Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

Box 60.6  Treatment

•Protect skin and eyes from excessive light

•Correct refractive errors

•Low-vision support

•Social support (www.positivexposure.org)

Box 60.7  Prognosis

•Gradual improvement in vision through infancy and childhood

•Stable vision thereafter

Not only is protection important, but it is in moderate light that vision is the best. Optimal correction of refractive errors is indicated. In early childhood, we favor full optical correction for refractive errors that are outside the prediction interval for normal20; this advice is modified if the child’s head position to damp the nystagmus precludes using the glasses lenses close to the optical center. Strabismus surgery may be considered when improvement in ocular alignment and anomalous head position is sought.21,22 Low-vision support, including use of optical and electronic devices, is needed to facilitate education and communication. Strategies for safe independent orientation and mobility may require specialized instruction. This is especially important outdoors in daylight hours when photophobia is an added impairment. The importance of social support cannot be overestimated (www.positivexposure.org) to counteract the negative press that albinism has suffered at times.

Prognosis

In general, individuals with OCA1A have more impaired vision than those with OCA1B, OCA2, and OA1. Newborns with OCA1A have very white skin and hair at birth, whereas newborns with OCA2 have more of a cream-colored complexion with yellow-tinted hair and those (males) with OA1 generally have normally pigmented skin and hair. During childhood, the developmental increases in acuity and visual performance that ordinarily occur may be further improved by a slight, gradual increase in pigmentation that is generally seen in many children with albinism. Vision is otherwise stable, although with the passing years, supports need modification to match demands of school and life (Box 60.7).

Pathology

Albinism is characterized by foveal hypoplasia, which clinicians attempt to identify by ophthalmoscopy (Box 60.8). Optical coherence tomography (OCT) performed by a number of groups supports the clinical impression of an absent or blunted foveal dimple.23–25 There has been a

Pathology

Box 60.8  Pathology

•Melanosomes with paucity of melanin granules

•Megamelanosomes (X-linked ocular albinism and Chédiak– Higashi syndrome)

•Reduced number of ganglion cells in the central retina

•Foveal hypoplasia

•Subclinical hypoplasia of optic nerve

•Small chiasm

•Relative deficit in nondecussated fibers at the chiasm

paucity of anatomic specimens and each has had limited generalizability due to comorbidities or advanced age of the donors.2,3,26 High-resolution OCT with eye-tracking capability may be the most promising approach to contemporary analysis of the neurovascular elements27 that must define the anomalies of the albino fovea.

Anomalies, that is, misrouting of the visual pathways, are commonly considered to be specified in the retina during development and are related to the deficiency in melanin. Fundus pigmentation, graded on a five-point scale, predicts the shift in the nasotemporal line of decussation in humans with albinism.28 Furthermore, the number of ganglion cells in the central retina is reduced in albinism, and anomalies of the visual pathway are limited to those brain regions representing the central retina.29,30 The mechanisms, however, by which melanin induces the spatial temporal defects in the immature ganglion cells that might give rise to the anomalous pathways, classically described as a paucity of nondecussated fibers, remain unknown.29,31,32 Another perspective is based on careful observations of well-pigmented patients with foveal hypoplasia who also had misrouting of the visual pathways demonstrated using visual evoked potential procedures.33 Thus, not only do foveal hypoplasia and misrouting of the visual pathways exist distinct from albinism, but they call into question the retinal specification of the misrouting. The fovea has a protracted course of development from approximately 24 weeks’ gestation to well into childhood. The decussation at the chiasm is established earlier. van Genderen and colleagues raise the possibility that retrograde processes govern the development of foveal hypoplasia in general, perhaps even in albinism.33

The photoreceptors, the last retinal cells to mature, are also abnormal in animal models of albinism. The rods are too few in number,34 rhodopsin content is low,35 and sensitivity is low.36 Each of these reports presents evidence that light damage to the rods does not account for the abnormalities of the photoreceptors. In some children with albinism there is an anecdotal report of mild deficits in rod sensitivity, as derived from analysis of the electroretinogram a-wave.37 Although the mechanisms leading to the reported changes in the rod photoreceptors34–37 have not been delineated, we note that signaling pathways involved in melanization appear to modulate oxygen consumption by the photoreceptors and the rate of photoisomerization in the rod outer segments. Furthermore, low melanin is associated with increased phagocytosis of rod outer segments by the retinal pigment epithelium.38

