By synchronizing the reproductive cycle of a group of cells and arresting the progression of the mitotic process, the chromosomes can be visualized microscopically, and specific chromosomal identification can be made on the basis of length and the use of stains such as trypsin-Giemsa (G-banding; Fig. 1.4) (11), quinacrine mustard (Q-banding) (12), “reverse” or R-banding (Giemsa staining following controlled denaturation by heat), silver (staining of nucleolar organizing regions), and C-banding (staining of the condensed chromosome material near the centromere and regions with heterochromatin). All methods identify bands or specific regions and are useful for studying the specific structure of chromosome(s). The number of chromosomes can be counted and the bands of each studied for deletions, duplications, and other rearrangements. This technique is called karyotyping.

Relatively new techniques for arresting the progression of mitosis earlier in the cell cycle have been developed. In late prophase or early metaphase, the chromosomes are longer and less condensed and bands are further subdivided; smaller deletions and/or duplications can be detected. Extended chromosome analysis, termed high-resolution banding, is much more time-consuming but is particularly useful if a specific chromosomal abnormality or rearrangement is suspected. As the method is more labor intensive and expensive, one would not order high-resolution banding for a routine sample.

A cytogenetic nomenclature system has been adopted to describe the human chromosomal complement and indicate deviations from normal. An extra chromosome is indicated by a plus (+) and a missing one by a minus (-); thus 47,XX, +21 is a female with trisomy 21 (three copies of chromosome 21). The short arm of a chromosome is called p and the long arm is q. Chromosomal bands are numbered according to landmarks starting from the centromere up the short arm and down the long arm. Chromosomal rearrangements are described by noting the rearrangement and indicating the breakpoint(s). For example, a female with a deletion of the short arm of chromosome 4 with breakpoint at band p15 has the karyotype: 46,XX,del (4)(p15). A ring is indicated as r (e.g., 46,XY,r[13]male with a ring chromosome 13) and translocation as t (e.g., 46,XX,t[3;9][p14; q21] female with a translocation between chromosomes 3 and 9 with breakpoints at band p14 on chromosome 3 and band q21 on chromosome 9). An inversion is indicated as inv (e.g., 46XY,inv[2][p12q12]male with an inversion of chromosome 2 with breakpoints at p12 and q12).

Recently, DNA probes have become available for all human chromosomes—greatly expanding the field of molecular cytogenetics. If the origin of a duplication or translocation is unknown, the DNA probes can be fluorescently labeled and “painted” onto the metaphase spread or interphase nuclei chromosomal preparation, a technique called fluorescent in situ hybridization (FISH); chromosomal abnormalities can be readily identified under the microscope.

Telomeres, the physical ends of linear eukaryotic chromosomes, are specialized nucleoprotein complexes that have important functions, primarily in the protection, replication, and stabilization of the chromosome ends. In most organisms studied, telomeres contain lengthy stretches of tandemly repeated simple DNA sequences composed of GC-rich strands (called terminal repeats). These terminal repeats are highly conserved; in fact, all vertebrates appear to have the same simple sequence repeat in telomeres: (TTAGGG)n. Often sequences adjacent to the telomeric repeats are highly polymorphic, are rich in DNA repetitive elements (termed subtelomeric repeats), and in some cases, genes have been found in the proterminal regions of chromosomes.

Telomerase is the reverse transcriptase enzyme responsible for the extension of telomeric repeat sequences in most species studied. If telomerase activity is diminished or absent, telomeres will shorten. Shortened telomeres appear to lead to cell senescence (13). Eventually, telomeric sequences can shorten to the point where they are not long enough to support the telomere-protein complex protecting the ends and the chromosomes become unstable. These shortened ends become “sticky” and promote chromosomal rearrangements (14,15). Some rearrangements may contribute to the development of cancers (16,17). Telomere testing has been shown to identify alterations in 7% to 10% of cases with moderate/severe mental retardation and cases of multiple congenital anomalies with mental retardation in the setting of normal karyotype testing (18). The analysis involves the detection of deletions, duplications, or cryptic translocations using subtelomere FISH probes on each chromosome (15).

Comparative genomic hybridization (CGH) was developed to screen the entire genome for DNA content differences by comparing a test sample to a control (19,20). Because metaphase chromosomes are used as the substrate for analysis, the detection of unbalanced alterations is limited to the resolution of the metaphase target (at the level of a 450-band karyotype, approximately 5- to 10-Mb change). DNA microarray CGH is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by cohybridizing differentially labeled test and control DNA samples to a microarray chip (21,22,23). The chip (small metallic platform with applied spots of known large-insert
DNA clones such as bacterial artificial chromosomes) technology offers higher resolution, is faster, and is highly sensitive. Because the DNA clones have a known map location and information, detected alterations are immediately linked to known genetic markers (pieces of DNA with a known chromosomal location and sequence), and the genetic location of a chromosomal abnormality such as a deletion or duplication can be determined by the map distances between known markers or by the length of the clones used.

Newer techniques of producing multiple copies of the coding regions of the genome (exomes), or the entire genome including exons and intronic sequence (whole genome) have revolutionized gene variant detection in disease (www.ambrygen.com/exome-sequencing). There are many factors that make exome sequencing superior to single-gene analysis including the ability to identify mutations in genes that were not tested due to an atypical clinical presentation, or the ability to identify clinical cases where mutations from different genes contribute to the different phenotypes in the same patient. This technology is particularly useful for gene discovery in those conditions in which mapping has been confounded by locus heterogeneity and uncertainty about the boundaries of diagnostic classification. Direct-to-consumer whole exome sequencing services are available to individuals without a physician intermediary through programs such as 23andMe (www.23andme.com/).

