicine and Genetics, University of Washington, Seattle, WA 98195, USA. P. E. Leo ´ n, Center for Research in Cellular and Molecular Biology and School of Medicine, University of Costa Rica, San Jose, Costa Rica. * To whom correspondence should be addressed. E-mail: eric@lynch.com or genemap@u.washington.edu Fig. 1. The M kindred of Costa Rica. Hearing loss in this kindred is autosomal dominant, progressive and fully penetrant by age 30 and not associated with any other phenotype. Individuals with hearing loss are indicated by filled symbols and unaffected individuals by open symbols. All living individuals on the pedigree are included in the analysis. The pedigree is altered slightly, omitting young unaffected individuals, in order to protect privacy. The three-generation family drawn separately is related to the kindred, but the exact genealogy is unclear. All 78 affected individuals in the kindred share the DFNA1 mutation, and all unaffected individuals over age 30 are wild type at the comparable site. REPORTS www.sciencemag.org  SCIENCE  VOL. 278  14 NOVEMBER 1997 1315