Diagnosis of primary congenital glaucoma

Methods of diagnosing primary congenital glaucoma, by detecting particular mutations in a human cytochrome P4501B1 (CYP1B1) gene, are disclosed. Methods include hybridization analysis, such as Southern or Northern analysis, which use hybridization of a mutant nucleic acid probe to the CYP1B1 gene; direct mutation analysis by restriction digest; sequencing of the CYP1B1 gene; hybridization of an allele-specific oligonucleotide with amplified genomic DNA; or identification of the presence of mutant proteins encoded by the CYP1B1 gene.

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. et 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. etal., 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.fwdarw.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.fwdarw.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.fwdarw.A transition) in codon 65, resulting in a change of the encoded 
amino acid from Ala65 to Glu; a single-base change (a T.fwdarw.A 
transition) in codon 81, resulting in a change of the encoded amino acid 
from Tyr81 to Asn; a single-base change (a T.fwdarw.G transition) in codon 
137, resulting in a change of the encoded amino acid from Tyr137 to Asp; a 
single-base change (a G.fwdarw.C transition) in codon 238, resulting in a 
change of the encoded amino acid from Gly238 to Arg; a single-base change 
(a G.fwdarw.C transition) in codon 242, resulting in a change of the 
encoded amino acid from Asp242 to His; a single-base change (a C.fwdarw.A 
transition) in codon 261, resulting in a change of the encoded amino acid 
from Phe261 to Leu; a single-base change (a T.fwdarw.G transition) in 
codon 356, resulting in a change of the encoded amino acid from Val356 to 
Gly; a single-base change (a G.fwdarw.A transition) in codon 368, 
resulting in a change of the encoded amino acid from Arg368 to His; a 
single-base change (a C.fwdarw.T transition) in codon 390, resulting in a 
change of the encoded amino acid from Arg390 to Cys; a single-base change 
(a G.fwdarw.A transition) in codon 393, resulting in a change of the 
encoded amino acid from Ser393 to Asn; a single-base change (a C.fwdarw.T 
transition) in codon 400, resulting in a change of the encoded amino acid 
from Pro400 to Ser; a single-base change (a C.fwdarw.G transition) in 
codon 443, resulting in a change of the encoded amino acid from Ala443 to 
Gly; a single-base change (a T.fwdarw.A transition) in codon 445, 
resulting in a change of the encoded amino acid from Phe445 to Ile; a 
single-base change (a T.fwdarw.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 sequence data. A sample of genomic DNA, RNA or cDNA 
from the test individual is obtained, and the sequence of the CYP1B1 gene, 
or a fragment of the gene, is determined. The sequence of the CYP1B1 gene 
from the individual is compared with the known sequence of the CYP1B1 gene 
(the control sequence). The presence of a mutation as described above in 
the gene of the individual is indicative of the presence of a mutation 
that is associated with primary congenital glaucoma. 
The invention additionally pertains to methods of diagnosing primary 
congenital glaucoma in an individual by detecting alterations in the 
composition of the protein encoded by the CYP1B1 gene. An alteration in 
the composition of the CYP1B1 protein is indicative of the disease. 
Alterations in composition of the protein can be assessed using standard 
techniques, such as Western blotting. 
The invention additionally pertains to antibodies (monoclonal or 
polyclonal) to proteins encoded by CYP1B1 genes having the mutations 
described above. These antibodies can also be used in methods of 
diagnosis. For example, a test sample which includes CYP1B 1 protein of 
interest is contacted with antibodies specific for a protein that is 
encoded by a CYP1B1 gene having a mutation described above. Specific 
binding of the antibody to the CYP1B1 protein of interest is indicative of 
a mutation associated with primary congenital glaucoma. 
The current invention facilitates identification of certain mutations in 
the CYP1B1 gene which are associated with primary congenital glaucoma, and 
thereby facilitates both better and earlier diagnosis and treatment of the 
disease. Identification of such mutations distinguishes one form of 
glaucoma from other forms, thereby enabling better treatment planning for 
affected individuals, as well as for other family members who may be 
affected individuals or disease carriers. 
