Introduction
Tremendous advances in the molecular genetics of human disease have been made in the past 20 years. Many genes responsible for inherited eye diseases have been isolated and characterized, and the chromosomal location of a number of additional genes has been determined. Identifying and characterizing genes responsible for human disease has led to DNA-based methods of diagnosis; novel therapeutic approaches, including gene therapy; and improved knowledge about the molecular events that underlie the disease processes. The disorders discussed in this chapter represent important examples of major advances in human ocular molecular genetics.
Although all inherited disorders are the result of gene mutations, the molecular consequences of a mutation are quite variable. The type of mutation responsible for a disease usually defines the inheritance pattern. For example, mutations that create an abnormal protein detrimental to the cell are typically autosomal dominant, because only one mutant gene is required to disrupt normal cell function. Mutations that result in proteins that have reduced biological activity (loss of function) may be inherited as autosomal dominant or autosomal recessive conditions, depending on the number of copies of normal genes (and the amount of normal protein) required. Disorders may be caused by mutations in mitochondrial DNA that result in a characteristic maternal inheritance pattern. Also, mutations in genes carried on the X chromosome result in characteristic inheritance patterns.
Dominant Corneal Dystrophies
The autosomal dominant corneal dystrophies are an excellent example of dominant negative mutations that result in the formation of a toxic protein. Four types of autosomal dominant dystrophies that affect the stroma of the cornea are well characterized :
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Groenouw (granular) type I.
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Lattice type I.
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Avellino (combined granular-lattice).
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Reis–Bücklers.
Although all four corneal dystrophies affect the anterior stroma, the clinical and pathological features differ. The granular dystrophies typically form discrete, white, localized deposits that may obscure vision progressively. Histopathologically, these deposits stain bright red with Masson trichrome and have been termed “hyalin.” In lattice dystrophy, branching amyloid deposits gradually opacify the visual axis. These deposits exhibit a characteristic birefringence under polarized light after staining with Congo red. Avellino dystrophy includes features of both granular and lattice dystrophies. Reis–Bücklers dystrophy appears to involve primarily Bowman’s layer and the superficial stroma.
All four dystrophies were mapped genetically to a common interval on chromosome 5q31, and mutations in a single gene, TGFB1 (also known as BIGH3 ), located in this region were found in affected individuals. The product of this gene, keratoepithelin, is probably an extracellular matrix protein that modulates cell adhesion. Four different missense mutations, which occur at two arginine codons in the gene, have been found ( Fig. 1.2.1 ). Interestingly, mutations at one of these arginine codons cause lattice dystrophy type I or Avellino dystrophy, the two dystrophies characterized by amyloid deposits. Mutations at the other arginine codon appear to result in either granular dystrophy or Reis–Bücklers dystrophy. The mutation analysis of this gene demonstrates that different mutations within a single gene can result in different phenotypes.
The mutation that causes Avellino and lattice dystrophies abolishes a putative phosphorylation site, which probably is required for the normal structure of keratoepithelin. Destruction of this aspect of the protein structure leads to formation of the amyloid deposits that are responsible for opacification of the cornea. Consequently, the mutant protein is destructive to the normal tissue. Mutations at the R555 (arginine at amino acid position 555) appear to result in either granular dystrophy or Reis–Bücklers dystrophy. These phenotype–genotype correlations demonstrate the variable expressivity of mutations in this gene and the significance of alteration of the arginine residues 124 and 555.
Aniridia, Peter’s Anomaly, Autosomal Dominant Keratitis
Some cellular processes require a level of protein production that results from the expression of both copies of a particular gene. Such proteins may be involved in a variety of biological processes. Certain disorders are caused by the disruption of one copy of a gene that reduces the protein level by half. Such a reduction is also called “haploinsufficiency.”
Mutations in the PAX6 gene are responsible for aniridia, Peter’s anomaly, and autosomal dominant keratitis. Most of the mutations responsible for these disorders alter the paired-box sequence within the gene ( Fig. 1.2.2 ) and result in inactivation of one copy of the PAX6 gene. The paired-box sequence is an important element that is necessary for the regulatory function of the protein. Losing half the normal paired-box sequence, and probably other regulatory elements within the gene, appears to be the critical event that results in the associated ocular disorders. The protein plays an important role in ocular development, presumably by regulating the expression of genes that are involved in embryogenesis of the eye. A reduction in the amount of active gene product alters the expression of these genes, which results in abnormal development. The genes that code for the lens crystallin proteins are one class of genes developmentally regulated by the PAX6 protein.