For some mutations, the loss of a functional protein product from one of the chromosomes is asymptomatic because the gene on the homologous chromosome produces a normal product. If 50% activity of a protein is sufficient for normal function, the individual with one mutation is called a carrier, the signs and symptoms of the disease would be evident only in those who have two abnormal genes. Such a disorder is called autosomal recessive and is evident in some (statistically 25%) of the offsprings of two carriers (Fig. 1.5). In general, there is a higher incidence of parental consanguinity associated with rare autosomal recessive disorders. For some diseases, carriers probably have a common ancestor because nearly all affected individuals have the identical mutation; an example of this founder effect is Tay-Sachs disease (OMIM 272800: infantile developmental delay, paralysis, dementia, blindness with lipid-laden ganglion cells leaving a central “cherry-red” spot funduscopically, premature death before the age of 5 years). Mutations of the implicated gene, the enzyme hexosaminidase, occur more commonly in the Ashkenazi Jewish population.

When the protein is structural or the organism requires 100% activity for normal structure or function, phenotypic abnormalities are evident with a mutation of only one of the two homologous chromosomes. Such a disorder is termed autosomal dominant because the presence of a mutation on one of the chromosomes results in a phenotypically identifiable disease state. An affected individual with an autosomal dominant trait has a 50% chance of passing the gene to each offspring (Fig. 1.6). Examples of autosomal dominant disorders include Marfan syndrome (OMIM 154700: dislocated lenses, dilated aortic root, increased height, disproportionately long limbs and digits, anterior chest deformity, joint laxity, narrow arched palate, scoliosis due to connective-tissue disorder; caused by mutations of the fibrillin-1 gene); neurofibromatosis type I (OMIM 162200: consistent features of cafe au lait spots, Sakurai-Lisch nodules of the iris, fibromatous skin tumors, occasional hamartomatous tumors found systemically; due to mutations in the neuronbromin gene); and Best disease (OMIM 153700: mutations in the bestrophin gene cause
juvenile-onset and adult vitelliform macular dystrophy with collections of lipofuscin-like material in the subretinal space creating macular lesions which resemble an egg yolk). Disease-causing genes located on the autosomes occur equally in males and females, regardless of dominant or recessive inheritance.

Mutations on the X chromosome are unique, as normal females have a pair, and normal males have only one X chromosome along with an unpaired Y chromosome. X-linked diseases may be recessive or, much less frequently, dominant. Therefore, one abnormal gene for which an individual requires only some of the protein product usually would not cause disease in a female, who is a heterozygote, but would in a male, who is a hemizygote. Such disorders are termed X-linked recessive and become clinically evident in the male as he has only one X chromosome copy. Occasionally, a heterozygous female carrier exhibits some manifestations of an X-linked recessive disease. The expression of the single recessive gene on the × chromosome in a female has been explained by the X inactivation theory, or Lyon hypothesis (25). During the second week of embryonic life, one of the two X chromosomes in each cell of the female fetus randomly becomes the “inactive X”; this X chromosome becomes condensed during interphase and appears as a darkly stained mass at the nuclear membrane (Barr body or sex chromatin; Fig. 1.7). Most of the genes on the inactive X are not expressed. Once this differentiation occurs, the same X chromosome continues to be the inactive one in all the linear descendants of that cell. Thus, the female is a mosaic of two cell lines, those in which the genes on the maternally inherited X are active and those in which the paternally inherited X are active. The Lyon hypothesis is invoked to explain the splotchy pigment epithelium and choroidal pigmentation in the fundus of a female carrier of ocular albinism (OA), or the tapetal reflex in the carrier of X-linked retinitis pigmentosa (ghr.nlm.nih.gov/glossary=xch romsomeinactivation).

The heritable inability to correctly perceive the color green, known as Daltonism (after the English chemist John Dalton, who himself was affected), was the first human trait mapped to the X chromosome (www.colblindor.com/2006/04/09/daltonism). X-linked disorders of the eye for which phenotypic evidence of the carrier state may be present in females include choroideremia (OMIM 303100: degeneration of the choriocapillaris and retina due to mutations in the Rab escort protein-1 gene [REP1]); Nance-Horan or cataract-dental syndrome (OMIM 302350: affected males have dense nuclear cataracts and frequent microcornea, and carrier females show posterior Y-sutural cataracts with small corneas and only slightly reduced vision; caused by mutations in the NHS gene) (26,27); blue cone monochromatism (OMIM 303700: affected males have poor central vision and color discrimination, infantile nystagmus, and nearly normal retinal appearance due to mutations in the locus control region upstream to the red and green cone pigment gene array) (28); and Lowe syndrome (OMIM 309000: affected males have cataracts, developmental delay, vitamin D-resistant rickets, and aminoaciduria, and carrier females have peripheral cortical lens opacities) (29). A carrier female has a 50% chance of passing a mutant gene on the X chromosome to any offspring; thus, the daughters have that same chance of being carriers and the sons of being affected with the disease (Fig. 1.8). Examples of X-linked recessive disorders include Duchenne muscular dystrophy (OMIM 310200: due to mutations of the dystrophin gene, affected males display progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves and severe cardiomyopathy; there is massive elevation of creatine kinase levels in the blood, myopathic changes by electromyography, and myofiber degeneration with fibrosis and fatty infiltration on muscle biopsy); and X-linked juvenile retinoschisis (OMIM 312700: affected males with RS gene mutations have intraretinal splitting due to degeneration). If the X-linked gene is dominant, both the heterozygous female and hemizygous male manifest
the mutant phenotype. It has been observed in the few X-linked dominant traits that a heterozygous female is more likely to have female offspring, and it has been postulated that there is fetal wastage of males. Presumably, the hemizygous state of the mutation may be lethal. Examples of X-linked dominant disorders include familial incontinentia pigmenti type II (OMIM 308300: mutations in the IKK-gamma gene causing skin defects of perinatal inflammatory vesicles, verrucous patches, dermal scarring, and retinal vascular anomalies) and Aicardi syndrome (OMIM 304050: affected females have flexion spasms and lacunar lesions of the choroid and retina).