DETAILED DESCRIPTION OF THE INVENTION 
The current invention relates to methods of diagnosing primary congenital 
glaucoma. As described herein, Applicant has identified certain mutations 
in the human cytochrome P4501B1 gene (CYP1B1 gene) that are associated 
with the presence of disease. The CYP1B1 gene is described by Sutter, T. 
R. et al., J. Biol. Chem. 269:13092 (1994), and the genomic structure of 
the introns and exons of the gene is described by Tang, Y. M. et al., J. 
Biol. Chem. 271:28324 (1996). The entire teachings of these references are 
incorporated herein by reference. The nucleotide sequence of the CYP1B1 
gene is available from GenBank, as accession number UO3688. 
The mutations in the CYP1B1 gene, as described herein, include mutations in 
certain codons of the CYP1B1 gene. The term "codon" indicates a group of 
three nucleotides which designate a single amino acid in the gene. The 
codons are numbered from the beginning of the coding sequence of the 
protein: the first three nucleotides which together designate the initial 
methionine residue in the protein, together are "codon 1". The CYP1B1 gene 
has 543 codons, flanked by non-encoding nucleotides. Nucleotides of the 
introns of the CYP1B1 gene and the non-coding 5' and 3' regions of the 
CYP1B1 gene are not included in the numbering of the codons. The mutations 
in the CYP1B1 gene as described herein also include mutations in certain 
specific nucleotides of the gene. The nucleotide numbering does include 
non-coding nucleotides (see, e.g., Sutter et al. and the Genbank 
submission cited supra). 
One mutation was a single-base change (a T.fwdarw.C transition) in codon 1. 
This mutation resulted in a change of the encoded amino acid (the 
initiation codon (Met1)) to Thr. A second mutation was a single-base 
change (a G.fwdarw.A transition) in codon 57, resulting in a change of the 
encoded amino acid from Trp57 to a stop codon, truncating the protein. 
Several mutations involved amino-acid substitutions in the N-terminal half 
of the CYP1B1 protein, which is involved in the substrate binding 
(mutation 3, a single-base change (a C.fwdarw.A transition) in codon 65, 
resulting in a change of the encoded amino acid from Ala65 to Glu; 
mutation 4, a single-base change (a T.fwdarw.A transition) in codon 81, 
resulting in a change of the encoded amino acid from Tyr81 to Asn; 
mutation 5, a single-base change (a T.fwdarw.G transition) in codon 137, 
resulting in a change of the encoded amino acid from Tyr137 to Asp; 
mutation 6, a single-base change (a G.fwdarw.C transition) in codon 238, 
resulting in a change of the encoded amino acid from Gly238 to Arg; 
mutation 7, a single-base change (a G.fwdarw.C transition) in codon 242, 
resulting in a change of the encoded amino acid from Asp242 to His; 
mutation 8, a single-base change (a C.fwdarw.A transition) in codon 261, 
resulting in a change of the encoded amino acid from Phe261 to Leu; and 
mutation 9, a single-base change (a T.fwdarw.G transition) in codon 356, 
resulting in a change of the encoded amino acid from Val356 to Gly). 
Several more mutations involved amino-acid substitutions in the C-terminal 
half of the CYP1B1 protein that contains the structures involved in 
heme-binding. These mutations included mutation 10, a single-base change 
(a G.fwdarw.A transition) in codon 368, resulting in a change of the 
encoded amino acid from Arg368 to His; mutation 11, a single-base change 
(a C.fwdarw.T transition) in codon 390, resulting in a change of the 
encoded amino acid from Arg390 to Cys; mutation 12, a single-base change 
(a G.fwdarw.A transition) in codon 393, resulting in a change of the 
encoded amino acid from Ser393 to Asn; mutation 13, a single-base change 
(a C.fwdarw.T transition) in codon 400, resulting in a change of the 
encoded amino acid from Pro400 to Ser; mutation 14, a single-base change 
(a C.fwdarw.G transition) in codon 443, resulting in a change of the 
encoded amino acid from Ala443 to Gly; mutation 15, a single-base change 
(a T.fwdarw.A transition) in codon 445, resulting in a change of the 
encoded amino acid from Phe445 to Ile; mutation 16, a single-base change 
(a T.fwdarw.C transition) in codon 464, resulting in a change of the 
encoded amino acid from Ser464 to Pro. In addition, several frame-shift 
mutations predicted to introduce premature stop codons were identified, 
including mutation 17, a deletion of nucleotide 4340 (G); mutation 18, a 
deletion of nucleotide 4634 (T); mutation 19, a deletion of nucleotide 
4681 (G); mutation 20, a deletion of nucleotide 8228 (C); and mutation 21, 
a deletion of nucleotides 8373-8378. 