The clinical disorders caused by mutations in PAX6 exhibit extensive phenotypic variability. Similar mutations may give rise to aniridia, Peter’s anomaly, or autosomal dominant keratitis. Variation in the phenotype associated with a mutation is termed “variable expressivity” and is a common feature of disorders that arise from haploinsufficiency. It is possible that the variability of the mutant phenotype results from the random activation of downstream genes that occurs when only half the required gene product is available.
Rieger’s Syndrome
Rieger’s syndrome is an autosomal dominant disorder of morphogenesis that results in abnormal development of the anterior segment of the eye. Typical clinical findings may include posterior embryotoxon, iris hypoplasia, iridocorneal adhesions, and corectopia. Approximately 50% of affected individuals develop a high-pressure glaucoma associated with severe optic nerve disease. The cause of the glaucoma associated with this syndrome is not known, although anomalous development of the anterior chamber angle structures is usually found.
Genetic heterogeneity of Rieger’s syndrome is indicated by the variety of chromosomal abnormalities that have been associated with the condition, including deletions of chromosome 4 and chromosome 13. Genes for Rieger’s syndrome are located on chromosomes 4q25, 13q14, and 6p25. Iris hypoplasia is the dominant clinical feature of pedigrees linked to the 6p25 locus, whereas pedigrees linked to 4q25 and 13q14 demonstrate the full range of ocular and systemic abnormalities found in these patients.
The genes located on chromosomes 4q25 and 6p25 have been identified. The chromosome 4q25 gene (PITX2) codes for a bicoid homeobox transcription factor. Like PAX6, this gene is expressed during eye development and is probably involved in the ocular developmental processes. The chromosome 6p25 gene FOXC1 (also called FKHL7 ) is a member of a forkhead family of regulatory proteins. FOXC1 is expressed during ocular development, and mutations alter the dosage of the gene product. There is some indication that the FOXC1 protein and the PITX2 protein interact during ocular development. The identification of other genes responsible for Rieger’s syndrome and anterior segment dysgenesis is necessary to determine whether these genes are part of a common developmental pathway or represent redundant functions necessary for eye development.
Juvenile Glaucoma
Primary juvenile open-angle glaucoma is a rare disorder that develops during the first two decades of life. Affected patients typically present with a high intraocular pressure (IOP), which ultimately requires surgical therapy. Juvenile glaucoma may be inherited as an autosomal dominant trait, and large pedigrees have been identified and used for genetic linkage analysis. One gene responsible for this condition, MYOC, codes for the myocilin protein and is located on chromosome 1q23 (GLC1A).
Myocilin has been shown to be expressed in the human retina, ciliary body, and trabecular meshwork. The protein has several functional domains, including a region homologous to a family of proteins called olfactomedins. Although the function of the protein and the olfactomedin domain is not known, nearly all the mutations associated with glaucoma have been found in the olfactomedin portion of the protein ( Fig. 1.2.3 ). Mutations in myocilin also have been associated with some cases of adult-onset primary open-angle glaucoma. Patients with only one copy of the myocilin gene (because of chromosomal deletion removing the second copy of the gene) or without any functional myocilin (caused by homozygosity of a stop-codon polymorphism in the first part of the gene) do not develop glaucoma. Collectively these results suggest that mutations in myocilin cause a gain-of-function or dominant negative effect rather than a loss-of-function or haploinsufficiency. The role of myocilin in IOP elevation is not completely known, but in vitro studies show that myocilin mutants are misfolded and detergent resistant. Myocilin mutations may be secretion incompetent and accumulate in the endoplasmic reticulum (ER) inducing ER stress. Recent studies using a transgenic mouse model indicate that compounds that relieve ER stress can also reduce the mutation-associated elevation of IOP.
