Primary open-angle glaucoma (POAG) is an important cause of irreversible blindness worldwide (Quigley 1996). The disease results in a characteristic degeneration of the optic nerve that is usually associated with an elevation of intraocular pressure. Pressure within the eye is dependent on the rate of production of a fluid (aqueous humor) by the ciliary body and on the rate of removal of the fluid by the trabecular meshwork. Relatives of POAG patients have an increased risk of developing glaucoma, which suggests that genetic factors are an important component of POAG susceptibility (Leske 1983). Adult-onset POAG is inherited as a non-Mendelian trait, whereas forms of juvenile-onset POAG exhibit autosomal-dominant inheritance (Wiggs et al. 1995). One locus for juvenile glaucoma was initially mapped to 1q23 (Sheffield et al. 1993; Richards et al. 1994; Wiggs et al. 1994) and was subsequently refined to a 3-cM interval (Belmouden et al. 1997). In recent studies, evaluation of candidate genes mapped to this region has led to the identification of mutations in the TIGR/Myocilin gene (Stone et al. 1997). This gene was originally cloned, from cultured trabecular meshwork cells, as a steroid-response protein, named “trabecular meshwork-induced glucocorticoid response protein”(TIGR; Nguyen et al. 1993). The gene was isolated subsequently from a retinal cDNA library and was shown to be localized to the cilium connecting the inner and outer segments of photoreceptor cells (named “myocilin”; Kubota et al. 1997). Mutations have been detected in the TIGR/myocilin gene in juvenile-and adult-onset glaucoma pedigrees, and in populations of sporadic adult-and juvenile-onset patients (Adam et al. 1997; Stone et al. 1997; Suzuki et al. 1997; Alward et al. 1998; Morissette et al. 1998). Because the prevalence of mutations in TIGR/myocilin has not yet been investigated in a large number of pedigrees affected by juvenile-and adult-onset glaucoma, we have performed SSCP and sequence analysis in 152 affected families and in 104 individuals with macular degeneration but with normal intraocular pressures and optic nerves. The pedigrees used for this study are all of North American origin and were ascertained and sampled at the New England Medical Center and the Duke University Medical Center. The diagnostic criteria for POAG included intraocular pressure 122 mm/Hg and glaucomatous optic-nerve damage with consistent visual-field loss. Gonioscopic evaluation showed open angles (at least grade III) without any associated abnormalities. Individuals were identified as affected by adult-onset POAG if onset of the disease occurred after age 35 years, and as affected by juvenile-onset POAG if onset occurred before age 35 years. All pedigrees included in this study had at least two affected individuals. The control population underwent a complete ocular examination and did not show evidence of elevation of intraocular pressure or of optic-nerve disease. For mutation detection, a BAC clone (244L10) containing the TIGR/myocilin gene was identified by the screen of an arrayed BAC library (Research Genetics, Inc.). The BAC DNA was used as a source for the genomic sequence, and three exons separated by two introns were identified. Oligonucleotide primers (sequences available on request from Janey L. Wiggs), developed from the intron sequences flanking the intron/exon boundaries and from the cDNA sequence, were used to selectively amplify overlapping fragments of the coding sequence and the exon/intron splice sites. Sixtyeight families (25 juvenile-onset and 43 adult-onset) were screened for mutations in the entire coding sequence of the gene …