Using methods such as those described herein, or other appropriate methods, 
it is now possible to diagnose primary congenital glaucoma by detecting a 
mutation or mutations in the CYP1B1 gene that are associated with 
glaucoma. 
In a first method of diagnosing primary congenital glaucoma, hybridization 
methods, such as Southern analysis, are used (see Current Protocols in 
Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including 
all supplements through 1997). For example, a test sample of genomic DNA, 
RNA, or cDNA, is obtained from an individual suspected of having (or 
carrying a defect for) primary congenital glaucoma (the "test 
individual"). The individual can be an adult, child, or fetus. The test 
sample can be from any source which contains genomic DNA, such as a blood 
or tissue sample, such as from skin or other organs. In a preferred 
embodiment, the test sample of DNA is obtained from a fibroblast skin 
sample, from hair roots, or from cells obtained from the oral cavity 
(e.g., via mouthwash). In another preferred embodiment, the test sample of 
DNA is obtained from fetal cells or tissue by appropriate methods, such as 
by amniocentesis or chorionic villus sampling. The DNA, RNA, or cDNA 
sample is examined to determine whether one of the mutations described 
above is present; the presence of the mutation is indicated by 
hybridization of the CYP1B1 gene in the genomic DNA, RNA, or cDNA to a 
nucleic acid probe. A "nucleic acid probe", as used herein, can be a DNA 
probe or an RNA probe. The nucleic acid probe hybridizes to at least one 
of the mutations described above. A fragment of such a nucleic acid probe 
can also be used, provided that the fragment hybridizes to the part of the 
CYP1B1 gene that contains the mutation. 
To diagnose primary congenital glaucoma by hybridization, a hybridization 
sample is formed by contacting the test sample containing the CYP1B1 gene 
with at least one nucleic acid probe. The hybridization sample is 
maintained under conditions which are sufficient to allow specific 
hybridization of the nucleic acid probe to the CYP1B1 gene. "Specific 
hybridization", as used herein, indicates exact hybridization (e.g., with 
no mismatches). Specific hybridization can be performed under high 
stringency conditions or moderate stringency conditions, for example. 
"Stringency conditions" for hybridization is a term of art which refers to 
the conditions of temperature and buffer concentration which permit 
hybridization of a particular nucleic acid to another nucleic acid in 
which the first nucleic acid may be perfectly complementary to the second, 
or the first and second nucleic acids may share only some degree of 
complementarity. For example, certain high stringency conditions can be 
used which distinguish perfectly complementary nucleic acids from those of 
less complementarity. "High stringency conditions" and "moderate 
stringency conditions" for nucleic acid hybridizations are explained in 
chapter 2.10 and 6.3, particularly on pages 2.10.1-2.10.16 and pages 
6.3.1-6 in Current Protocols in Molecular Biology, supra, the teachings of 
which are hereby incorporated by reference. The exact conditions which 
determine the stringency of hybridization depend on factors such as length 
of nucleic acids, base composition, percent and distribution of mismatch 
between the hybridizing sequences, temperature, ionic strength, 
concentration of destabilizing agents, and other factors. Thus, high or 
moderate stringency conditions can be determined empirically. In one 
embodiment, the hybridization conditions for specific hybridization are 
moderate stringency. In a particularly preferred embodiment, the 
hybridization conditions for specific hybridization are high stringency. 
Specific hybridization, if present, is then detected using standard 
methods. If specific hybridization occurs between the nucleic acid probe 
and the CYP1B1 gene in the test sample, then the CYP1B1 gene has a 
mutation associated with primary congenital glaucoma. More than one 
nucleic acid probe can also be used concurrently in this method. Specific 
hybridization of any one of the nucleic acid probes is indicative of a 
mutation that is associated with primary congenital glaucoma, and is 
therefore diagnostic for the disease. 