Congenital Glaucoma
Congenital glaucoma is a genetically heterogeneous condition, with both autosomal recessive and autosomal dominant forms reported. Two genes responsible for autosomal recessive congenital glaucoma have been identified, CYP1B1, a member of the cytochrome P-450 family of proteins (cytochrome P-4501B1) and LTBP2 (latent transforming growth factor beta binding protein 2). Mutations in CYP1B1 have been identified in patients with autosomal recessive congenital glaucoma from all over the world but especially in areas where consanguinity is a custom. Responsible mutations disrupt the function of the protein, implying that a loss of function of the protein results in the phenotype. Recurrent mutations are likely to be the result of founder chromosomes that have been distributed to populations throughout the world. Because the defects responsible for congenital glaucoma are predominantly developmental, cytochrome P-4501B1 and latent transforming growth factor beta binding protein 2 must play a direct or indirect role in the development of the anterior segment of the eye.
Nonsyndromic Congenital Cataract
At least one-third of all congenital cataracts are familial and are not associated with other abnormalities of the eye or with systemic abnormalities. A number of different genes can contribute to congenital cataract, including some that code for the crystallin proteins. The human γ-crystallin genes constitute a multigene family that contains at least seven highly related members. All seven of the γ-crystallin genes have been assigned to chromosome 2q34-q35. Of the genes mapped to this region, only two of them, γ-C and γ-D, encode abundant proteins. Two of the genes, γ-E and γ-F, are pseudogenes, which means they are not expressed in the normal lens. A pedigree affected by the Coppock cataract, a congenital cataract that involves primarily the embryonic lens, was shown to be linked genetically to the region that contains the γ-crystallin genes. In individuals affected by the Coppock cataract, additional regulatory sequences have been found in the promoter region of the γ-E pseudogene. This result implies that the γ-E pseudogene is expressed in affected individuals and that expression of the pseudogene is the event that leads to cataract formation. A number of other genes have been associated with hereditary cataract. A useful collection of mutations and phenotypes can be found at the OMIM website ( Table 1.2.1 ).
| NCBI | National Center for Biotechnology Information | http://www-ncbi-nlm-nih-gov.easyaccess1.lib.cuhk.edu.hk/ |
| OMIM | Online Mendelian Inheritance in Man | http://www-ncbi-nlm-nih-gov.easyaccess1.lib.cuhk.edu.hk/omim |
| RetNet | Retinal disease genes | http://www.sph.uth.tmc.edu/Retnet/ |
| Genes and Disease (NCBI Bookshelf) | Systemic inherited disorders | http://www-ncbi-nlm-nih-gov.easyaccess1.lib.cuhk.edu.hk/books/NBK22183/ |
| UCSC | Human genome sequence browser | http://www.genome.ucsc.edu |
Retinitis Pigmentosa
The molecular genetics of retinitis pigmentosa (RP) is exceedingly complex. The disease can exhibit sporadic, autosomal dominant, autosomal recessive, X-linked, or digenic inheritance. At least 200 genes are known to be associated with RP, and a number of genes have been mapped but not yet found. Most of these genes are expressed preferentially in the retina, but some are expressed systemically. A useful resource listing genes responsible for various forms of retinal diseases, including retinitis pigmentosa, can be found at the RetNet website ( http://www.sph.uth.tmc.edu/Retnet/ ).
Mutations in rhodopsin can cause an autosomal dominant form of RP that provides an interesting example of how mutant proteins can interfere with normal cellular processes. Initially, one form of autosomal dominant RP was mapped to chromosome 3q24. With a candidate gene approach, the rhodopsin gene was identified as the cause of the disease in affected families. Many of the first mutations detected in the rhodopsin protein were missense mutations located in the C-terminus of the gene ( Fig. 1.2.4 ). To explore the pathogenical mechanisms of these mutations, transgenic mice were created that carried mutant copies of the gene. Histopathological studies of these mice showed an accumulation of vesicles that contained rhodopsin at the junction between the inner and outer segments of the photoreceptors. The vesicles probably interfere with the normal regeneration of the photoreceptors, thus causing photoreceptor degeneration. Because the C-terminus of the nascent polypeptide is involved in the transport of the maturing protein, the accumulation of rhodopsin-filled vesicles is likely to result from abnormal transport of the mutant rhodopsin to the membranes of the outer segments.