An additional source of genetic information in the cell lies in the mitochondria (30). Each cell contains hundreds of mitochondria, each of which contains multiple copies of a 16,569base pair circular, double-stranded DNA molecule. This DNA encodes 13 peptides that are subunits of proteins required for oxidative phosphorylation. In addition, there is a complete set of 22 tRNAs and two rRNAs. These RNAs are involved in translation of mitochondrial-encoded proteins within the mitochondrion. Mitochondria are responsible for the generation of ATP via aerobic metabolism. Most mitochondrial proteins are encoded by nuclear genes; however, some are encoded by mitochondrial genes, and mutations can lead to energy failure. The mitochondrial genome is subject to a number of peculiarities of inheritance including maternal transmission and a phenomenon known as heteroplasmy, resulting in distinctive patterns of familial disease. Heteroplasmy determines expression variability of the disease within a family. Different cells in an individual and different individuals in a family contain different proportions of mutant and wild-type mitochondria (31).

Mitochondrial mutation disorders display maternal genetic transmission. There may be the appearance of transmission directly from generation to generation, suggesting dominant inheritance. Both males and females may be affected, but men never transmit the disorder to any of their offspring. Women, on the other hand, pass the trait to all of their children, although expression may be more severe in some than in others. At the time of fertilization, the sperm sheds its tail, including all of its mitochondria. Only the sperm head, containing nuclear DNA, enters the egg. Therefore, all the mitochondria for the next generation are contributed by the egg cell. Hence, mitochondrial genes are exclusively maternally derived, explaining the pattern of maternal transmission.

Major mitochondrial gene mutation syndromes include Kearns-Sayre (OMIM 530000: external ophthalmoplegia, pigmentary retinopathy, heart block, ataxia, increased cerebrospinal fluid protein); myoclonic epilepsy with ragged red fibers or MERRF (OMIM 545000: myoclonic epilepsy, myopathy, dementia); mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes or MELAS (OMIM 540000: lactic acidosis, stroke-like episodes, myopathy, seizures, dementia); Leber hereditary optic neuropathy (OMIM 535000: blindness, cardiac conduction defects); and Leigh syndrome (OMIM 256000: movement disorder, respiratory dyskinesia, regression) (32,33,34,35,36).

The clinical features of single-gene mutations vary from one individual to another. Penetrance is the percentage of known carriers manifesting the phenotype and reflects our ability to identify an individual with a mutant gene; reduced penetrance means that some individuals with the gene may not exhibit clinical evidence of the disease. Nonpenetrance is defined as the absence of phenotype in a person known to carry a specific mutant gene. Nonpenetrance has been demonstrated to occur with many genetic traits and can be most easily inferred when a grandparent and child have a disorder that does not appear to be expressed in the parent. For example, some individuals without evidence of colobomatous microphthalmia have an affected ancestor and an affected child (Fig. 1.9). The only potential explanation is that the apparently unaffected individual has the mutant gene in his or her genome but that the clinicians cannot find the evidence by ocular examination. If four family members descended from the same affected ancestor had affected progeny but one was free of the disease, the gene would be 75% penetrant.

Expressivity refers to the degree of phenotypic expression of a genetic trait. A disease may exhibit different manifestations among individuals in the same family. Such variable expressivity is common, particularly in autosomal dominant disorders. For example, some individuals with Marfan syndrome (OMIM 154700), an autosomal dominant disease of the connective-tissue gene fibrillin, may be tall and have dislocated lenses; others may be of normal stature and show no evidence of a dislocated lens but have an aneurysm of the ascending aorta.

Autosomal recessive disorders can exhibit variable expressivity, particularly if the mutation occurs at different sites in the protein. For example, there are numerous hemoglobinopathies with distinctive clinical manifestations that are caused by different mutations of the hemoglobin gene.

Different gene defects, modes of inheritance, and chromosomal abnormalities can produce similar clinical phenotypes, a phenomenon termed genetic heterogeneity (ghr.nlm. nih.gov/glossary=geneticheterogeneity). Examples of genetic heterogeneity are common in ophthalmology. Classification of diseases is thus most reliably made on the basis of specific gene mutations or chromosomal aberrations; doing so allows for directed therapies.

Epigenetics is the study of changes in gene expression or cellular phenotype caused by mechanisms other than changes in the original DNA sequence (www.pbs.org/wgbh/nova/body/epigenetics.html, and learn.genetics.utah.edu/content/epi genetics/). The development and maintenance of an organism is orchestrated by a set of chemical reactions that switch parts of the genome off and on at strategic times and locations. Epigenetics is the study of these reactions and the factors that influence them. In epigenetic modification, a methyl group (-CH3) is added to specific cytosine bases of the DNA. This enzymatic process, called DNA methylation, is known to play a key role in both development and disease. Methylation is a physical modification to the DNA that affects the way the molecule is shaped and, consequently, regulates which genes are transcriptionally active. Another epigenetic modification of DNA is the addition of a hydroxymethyl group (-CH2-OH) to specific cytosine bases of DNA. In eukaryotic cells, genomic DNA is wrapped around histone proteins to form nucleosomes, which together form chromatin fibers that can be further compacted into dense coils. Epigenetic modifications to histone proteins can either inhibit or promote coiling or condensation of the chromatin, leading to chromatin organizational changes, which in turn affects how the associated genes are expressed.