For example, in the diagnosis of primary congenital glaucoma, a nucleic 
acid probe can be prepared that hybridizes to a part of the CYP1B1 gene 
having a T.fwdarw.C transition in codon 1. If this nucleic acid probe 
specifically hybridizes with the CYP1B1 gene in the test sample, a 
diagnosis of primary congenital glaucoma is made. Alternatively, a nucleic 
acid probe can be prepared that hybridizes to a CYP1B1 gene having one of 
the other mutations described above. Specific hybridization of such a 
nucleic acid probe with the CYP1B1 gene in the test sample is indicative 
of primary congenital glaucoma. 
In another hybridization method, Northern analysis (see Current Protocols 
in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra) 
is used to identify the presence of a mutation associated with glaucoma. 
For Northern analysis, a sample of RNA is obtained from the test 
individual by appropriate means. Specific hybridization of a nucleic acid 
probe, as described above, to RNA from the individual is indicative of a 
mutation that is associated with primary congenital glaucoma, and is 
therefore diagnostic for the disease. 
For representative examples of use of nucleic acid probes, see, for 
example, U.S. Pat. Nos. 5,288,611 and 4,851,330. 
Alternatively, a peptide nucleic acid (PNA) probe can be used instead of a 
nucleic acid probe in the hybridization methods described above. PNA is a 
DNA mimic having a peptide-like, inorganic backbone, such as 
N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) 
attached to the glycine nitrogen via a methylene carbonyl linker (see, for 
example, Nielsen, P. E. et al., Bioconjugate Chemistry, 1994, 5, American 
Chemical Society, p. 1 (1994). The PNA probe can be designed to 
specifically hybridize to a CYP1B1 gene having a mutation associated with 
glaucoma. Hybridization of the PNA probe to the mutant CYP1B1 gene is 
diagnostic for the disease. 
In another method of the invention, mutation analysis by restriction 
digestion can be used to detect mutations, if the mutation in the gene 
results in the creation or elimination of a restriction site. A test 
sample containing genomic DNA is obtained from the test individual. 
Polymerase chain reaction (PCR) or Ligase chain reaction (LCR) can be used 
to amplify the CYP1B1 gene (and, if necessary, the flanking sequences) in 
a test sample of genomic DNA from the test individual. RFLP analysis is 
conducted as described (see Current Protocols in Molecular Biology, 
supra). The digestion pattern of the relevant DNA fragment indicates the 
presence or absence of the mutation associated with primary congenital 
glaucoma. 
Sequence analysis can also be used to detect specific mutations in the 
CYP1B1 gene. A test sample of DNA is obtained from the test individual. 
PCR or LCR can be used to amplify the gene, and/or its flanking sequences. 
The sequence of the CYP1B1 gene, or a fragment of the gene, is determined, 
using standard methods. The sequence of the gene (or gene fragment) is 
compared with the known nucleic acid sequence of the gene. The presence of 
any of the mutations associated with glaucoma as described above indicates 
that the individual is affected with, or is a carrier for, primary 
congenital glaucoma. 
Allele-specific oligonucleotides can also be used to detect the presence of 
a mutation associated with glaucoma, through the use of dot-blot 
hybridization of amplified gene products with allele-specific 
oligonucleotide (ASO) probes (see, for example, Saiki, R. et al., (1986), 
Nature (London) 324:163-166). An "allele-specific oligonucleotide" (also 
referred to herein as an "allele-specific oligonucleotide probe") is an 
oligonucleotide of approximately 10-50 base pairs, preferably 
approximately 15-30 base pairs, that specifically hybridizes to the CYP1B1 
gene. An allele-specific oligonucleotide probe that is specific for 
particular mutations in the CYP1B1 gene can be prepared, using standard 
methods (see Current Protocols in Molecular Biology, supra). To identify 
the presence or absence of mutations that are associated with glaucoma, a 
test sample of DNA is obtained from the test individual. PCR or LCR can be 
used to amplify all or a fragment of the CYP1B1 gene, and its flanking 
sequences. The DNA containing the amplified CYP1B 1 gene (or fragment of 
the gene) is dot-blotted, using standard methods (see Current Protocols in 
Molecular Biology, supra), and the blot is contacted with the 
oligonucleotide probe. The presence of specific hybridization of the probe 
to the amplified CYP1B1 gene is then detected. Specific hybridization of 
an allele-specific oligonucleotide probe to DNA from the individual is 
indicative of a mutation in the CYP1B1 gene that is associated with 
primary congenital glaucoma, and is therefore diagnostic for the disease. 