Tyr

P

OCA1

OCA3

Megamelanosome

Box 60.10  Specific cellular features

Melanosomes are derived from the endosomal lysosomal system

Stage I (premelanosomes)

•Spherical

•Originate from smooth endoplasmic reticulum

•Contain transmembrane protein, gp 100

Stage II

•Ovoid

•Internal fibrillar matrix, arising from cleavage of gp100

Stage III

•Beginning of melanin deposition

Stage IV (mature)

Filled with melanin

In albinism, low amount of melanin in melanosomes due to:

•Defect in melanin biosynthesis

•Abnormal biogenesis of melanosome

OCA1, OCA3

•Tyrosinase (OCA1) and tyrosinase-related protein (OCA3) retained in rough endoplasmic reticulum

OCA2

•Abnormal P-protein prevents targeting of tyrosinase and other proteins to the melanosome immediately post-Golgi

OCA4

•Membrane-associated transporter protein (MATP) interferes with processing and trafficking of tyrosinase to the melanosome and results in secretion of early melanosomes

melanocyte-specific protein whose precise function is not well understood. Various roles may include stabilizing tyrosinase, regulating melanin production through peroxide levels, and determining the shape of melanosomes. Normally, tyrosinase and tyrosinase-like protein are synthesized in the rough endoplasmic reticulum, transported through the Golgi apparatus, and targeted to small vesicles that pass through the endosomal–lysosomal compartment and fuse with melanosomes (Figure 60.3). In OCA1 and OCA3, the abnormal proteins are instead largely retained within the rough endoplasmic reticulum and targeted for disposal in the proteosome.40–42

OCA2

The P-gene (human homolog of the murine pink-eyed dilution gene, p), is located on chromosome 15q11-13 and encodes a transmembrane protein of the melanosome membrane. In cultured primary melanocytes from mice with p-gene mutations, abnormal p-protein prevents the efficient targeting of tyrosinase and other melanosomal proteins immediately post-Golgi from small vesicles to melanosomes (Figure 60.3).43–45 Instead, the proteins are retained in small vesicles and mainly secreted from melanocytes, such that only 20% of the synthesized tyrosinase is localized in melanosomes. Another and perhaps related role of the

Pathophysiology

P-protein is the acidification of melanosomes and their precursors, which is required for melanin biosynthesis and possibly the sorting and localization of tyrosinase.46

Approximately 1% of patients with Angelman and Prader– Willi syndromes have a form of albinism related to OCA2. This low frequency is consistent with the carrier frequency of P-gene mutations in the general population. These two neurodevelopmental disorders are caused by the loss of either maternally expressed genes (Angelman) or paternally expressed genes (Prader–Willi) within imprinted regions of chromosome 15q11-13. Since imprinted genes are expressed from only one parental chromosome, the presence of a deletion on that chromosome leads to the total lack of expression of the imprinted genes from both chromosomes. In a few such individuals with albinism, it has been shown molecularly that one copy of the P-gene is lost as part of the chromosome 15q11-13 deletion responsible for Angelman or Prader–Willi syndrome, while the other copy of the P-gene carries a mutation by chance.47,48 Even individuals with Angelman or Prader–Willi syndrome who do not have a second P-gene mutation often appear less pigmented than their relatives, suggesting that there are other pigmentmodifying genes located in this chromosomal region.

OCA4

Like the P-protein, membrane-associated transporter pro­ tein (MATP) contains 12 membrane-spanning domains and shares homology with transport proteins. In a cultured primary murine melanocyte model of OCA4 containing the underwhite (uw) mutation, lack of MATP interfered with the processing and intracellular trafficking of tyrosinase to melanosomes (Figure 60.3).49 Tyrosinase was abnormally secreted from cells in immature melanosomes, thereby disrupting the normal maturation process of these organelles.

OA1

Abnormalities in the OA1 protein, GRP143, lead to a defect in melanosome biogenesis and the regulation of melanosome size; consequently, stage III and IV melanosomes are decreased in number and unusually large (megamelanosomes). They are found in most carrier females in a mosaic pattern in tissues because of random X-linked inactivation, which allows for only one X chromosome to be expressed in a given cell.

