Diagnosis of primary congenital glaucoma

Details Diagnosis of primary congenital glaucoma Diagnosis of primary congenital glaucoma

2000-06-28

2001-03-27

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S006120, C514S002600, C530S356000

Reexamination Certificate

active

06207394

ABSTRACT:

BACKGROUND OF THE INVENTION
Glaucoma is a group of ocular disorders, characterized by degeneration of the optic nerve. It is one of the leading causes of blindness worldwide. One major risk factor for developing glaucoma is family history: several different inherited forms of glaucoma have been described.
Primary congenital or infantile glaucoma (gene symbol:GLC3) is an inherited disorder that accounts for 0.01-0.04% of total blindness. It is characterized by an improper development of the aqueous outflow system of the eye, which leads to elevated intraocular pressure, enlargement of the globe or cornea (i.e., buphthalmos), damage to the optic nerve, and eventual visual impairment. Pathogenesis of GLC3 remains elusive despite efforts to identify a single anatomic defect. At least two chromosomal locations associated with the disease have been identified: one locus at 2p21 (GLC3A) (Sarfarazi, M. el al., Genomics 30:171-177 (1995); and a second locus at 1p36 (GLC3B) (Akarsu, A. N. et al., Hum. Mol. Gen. 5(8):1199-1203 (1996)). Other specific loci, including a region of 6p and chromosome 11, have been excluded (Akarsu, A. N. et al., Am. J. Med. Genet. 61:290-292 (1996)).
Primary open angle glaucoma (gene symbol: GLC1) is a common disorder characterized by atrophy of the optic nerve resulting in visual field loss and eventual blindness. GLC1 has been divided into two major groups, based on age of onset and differences in clinical presentation.
Juvenile-onset primary open angle glaucoma (GLC1A) usually manifests in late childhood or early adulthood. The progression of GLC1A is rapid and severe with high intraocular pressure, is poorly responsive to medical treatment, and is such that it usually requires ocular surgery. GLC1A was initially mapped to the q21-q31 region of chromosome 1 (Sheffield, V. C. et al., Hum. Mol. Genet. 4:1837-1844 (1995)); mutations in the gene for trabecular meshwork inducible glucocorticoid response (TIGR) protein, located a chromosome 1q24, have been identified as associated with GLC1A glaucoma (Stone, E. M. et al., Science 275:668-670 (1997); Stoilova, D. et al., Opthamalic Genetics 18(3):109-118 (1997); Adam, M. F. et al., Hum. Mol. Genet. 6:2091-2097 (1997); Michels-Rautenstrauss, K. G., et al., Hum. Genet. 102:103-106 (1998); Mansergh, F. C. et al., Hum. Mutat. 11:244-251 (1998)).
Adult- or late-onset primary open angle glaucoma (GLC1B) followed by direct mutation analysis by restriction enzyme digestion is the most common type of glaucoma. It is milder and develops more gradually than juvenile-onset primary open angle glaucoma, with variable onset usually after the age of 40. GLC1B is associated with slight to moderate elevation of intraocular pressure, and often responds satisfactorily to regularly monitored medical treatment. However, because the disease progresses gradually and painlessly, it may not be detected until a late stage when irreversible damage to the optic nerve has already occurred. Linkage, haplotype and clinical data have assigned a locus for GLC1B to the 2cen-q13 region as well as a new locus 3q21-q22 (Stoilova, D. et al., Genomics 36:142-150 (1996)). Further evidence has identified several additional loci for primary open angle glaucoma. GLC1C, an adult-onset POAG gene, has been mapped to 3q (Wirtz, M. K. et al., Am. J. Hum. Genet. 60:296-304 (1997)); GLC1D has been mapped to 8q23 (Trifan, O. C. et al., Am. J Ophthalmol. 126:17-28 (1998)); GLC1E has been mapped to 10p15-p14 (Sarfarazi, M. et al., Am. J Hum. Genet. 62: 641-652 (1998)).
Because of the insidious nature of glaucoma, a need remains for a better and earlier means to diagnose or predict the likelihood of development of glaucoma, so that preventative or palliative measures can be taken before significant damage to the optical nerve occurs.
SUMMARY OF THE INVENTION