Diagnosis of primary congenital glaucoma can also be made by examining the 
composition of the protein encoded by the CYP1B1 gene. A test sample from 
an individual is assessed for the presence of an alteration in the 
qualitative protein expression (i.e., the composition of the protein), or 
both. An "alteration" in the protein composition, as used herein, refers 
to an alteration in composition of CYP1B1 protein in a test sample, as 
compared with the known composition of the non-mutant CYP1B1 protein 
(e.g., CYP1B1 protein in a control sample). A control sample is a sample 
that corresponds to the test sample (e.g., is from the same type of 
cells), and is from an individual who is not affected by primary 
congenital glaucoma. An alteration in the composition of the protein in 
the test sample is indicative of primary congenital glaucoma. Various 
means of examining the composition of protein encoded by the CYP1B1 gene 
can be used, including spectroscopy, colorimetry, electrophoresis, 
isoelectric focusing, and immunoblotting (see Current Protocols in 
Molecular Biology, particularly chapter 10). For example, Western blotting 
analysis, using an antibody that specifically binds to a protein encoded 
by CYP1B1 gene having one of the mutations described above, can be used to 
identify the presence in a test sample of a protein encoded by a mutant 
CYP1B1 gene. The presence of a protein encoded by a mutant CYP1B1 gene, is 
diagnostic for glaucoma. 
The invention also relates to antibodies to mutant proteins encoded by a 
CYP1B1 gene having one or more of the mutations described herein. 
Antibodies can be raised to the mutant protein or fragment of the mutant 
protein using standard methods (see, for example, Current Protocols in 
Molecular Biology, supra). The term "antibody", as used herein, 
encompasses both polyclonal and monoclonal antibodies, as well as mixtures 
of more than one antibody reactive with the protein or protein fragment 
(e.g., a cocktail of different types of monoclonal antibodies reactive 
with the mutant protein or protein fragment). The term antibody is further 
intended to encompass whole antibodies and/or biologically functional 
fragments thereof, chimeric antibodies comprising portions from more than 
one species, humanized antibodies, human-like antibodies, and bifunctional 
antibodies. Biologically functional antibody fragments are those fragments 
sufficient for binding of the antibody fragment to the mutant protein of 
interest. 
Monoclonal antibodies (mAb) reactive with a mutant protein encoded by a 
CYP1B1 gene can be produced using somatic cell hybridization techniques 
(Kohler and Milstein, Nature 256:495-497 (1975)) or other techniques. In a 
typical hybridization procedure, a crude or purified mutant protein 
encoded by a CYP1B1 gene having one or more of the mutations described 
herein can be used as the immunogen. An animal is immunized with the 
immunogen to obtain antibody-producing spleen cells. The species of animal 
immunized will vary depending on the specificity of mAb desired. The 
antibody producing cell is fused with an immortalizing cell (e.g., a 
myeloma cell) to create a hybridoma capable of secreting antibodies to the 
mutant protein of the invention. The unfused residual antibody-producing 
cells and immortalizing cells are eliminated. Hybridomas producing desired 
antibodies are selected using conventional techniques and the selected 
hybridomas are cloned and cultured. 
Polyclonal antibodies can be prepared by immunizing an animal in a similar 
fashion as described above for the production of monoclonal antibodies. 
The animal is maintained under conditions whereby antibodies are produced 
that are reactive with the mutant protein encoded by the CYP1B1 gene. 
Blood is collected from the animal upon reaching a desired titer of 
antibodies. The serum containing the polyclonal antibodies (antisera) is 
separated from the other blood components. The polyclonal 
antibody-containing serum can optionally be further separated into 
fractions of particular types of antibodies (e.g., IgG, IgM). 
