EYA1 mutations cause branchio-oto-renal (BOR) syndrome. These mutations include single nucleotide transitions and transversions, small duplications and deletions, and complex genomic rearrangements. The last cannot be detected by coding sequence analysis of EYA1. We sought to refine the clinical diagnosis of BOR syndrome by analyzing phenotypic data from families segregating EYA1 disease-causing mutations. Based on genotype-phenotype analyses, we propose new criteria for the clinical diagnosis of BOR syndrome. We found that in approximately 40% of persons meeting our criteria, EYA1 mutations were identified. Of these mutations, 80% were coding sequence variants identified by SSCP, and 20% were complex genomic rearrangements identified by a semiquantitative PCR-based screen. We conclude that genetic testing of EYA1 should include analysis of the coding sequence and a screen for complex rearrangements.

All plants studied in natural ecosystems are symbiotic with fungi (1), which obtain nutrients while either positively, negatively, or neutrally affecting host fitness (2). Plant adaptation to selective pressures is considered to be regulated by the plant genome (3). To test whether mutualistic fungi contribute to plant adaptation, we collected 200 Dichanthelium lanuginosum plants from geothermal soils at 10 sites in Lassen Volcanic (LVNP) and Yellowstone ( YNP) National Parks. These soils have annual temperature fluctuations ranging from about 20°to 50°C (4 ).Plants and their roots were removed and assessed for fungal colonization (5). A fungal endophyte was isolated from the roots, crowns, leaves, and seed coats of all plants collected. Cultures established from single spores were analyzed by morphological (6) and rDNA sequence analyses (7) that suggested that the endophyte may be a new species of Curvularia (5). Soils from the base of 30 plants in YNP were devoid of the Curvularia sp., although other fungi were abundant (4). Moreover, axenically cultured Curvularia sp. was incapable of mycelial growth, spore germination, or survival at Ն40°C (5). Because geothermal soils were above 40°C all summer (4) and devoid of the fungus, we conclude that this Curvularia sp., like all known Curvularia species, is exclusively associated with plants.To assess the effect of the endophyte on the thermotolerance of D. lanuginosum, we removed seed coats and surface sterilized seeds (8) to generate endophyte-free plants. Treated seeds were planted in sterile magenta boxes containing sand, and after 1 month, plants were either mock-inoculated or inoculated with Curvularia sp. by pipetting 10 5 spores between the crown and first leaf. In the absence of thermal stress, endophyte-colonized (symbiotic) and endophyte-free (nonsymbiotic) plants showed no measurable growth or developmental differences. When root zones were heated with thermal tape (Fig. S1), nonsymbiotic plants (45/45) became shriveled and chlorotic at 50°C (Fig. 1A). In contrast, symbiotic plants (45/45) tolerated constant 50°C soil temperature for 3 days and intermittent soil temperatures as high as 65°C for 10 days. All nonsymbiotic plants (45/45) died during the 65°C heat regime, whereas symbiotic plants (45/45) survived. The endophyte was reisolated from surface sterilized roots and leaves of all surviving plants, indicating that both the fungus and the host were protected from thermal stress.We also field-tested symbiotic and nonsymbiotic seedlings in pasteurized geothermal soil collected and returned to Amphitheater Springs ( YNP) in May 2001 (Fig. 1B). By May 2002, symbiotic plants were greener with greater root and leaf masses (Table S2) than those of nonsymbiotic plants in soils Յ40°C. In soils above 40°C, nonsymbiotic plants did not survive while symbiotic plants thrived. The beneficial effect of fungal symbiosis increased with soil temperatures, demonstrating that Curvularia sp. provided thermal protection for D. lanuginosum. We reisolated Curvularia sp. fr...

Branchio-Oto-Renal (BOR) syndrome is transmitted as an autosomal dominant disorder, affects an estimated 2% of profoundly deaf children, and is caused by mutations in the human EYA1 gene. However, in up to half of the reported cases, EYA1 mutation screening is negative. This finding has been taken as evidence of genetic heterogeneity. Mutation screening of the coding region of EYA1 in a panel of families linked to chromosome 8 was conducted using SSCP and direct sequencing. Only one point mutation in five probands was detected. However, complex rearrangements, such as inversions or large deletions, were discovered in the other four patients using Southern blot analysis. These data suggest that more complex rearrangements may remain undetected in EYA1 since SSCP and sequencing were commonly used to detect mutations in this gene.