The invention pertains to methods of diagnosing primary congenital glaucoma, by detecting the presence of certain mutations in the human cytochrome P4501B1 gene (CYP1B1 gene). The mutations include a single-base change (a T→C transition) in codon 1, resulting in a change of the encoded amino acid (the initiation codon (Met1)) to Thr; a single-base change (a G→A transition) in codon 57, resulting in a change of the encoded amino acid from Trp57 to a stop codon; a single-base change (a C→A transition) in codon 65, resulting in a change of the encoded amino acid from Ala65 to Glu; a single-base change (a T→A transition) in codon 81, resulting in a change of the encoded amino acid from Tyr81 to Asn; a single-base change (a T→G transition) in codon 137, resulting in a change of the encoded amino acid from Tyr137 to Asp; a single-base change (a G→C transition) in codon 238, resulting in a change of the encoded amino acid from Gly238 to Arg; a single-base change (a G→C transition) in codon 242, resulting in a change of the encoded amino acid from Asp242 to His; a single-base change (a C→A transition) in codon 261, resulting in a change of the encoded amino acid from Phe261 to Leu; a single-base change (a T→G transition) in codon 356, resulting in a change of the encoded amino acid from Val356 to Gly; a single-base change (a G→A transition) in codon 368, resulting in a change of the encoded amino acid from Arg368 to His; a single-base change (a C→T transition) in codon 390, resulting in a change of the encoded amino acid from Arg390 to Cys; a single-base change (a G→A transition) in codon 393, resulting in a change of the encoded amino acid from Ser393 to Asn; a single-base change (a C→T transition) in codon 400, resulting in a change of the encoded amino acid from Pro400 to Ser; a single-base change (a C→G transition) in codon 443, resulting in a change of the encoded amino acid from Ala443 to Gly; a single-base change (a T→A transition) in codon 445, resulting in a change of the encoded amino acid from Phe445 to Ile; a single-base change (a T→C transition) in codon 464, resulting in a change of the encoded amino acid from Ser464 to Pro; a deletion of nucleotide 4340 (G); a deletion of nucleotide 4634 (T); a deletion of nucleotide 4681 (G); a deletion of nucleotide 8228 (C); and a deletion of nucleotides 8373-8378.
More than one of these mutations can be present in the CYP1B1 gene. The mutations can be identified by numerous methods, such as Southern analysis of genomic DNA; amplification of genomic DNA followed by direct mutation analysis by restriction enzyme digestion; Northern analysis of RNA; gene isolation and direct sequencing; or analysis of the CYP1B1 protein.
For example, a sample of DNA containing the CYP1B1 gene is obtained from an individual suspected of having primary congenital glaucoma or of being a carrier for primary congenital glaucoma (the test individual). The DNA is contacted with at least one mutant nucleic acid probe under conditions sufficient for specific hybridization of the CYP1B1 gene to the mutant nucleic acid probe. The mutant nucleic acid probe comprises DNA, cDNA, or RNA of the gene, or a fragment of the gene, having at least one of the mutations described above, or an RNA fragment corresponding to such a cDNA fragment. The presence of specific hybridization of the gene to the mutant nucleic acid probe is indicative of a mutation that is associated with primary congenital glaucoma. In another example, the DNA is contacted with a PNA probe under conditions sufficient for specific hybridization of the gene to the PNA probe; the presence of specific hybridization is indicative of a mutation that is associated with primary congenital glaucoma.
Alternatively, direct mutation analysis by restriction digest of a sample of genomic DNA, RNA or cDNA from the test individual can be conducted, if the mutation results in the creation or elimination of a restriction site. The digestion pattern of the relevant DNA, RNA or cDNA fragment indicates the presence or absence of the mutation associated with primary congenital glaucoma.
The presence of a mutation associated with primary congenital glaucoma can also be diagnosed by

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