Antibodies that specifically bind to a protein or protein fragment encoded 
by the mutant CYP1B1 gene (i.e., those that bind to the protein or protein 
fragment encoded by the mutant gene, but not to protein encoded by a 
non-mutant copy of the gene) can also be used in methods of diagnosis. A 
test sample containing the protein encoded by the CYP1B1 gene is contacted 
with the antibody; binding of the antibody to the protein is indicative of 
the presence of a protein encoded by the mutant gene, and is diagnostic 
for disease. 
The present invention also includes kits useful in the methods of the 
invention. The kits can include a means for obtaining a test sample; 
nucleic acid probes, PNA probes, or allele-specific oligonucleotide 
probes; appropriate reagents; antibodies to mutant proteins encoded by a 
CYP1B1 gene having a mutation as described herein; instructions for 
performing the methods of the invention; control samples; and/or other 
components. In a preferred embodiment, the kit comprises at least one 
reagent useful for identifying a mutation in the CYP1B1 gene, and 
instructions for performing the methods described herein.

The invention is further illustrated by the following Example. 
EXAMPLE 
Identification of a CYP1B1 Gene Mutations Associated with Primary 
Congenital Glaucoma 
Methods used to identify the mutations are described in U.S. Pat. No. 
5,830,661 to Sarfarazi. Briefly, for rapid mutation screening, fragments 
containing portions of the CYP1B1 gene were amplified from genomic DNA 
with primers, using polymerase chain reaction (PCR), and the PCR products 
were analyzed on polyacrylamide minigels consisting of 5% Acrylamide/Bis 
solution (19:1), 15% urea, and 1.times.TBE. The mutations described herein 
were either sporadic or familial, and found in one or more individuals or 
families from varying geographical populations (families of Israeli, 
United Kingdom, Turkey, United States of America, Bulgaria, Lebanon, and 
Brazil origin). 
The mutations described above are summarized in the Table. 
TABLE 
______________________________________ 
Mutations Associated with Primary Congenital Glaucoma 
Muta- Nucleic Acid 
Amino Acid 
tion Change Change Origin Note 
______________________________________ 
1 ATG--&gt;ACG Met1--&gt;Thr Israel Affects initiation 
codon 
2 TGG--&gt;TGA Trp57--&gt;Stop UK Hinge Region 
3 GCG--&gt;GAG Ala65--&gt;Glu UK These mutations 
4 TAC--&gt;AAC Tyr81 --&gt;Asn UK are located in 
5 TAC--&gt;GAC Tyr137--&gt;Asp Turkey the N-terminal 
6 GGC--&gt;CGC Gly238--&gt;Arg UK half of the 
7 GAC--&gt;CAC Asp242--&gt;His UK protein, which 
8 TTC--&gt;TTA Phe261--&gt;Leu USA is involved 
9 GTG--&gt;GGG Va1356--&gt;Gly UK in substrate 
binding. 
10 CGT--&gt;CAT Arg368--&gt;His Bulgaria, These mutations 
UK located in the C- 
11 CGC--&gt;TGC Arg390--&gt;Cys Bulgaria terminal half of 
12 AGC--&gt;AAC Ser393--&gt;Asn Turkey the protein, 
13 CCT--&gt;TCT Pro400--&gt;Ser USA which contains 
the structures 
involved 
14 GCT--&gt;GGT Ala443--&gt;Gly Lebanon in heme binding. 
15 TTC--&gt;ATC Phe445--&gt;Ile Bulgaria 
16 TCA--&gt;CCA Ser464--&gt;Pro UK 
17 4340 del G frame-shift Brazil premature stop 
codon 
18 4634 del T frame-shift Brazil premature stop 
codon 
19 4681 del G frame-shift UK premature stop 
codon 
20 8228 del c frame-shift Lebanon premature stop 
codon 
21 del 8373- frame-shift UK premature stop 
8378 codon 
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The relevant teachings of the references cited herein are incorporated by 
reference in their entirety. 
While this invention has been particularly shown and described with 
references to preferred embodiments thereof, it will be understood by 
those skilled in the art that various changes in form and details may be 
made therein without departing from the spirit and scope of the invention 
as defined by the appended claims.