The autosomal recessive Hurler syndrome or mucopolysaccharidosis type IH (OMIM 607014: corneal clouding, coarse facies, developmental delay, hepatosplenomegaly, and hernia) and X-linked recessive Hunter syndrome or mucopolysaccharidosis type II (OMIM 309900: no corneal clouding, coarse facies, dwarfism, hepatosplenomegaly, and deafness) may be difficult to differentiate clinically on the basis of physical findings in a young boy with no family history. As the inheritance patterns are different, the gene defects are distinctive (http://ghr.nlm.nih.gov/condition/mucopolysaccharidosis-type-i). Conversely, the clinical features of the Hurler and Scheie (OMIM 607016: stiff joints, clouding of the peripheral cornea, and aortic regurgitation) syndromes can be differentiated by the mental retardation and early death that are features of the Hurler syndrome. Life expectancy in the Scheie form is nearly normal and mental retardation is very rare. Originally the two were classified as different diseases. With the identification of the enzymatic defect, it became clear that the disorders were allelic as the same gene is mutated in both, causing a deficiency in the enzyme alpha-1-iduronidase.

Leber congenital amaurosis (LCA; OMIM 204000) is a group of autosomal recessive retinal dystrophies. It is the most common genetic cause of congenital retinal disorders in infants and children, and results in significant and often severe vision loss at an early age. Its incidence is 2 to 3 per 100,000 births and it accounts for 10% to 18% of cases of congenital blindness. At least 17 genes contribute to this disorder (37). The observed genetic heterogeneity is the result not only of the number of genes that have been implicated in LCA, but also the consequences of the different mutations in these genes. Mutations in at least two of these genes—RPE65 and CRX—not only cause an LCA clinical presentation, but also lead to other late-onset retinal dystrophies such as retinitis pigmentosa and cone-rod dystrophy (38,39). Gene therapy was used to successfully recover vision in a canine model of LCA (40). The researchers designed an adeno-associated virus vector to use in retinal transgene delivery. This vector was used to
carry wild-type canine RPE65 complementary DNA and was injected into the subretinal space of dog eyes. The LCA dogs showed visual recovery in performing visual function and behavioral tests. Multiple gene therapy trials are in progress to treat humans with the same genetic disease (37).

Albinism comprises a group of heterogeneous inherited abnormalities of melanin synthesis characterized by a congenital reduction or absence of melanin pigment associated with specific developmental changes of the visual system (www. albinism.org). Oculocutaneous albinism (OCA) involves two regions of the body: the skin and hair and the optic system, including the eye and optic nerves. Ocular albinism (OA) has similar changes in the visual system by reducing mainly the pigment in the retinal pigment epithelium (RPE) of the eye, usually with no clinical difference in the color of the skin and hair. Ophthalmologic signs, although variable, include nystagmus, hypopigmentation of the uveal tract and RPE, iris transillumination, foveal hypoplasia, and abnormal decussation of the optic nerve fibers at the optic chiasm.

The formation of melanin pigment is a complex event requiring several enzymes and proteins and the pigmentcontaining subcellular organelle, the melanosome. Melanin pigment is produced in the melanocyte, which is found in the skin, hair follicles, iris, and RPE of the eye. Melanin biosynthesis begins with hydroxylation of the amino acid 1-tyrosine to dihydroxylphenylalanine (DOPA) and the oxidation of DOPA to DOPA quinone by the copper-containing enzyme tyrosinase, resulting in either black-brown eumelanin, or in the presence of sulfhydryl compounds, red-yellow pheomelanin. The resulting pigment polymer is deposited on a protein matrix within the melanosome. In the skin and hair follicles, the melanosome is then transferred to keratinocytes via the dendrites of the melanocyte.

At present, four genetic loci responsible for human albinism have been mapped (OCA1: OMIM 203100, OCA2: OMIM 203200, Hermansky-Pudlak syndrome: OMIM 203300, and OA1: OMIM 300500); three of the genes have been isolated and pathologic mutations identified (OCA1, OCA2, and OA1). The classic “tyrosinase-negative” albinism results from tyrosinase gene mutations that inactivate the encoded enzyme and is categorized as OCA1 (41). Mild forms of OCA1, initially described as autosomal recessive OA, result from mutations of the tyrosinase gene that produce an enzyme with residual activity. OCA in which hairbulbs form melanin upon incubation in DOPA or tyrosine (tyrosinase-positive OCA) can be caused by mutations at several loci, including OCA1; OCA2, which is associated with the human homolog (P) of the mouse p gene— a melanosomal transport protein (42); and the rare Hermansky-Pudlak syndrome (OCA with platelet dysfunction).

Magnetic resonance imaging (MRI) size and configuration comparisons of the optic pathways in normal versus albinism phenotypes revealed significantly smaller optic nerves and tracts, smaller chiasmatic widths, and wider angles between nerves and tracts in the albino group than in the control group (43). The chiasms of the albinos are shaped like an X, whereas the chiasm in the controls was shaped like two back-to-back parentheses, that is, “)(.” These differences reflect the atypical crossing of optic fibers. MRI can be an important diagnostic tool in patients with equivocal albinotic presentations.

X-linked OA1 is caused by mutations in the OA1 gene at chromosome Xp22.3-p22.2, which encodes a membrane glycoprotein localized to melanosomes. Approximately 48% of the reported mutations in the OA1 gene are intragenic deletions and about 43% are point mutations. Faugere and associates (44) report three OA1 unrelated families with an initial diagnosis of congenital nystagmus. They identified three novel OA1 mutations consisting of two intragenic deletions and a point mutation. Direct testing of carrier females is advocated and can be performed by direct sequencing or by scanning methods such as denaturing gradient gel electrophoresis or denaturing high-performance liquid chromatography. The real-time fluorescent PCR gene-dosage assay is an accurate, nonradioactive, and fast method for gene carrier assessment that can be applied to any type of OA1 gene deletion. A similar two-tiered diagnostic test strategy for mutation screening for OA1 has been proposed (45).

A single-gene mutation that is expressed in many different tissues or can affect more than one organ system is termed pleiotropic. Waardenburg syndrome (Ptosis-epicanthus syndrome) is an example of a disorder in which the mutation of one gene has multiple organ effects. This dominantly inherited disorder includes displaced canthi, heterochromia of the irides, white forelock, broad nasal root, and deafness (Fig. 1.6); the syndrome is divided into four types (I, II [A, B, and C], III, and IV) with the most severe being associated with upper limb defects (type III; OMIM 148820) and a ganglionic megacolon (Hirschsprung disease) (type IV; OMIM 277580). Dystopia canthorum, the lateral displacement of the inner canthi, distinguishes type I (OMIM 193500) (which shows this clinical phenotype) from type II. Types I and III (OMIM 148820) are caused by mutations in the PAX3 gene, which was first identified in Drosophila (46,47). Mutations in the transcription factor MITF have been implicated for Waardenburg syndrome type IIA (OMIM 193510) (48,49,50); digenic inheritance of type IIA and autosomal recessive OA has been proposed (51). Type IV or Waardenburg-Shah syndrome is a disorder of the embryonic neural crest that combines clinical features of Waardenburg syndrome and Hirschsprung disease with colonic aganglionosis.

Alagille syndrome (OMIM 118450) is an autosomal dominant disorder characterized by cholestatic liver disease, pulmonic valvular and peripheral arterial stenosis, “butterfly” vertebrae, posterior embryotoxon (anterior displacement of Schwalbe’s
line) with retinal pigmentary changes in the eye in some individuals, and unusual facies with broad forehead, pointed mandible, and bulbous nose tip (http://digestive.niddk.nih.gov/ddiseases/pubs/alagille/). The syndrome is due to mutations in the Notch signaling JAG1 gene on chromosome 20p12 (52,53).

Cornelia de Lange syndrome (CDLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation, abnormalities of the upper limbs, gastroesophageal dysfunction, hirsutism, cardiac and genitourinary anomalies, and characteristic facial features (54,55,56) (www.cdlsusa.org/). Ophthalmic features include ptosis, nasolacrimal duct obstruction, arched eyebrows with synophrys, long eyelashes, refractive error, and infrequent glaucoma (57). The prevalence is estimated to be as high as 1 in 10,000. Gene mutations in the NIPBL gene on chromosome 5p13.1 have recently been associated with this disorder (58,59). The jly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in D. melanogaster (60).

Marfan syndrome (OMIM 154700) is an autosomal dominant connective-tissue disorder with an estimated incidence of 1 in 5,000 with approximately 25% being sporadic cases (www.marfan.org/). Three systems are predominantly involved: the skeleton, heart, and eye. Common and major manifestations of the disease include subluxation of the crystalline lens; dilatation of the aortic root and aneurysm of the ascending aorta; and skeletal abnormalities such as pectus excavatum and kyphoscoliosis and an upper segment/lower segment ratio of two standard deviations below the mean for age. Other criteria such as myopia, mitral valve prolapse, arachnodactyly, joint laxity, tall stature, pes planus, pneumothorax, and dural ectasia are also contributory. The diagnostic criteria have been recently revised, requiring involvement of three systems with two major diagnostic manifestations (61). The gene defect has been identified: Fibrillin (FBN1) maps to chromosome 15q15-q21.1. It is a large gene of more than 230 kb and highly fragmented with 65 exons. The protein is a 350-kDa glycoprotein and is the principal structural component of connective-tissue microfibrils found ubiquitously in all extracellular matrices. Fibrillin structures serve as scaffolds for the deposition of elastin in elastic tissues (61).

To date, more than 500 mutations have been identified in the FBN1 gene in Marfan syndrome patients and related diseases (61). Presently, no definite genotype-phenotype correlations have been identified except for neonatal mutations. An association with a subset of mutations in exons 24 to 32 and neonatal MFS appears correlative, thus molecular diagnostic testing can be performed (62). With a few exceptions, almost all mutations found in fibrillinopathies other than classic Marfan syndrome have been unique to one affected individual or family, which has hampered the delineation of potential genotype-phenotype correlations (62).

Comeglio and associates (63) characterized the incidence and class of FBN1 mutations in the largest series reported to date, a group of 11 consecutive British patients affected predominantly by ectopia lentis (EL). The investigators identified six causative mutations in the fibrillin 1 gene (FBN1)—three mutations are novel and one was recurrent in two patients—thus establishing an FBN1 mutation incidence of 63% (7/11). These results are within the 23% to 86% range of mutation identification rate in Marfan syndrome and related patients in recent investigations. All mutations were within the first 15 exons of the fibrillin gene, while database citations of Marfan mutations are distributed throughout the gene. A different type of FBN1 mutation presents in this group of patients, with arginine to cysteine substitutions appearing frequently. Patients with predominant EL should be screened for FBN1 mutations. Echocardiography is recommended initially and at regular intervals throughout the patients’lifetimes, as there is a tendency for late-onset aortic dilatation and/or dissection to develop.

Lens subluxation is the diagnostic ocular abnormality in this disease. It is present in 65% to 70% of patients and varies from a mild superotemporal displacement of the lens seen only in postpupillary dilation to significant subluxation that places the equator of the lens in the pupillary axis. Subluxation of the lens due to stretched or absent zonular fibers is slowly progressive in the first two decades of life. Further displacement at a later age is uncommon. Total dislocation into the vitreous cavity is uncommon and may be complicated by phacolytic uveitis and glaucoma. Anterior displacement of the lens into the anterior chamber or within the pupillary space is rare in Marfan syndrome with EL. Premature cataracts are common, developing 10 to 20 years earlier in patients with Marfan syndrome than in the general population.

Paired box gene 6 (PAX6) is the prototype for an eye master-control gene. It is one of the family of genes that encode transcription factors with a homeodomain and a paired domain. Loss of function leads to the eyeless (ey) phenotype in Drosophila and also causes severe ocular defects in many other animals (64). Interestingly, ectopic (at a different location other than normal) expression of Pax6 in Drosophila, mouse, and frog (Xenopus) causes the formation of a functional eye. This supports two concepts: (a) homologous genes in different species have the same function—to “switch on” eye development; (b) there is only one way to make the eye, by a cascade of signals initiated by Pax6 expression (65).

In humans, PAX6 mutations mainly cause aniridia (OMIM 106210), which is a panocular disorder, and less commonly cause isolated cataracts and macular hypoplasia (failure of retinal foveal development; OMIM 136520), keratitis (OMIM 148190), and Peters’ anomaly (central corneal opacity which is frequently associated with adhesion between the cornea and the lens; OMIM 603807) (66). As in the mouse, the homozygous loss (both parental alleles have mutations) of PAX6 function in humans affecting all expressing tissues is lethal.

Targets of PAX6 encode other transcription factors or structural proteins of the lens (crystallins) and cornea (keratins 1-12). Many PAX6-regulated transcription factors (such as SIX3, FOXE3, MAF, MITF, PROX1, LHX2, and PITX3) are involved in the formation of the cornea and lens; others (such as PAX2, CHX10, and EYA1) are involved in retina and optic nerve development (67).

Only a few transcription factors or signaling molecules (e.g., BMP4, BMP7, RX, and SHH) are known to regulate PAX6. Among them, SHH might be highest in the hierarchy: knockout mice and humans with mutations in SHH suffer from holoprosencephaly with ocular manifestations that range from microphthalmia to cyclopia, which indicates a disruption in the earliest event in eye development, separation of the central eye field (68).

Genotype and phenotype information for human PAX6 mutations is freely available from the PAX6 mutation database (http://pax6.hgu.mrc.ac.uk).

Sex-determining region Y-related high-mobility group box gene 2 (SOX2) is expressed in the developing lens placode, lens pit, optic cup, neural retina, lens, brain, and ear; heterozygous mutations of the gene result in anophthalmia in humans. Interestingly, all of these mutations seem to occur de novo (spontaneously) and are inherited as dominant alleles (69).

The retina and anterior neural fold homeobox gene (RX) encodes a homeodomain transcription factor and is one of the first retinal patterning genes to be expressed during development (70). Rx-deficient mouse embryos lack eye anlagen (simple preliminary organ structures in the embryo) and do not express Pax6 in the eye field, which indicates that Pax6 upregulation in these tissues is dependent on functional Rx expression (71). It has recently been shown that the spontaneous mouse mutant eyeless (analogous to anophthalmia in humans) is caused by a point mutation in an alternative translation-initiation codon of Rx (72).

Microphthalmia (OMIM 309700), a term derived from the Greek micro (small) and ophthalmos (eye), refers to a congenital malformation in which the volume of the eye is reduced; the spectrum ranges from mild reduction in the anteroposterior axis to histologically documented anophthalmia (OMIM 206900) (www.ncbi.nlm.nih.gov/books/NBK1378/). Nanophthalmia (OMIM 600165 and 605738) is used to describe microphthalmia with normal intraocular structures, and refers to a refractive error of +8.00 D or greater hyperopia. Although microcornea or high hyperopia may be a useful clinical clue, the diagnosis frequently can be made by inspection alone; however, as microcornea can occur in the absence of microphthalmia (73,74) and conversely, microphthalmia in association with a normal-sized cornea (75,76,77), the clinical diagnosis may be inaccurate (http://eyepathologist.com/disease.asp?IDNUM=308040). As the spectrum of microphthalmia varies from slightly reduced axial length to histologically documented anophthalmia, ultrasonography with precise measurement of the anteroposterior axis is essential for the diagnosis. Since most normal adult eyes range from 21.50 to 27.00 mm, an adult axial length of less than 20 mm should be considered abnormal (78,79). Despite the use of this technologically advanced tool, extreme microphthalmia may be difficult to distinguish from anophthalmia.

Microphthalmia appears to be a relatively common ocular malformation in all races. The high prevalence, which would be unusual for a disorder caused by a single gene, suggests multiple causes. Few studies have documented the prevalence in the general population. In a prospective study of more than 50,000 pregnancies in the United States in the late 1960s, the incidence of anophthalmia or microphthalmia was found to be 0.22 per 1,000 births and that of coloboma to be 0.26 per 1,000 (80). Prevalence among blind adults varies from 0.6% to 1.9% (81,82). In the pediatric age group, it accounts for 3.2% to 11.2% of cases of blindness (82,83,84). These differences are not readily explainable
but may reflect the race or population studied; the highest prevalence (11.2%) was found in the 1980 survey on the causes of blindness among Japanese schoolchildren.

Visual impairment in microphthalmia varies from little or no loss to absence of vision, as would be found in cases of anophthalmia. The degree of visual loss best correlates with the associated abnormalities and degree of microphthalmia; cataracts, optic nerve hypoplasia, and/or colobomata of the macula or optic nerve may cause significant visual impairment.

Cases of microphthalmia may be divided into two general forms: colobomatous and noncolobomatous. Although uveal colobomata may occur in the absence of microphthalmia, the two are frequently associated and presumably etiologically related. A coloboma may occur in the iris, choroid, optic nerve, or any combination thereof (Figs. 1.10 and 1.11). The colobomata result from incomplete closure of the fetal fissure, a process normally completed by the 6th week of gestation (85). The embryonic processes that determine the size of the eye are poorly understood. Congenital cystic microphthalmia and anophthalmia are extreme forms of this dysembryogenesis (86,87). As extreme microphthalmia may be difficult to distinguish from anophthalmia, serial sectioning of the orbit may be necessary to differentiate them. The malformation may be unilateral or bilateral, and asymmetry is common.

Microphthalmia, with or without colobomata, may be a manifestation of many different disorders: genetic, environmental, and those of unknown cause. The evaluation of a patient is interdisciplinary. Aside from a complete ocular examination, all patients should have a careful history, including a genetic pedigree and physical examination. In certain cases, family members should also be examined.

Isolated colobomatous microphthalmia (OMIM 300345) may occur as an autosomal dominant disorder with incomplete penetrance; expressivity varies from small colobomata of the eye to microphthalmia and even anophthalmia (Fig. 1.9). Microphthalmia without colobomata also may be inherited as an autosomal dominant disease; more commonly, associated congenital cataracts or other ocular malformations are present (88,89). Microphthalmia with congenital retinal detachment or congenital cataracts may be inherited as an autosomal recessive disorder (90,91).

Microphthalmia (Mi) was one of the first mouse mutant genes to be described in which the development of the retina is affected. An interesting allelic series ranging from weak recessive to severe dominant phenotype has been compiled and genetically characterized. The eyes of the mutants develop poorly because of the defects in the RPE. The mutated gene, microphthalmia-associated transcription factor (Mitf) is a member of the basic-helix-loop-helix leucine zipper family of transcription factors. Mutations in the
human homolog MITF (OMIM 156845) cause 20% of cases of Waardenburg syndrome type IIA (OMIM 193510) (92).

Human mutations in SIX3 lead to holoprosencephaly; in some cases, the phenotype is milder and manifests as microphthalmia and iris coloboma (93). Several reports indicate that SIX3 might function as a repressor of some developmental processes in the eye (94,95). In the mouse, Six3 is activated by Pax6 and Prox1 (a transcription factor that is important for the differentiation of lens fiber cells and alpha crystalline expression) but is regulated by its own negative feedback loop (96). Transgenic mouse experiments have shown that Pax6 and Six3 regulate the transcription of each other (97).

The CHARGE syndrome (OMIM 214800) is characterized by the nonrandom association of colobomatous microphthalmia, heart defects, atresia choanae, retarded growth, genital anomalies, and ear anomalies or deafness; at least two of the features are necessary for the diagnosis (98,99,100,101,102,103,104) (www. chargesyndrome.org/). The ocular malformation is a common feature; the cardiac defects described in the syndrome are varied and may be lethal. The phenotypes of the chromosomal trisomy syndromes (13,18), 4p— (Wolf-Hirschhorn; deletion of the short arm of chromosome 4) syndrome, and cat-eye syndrome may be similar to the features of CHARGE association, and chromosomal analysis may be necessary to clarify the diagnosis (see section “Chromosomal Rearrangements”).

Two genetic loci have been identified in conjunction with isolated high hyperopia, autosomal dominant nanophthalmos (NN 01; OMIM 600165) on chromosome 11p (105) and NNO2 (OMIM 605738), which maps to chromosome 15q12-15. The phenotype for NNO2, however, is not of isolated, nonsyndromic high hyperopia. Rather it is that of unilateral or bilateral microphthalmos with variable expressivity such as corneal clouding and iridocorneal synechiae resembling Peters’ anomaly or no microphthalmia with optic nerve agenesis.

The development of the eyelids and ocular adnexal structures is closely related to the formation of the eyes themselves. Complete failure of the eyelids to form results in cryptophthalmos (OMIM 123570), a condition in which the skin extends from the forehead to the cheeks uninterrupted, but attached to a usually malformed globe underneath. It may occur in isolation or as part of Fraser syndrome (OMIM 219000), which is autosomal recessive. A review of 27 cases of isolated cryptophthalmos revealed 11 of those to be familial, inherited in a dominant fashion (106). A gene has not yet been identified. Treatment depends upon the functional capability of the underlying globe, and this can be measured using imaging modalities in conjunction with visually evoked response and electroretinogram testing. If visual potential can be demonstrated, surgery may be attempted with the goal of creating a clear visual axis, possibly via a keratoprosthesis, as well as creating functioning lid structures. There is a clinical trial for this and a closely related disorder, Fryns syndrome (clinicaltrials.gov/ct2/show/NCT00032877).

Eyelid formation during development requires the eyelid folds to initially fuse and then to separate into upper and lower lids. The failure of this separation results in ankyloblepharon (OMIM 106250 and 106260), a condition in which the eyelids are completely or partially joined. Filiforme adnatum is a unique condition characterized by multiple strands of tissue connecting the two eyelids.

Structural maldevelopment of the eyelid may result in an eyelid coloboma or a disruption in the margin of the eyelid. Isolated eyelid colobomata range from a near total absence of the lid to the appearance of a small notch in the lateral aspect of the lid. Colobomata of the eyelid may also be part of a syndrome such as Treacher Collins syndrome (OMIM 154500) or Goldenhar syndrome (OMIM 164210) or may occur in association with cleft palate, dermoid, cleft lip, microphthalmia, iris colobomata, brow colobomata, and osseous facial clefts.

Congenital ptosis is due to a deficiency of the striated muscle fibers in the levator muscle. This abnormality may occur in three main forms: simple hereditary ptosis, with external ophthalmoplegia, and in the blepharophimosis syndrome (horizontal narrowing of the palpebral fissure; OMIM 110100). Simple congenital ptosis, unilateral or bilateral, may be inherited in an autosomal dominant fashion with incomplete penetrance (107). A gene for hereditary congenital ptosis type 1 (OMIM 178300) has been mapped to 8q21.12 (108). A case of bilateral isolated ptosis was also reported in a male patient found to have a de novo balanced translocation t(1;8) (p34.3;q21.12). The cytogenetic breakpoints were refined, and the chromosome 8 breakpoint was found to disrupt a gene homologous to the murine zfh4 gene, which encodes for a transcription factor expressed in muscle and nerve tissue. This suggests the human ZFH4 may be a candidate gene for congenital bilateral isolated ptosis (109). Analysis of a large white English pedigree revealed an X-linked dominant form of congenital isolated ptosis that mapped to chromosome Xq24q27.1. This was named hereditary congenital ptosis type 2 (OMIM 300245) (110). Treatment for isolated congenital ptosis is surgical and may be performed on patients at any age; however, if the ptosis is significant, early surgery is necessary to prevent occlusion amblyopia. The choice of surgical procedure is dictated by the degree of ptosis, amount of levator muscle function, and clinical response to 2.5% phenylephrine ophthalmic solution. Patients who have a mild to moderate amount of ptosis which improves within 5 minutes after the administration of 2.5% phenylephrine ophthalmic drops into the upper eyelid fornix may benefit from a Mueller’s muscle resection, while those with minimal response may require a levator muscle resection. In the absence or near absence of levator muscle function, a frontalis sling procedure is performed.

The association of blepharophimosis with ptosis and epicanthus inversus (BPES) is usually inherited in an autosomal dominant pattern (111). There are two types of BPES— type I is associated with ovarian failure and type II is not. The gene, FOXL2, is a winged helix/forkhead transcription factor and maps to chromosome 3q22-q23 (112,113,114). FOXL2 is mutated to produce truncated proteins in type I
families and larger proteins in type II. In this disorder, the palpebral fissures are narrowed both horizontally and vertically, and the ptosis is characterized by poor levator muscle function and the absence of a lid crease. Treatment involves correcting the ptosis with a frontalis sling procedure due to the poor levator muscle function. Surgical repair of the epicanthus inversus may also be undertaken in some cases.

Fibrosis of the extraocular muscle syndrome (FEOM) is a congenital disorder of innervation to the extraocular and eyelid muscles, which in turn affects muscular development (115). Affected individuals have a nonprogressive inability to move some or all of the extraocular muscles due to fibrotic and scarred muscles, adhesions between muscles and Tenon’s capsule, and adhesions between Tenon’s capsule and the globe. The eyes are usually fixed in a downward gaze, and as a result the patient assumes a chin-up head posture. The adhesions may involve the levator muscle, resulting in bilateral ptosis. FEOM1 (OMIM 135700) is caused by a mutation in the KIF21A gene (116), typically maps to chromosome 12q12, and shows neuropathologic changes suggesting a primary defect in the development of the superior division of the oculomotor nerve (117,118). FEOM2 (OMIM 602078) is associated with bilateral ptosis, with the eyes fixed in an exotropic position. This autosomal recessive phenotype maps to chromosome 11q13.3—q13.4 and results from mutations of the ARIX gene (119). In families with FEOM3 (OMIM 600638), one or more affected individuals do not have the classic findings of the disorder. Their eyes may be not be infraducted or may elevate above midline, or the individual may be unilaterally affected. Ptosis may not be present (120).

Congenital entropion and ectropion refer to a malposition of the eyelid where the eyelashes are rotated towards and away from the globe, respectively. Congenital entropion results from imbalances in preseptal and pretarsal orbicularis. Congenital ectropion may result from inflammatory conditions such as Chlamydia infection and may be associated with noninflammatory conditions such as Down syndrome and ichthyosis.

Distichiasis, an anomaly in which two rows (instead of one row) of lashes are present at the lid margin, may be autosomal dominant. During development, eyelashes differentiate in association with the glands of Zeis in the eyelids during the invagination of ectoderm to form pilosebaceous units. Distichiasis results from maldevelopment in which cilia formation occurs in association with Meibomian glands. Treatment is directed toward protecting the corneal epithelium. If the lashes do not touch the epithelium, no treatment is required. Electrolysis, cryotherapy, or a surgical eyelid-splitting procedure to remove the hair follicles may be performed when the lashes contact the corneal epithelial surface.

Many genetic disorders manifest themselves in the conjunctiva. Patients with ataxia telangiectasia (OMIM 208900: progressive cerebellar ataxia of childhood, oculomotor apraxia, absent optokinetic nystagmus, oculocutaneous telangiectases, immune defects, and malignancy predisposition) have significant dilatation and tortuosity of the conjunctival vessels. A characteristic oculomotor apraxia, that is, difficulty in the initiation of voluntary eye movements, frequently precedes the development of the telangiectases. The ataxia telangiectasia mutated gene on chromosome 11q23.3 (121) encodes a large protein kinase that regulates the cell cycle (122,123).

Congenital or early adult pterygium of the conjunctiva and cornea (OMIM 178000) is inherited as a dominant trait with 70% penetrance (124,125).

Pingueculae are evident in all forms of Gaucher disease (OMIM 230800, 230900, and 231000), one of the glycogen storage disorders (126).