Most of the complex forms of albinism involve not only melanosomes, but other organelles as well. Hermansky– Pudlak syndrome is a genetically heterogeneous group of disorders, all of which involve abnormalities of vesicles of the lysosomal lineage that form melanosomes, lysosomes, and platelet dense bodies. Decreased or absent platelet dense bodies can be demonstrated by electron microscopy of fresh blood. Chédiak–Higashi syndrome is caused by a defect in the CHS1 (LYST) gene, which is thought to play a role in vesicular formation and transport. As in OA1, megamelanosomes are present in Chédiak–Higashi syndrome.

Pathophysiology

In patients with OCA1, two mutations are identified in less than 50% of cases, suggesting that noncoding and regulatory regions of the gene harbor a substantial proportion of

the mutations.50 Most patients are compound heterozygotes, with only a few mutations being common (e.g., T373R and P81L in Caucasians). Patients with OCA1A have mutations that cause complete absence of tyrosinase activity, whereas mutations that cause OCA1B encode tyrosinase that has 5–10% residual enzymatic activity. R402Q is a temperature-sensitive mutation that renders tyrosinase partially active at 31°C and completely inactive above 37°C. These individuals typically have ocular features of albinism (because of high core body temperature) but then develop some pigmentation in skin of the extremities and their hair. Patient mutations tend to cluster near functional domains of the enzyme, specifically near the copper-binding regions in proximity to the active site, the N-terminus that is important for targeting to the rough endoplasmic reticulum, and the C-terminus that is important for sorting to melanosomes. Many of the mutations cause misfolding or instability of the nascent protein with retention in the rough endoplasmic reticulum and rerouting to the proteosome.

Less is known about mutations that cause OCA2 because of the large size of the gene; this should change with gene sequencing becoming available clinically. Of the approximately 200 individuals who have been genotyped, most have missense mutations that are spread throughout the gene, often present in the loops between the membranespanning domains.50 A 2.7-kb deletion accounts for 75–80% of P-gene mutations in sub-Sarahan Africans, where the frequency of OCA2 is as high as 1 in 1400 in Tanzania and 1 in 3900 in South Africa.

OCA3, rufous or red albinism, is associated with red hair and reddish-brown skin and has been reported mainly in South Africa and New Guinea. Of the small number of patients who have been genotyped, a variety of mutations has been observed (http://albinismdb.med.umn.edu/ oca3mut.html).

OCA4, which is clinically similar to OCA2, appears to be an uncommon form of OCA except in the Japanese population, where it accounts for 24% of OCA patients.51 A common missense mutation, D157N, was present in 39% of alleles, and the mutation was present on the same haplotype in both Japanese and Korean patients, indicating a founder effect in Asia.52

A variety of mutations have been identified in the OA1 gene, and of note, large intragenic deletions appear to be particularly common.53 The high rate of deletions is partly explained by the presence of Alu-repeat sequences within the first two introns, which makes these regions prone to unequal recombination during meiosis. Curiously, the rate of large

deletions in patients varies by geographic region, accounting for more than 50% of North American mutations but less than 10% of European mutations. Given the heterogeneous breakpoints of the deletions, this difference cannot be due to a single founder mutation, nor, given the lack of geno- type–phenotype correlations, can the difference be attributed to environmental selection; the different frequencies may simply be incidental.53 While genetic testing of the OA1 gene by the polymerase chain reaction is straightforward in males, deletions cannot be reliably detected in carrier females because of the presence of the second X chromosome. In suspected carriers, the presence of megamelanosomes on skin biopsy and uneven pigmentation in the retina is useful for clinical diagnosis.

Hermansky–Pudlak syndrome has its highest prevalence in Puerto Rico (1 in 1800 individuals), but is rare elsewhere in the world. Of the eight known genetic causes, HPS1 is the most common. HPS1 has two common founder mutations:

a 16-basepair duplication mutation in Puerto Rican pati­ ents and a splice-site mutation (IVS5+5G>A) in Japanese

patients.50 HPS3 also has a founder mutation in Puerto Rican patients, involving a deletion of exon 1.

Chédiak–Higashi syndrome is caused by mutations in the CHS1 (LYST) gene. Genotype–phenotype correlations in a limited number of patients suggest that childhood disease is associated with null mutations, while juvenile and adult forms are associated with missense mutations that retain some residual functional activity.54

Conclusion

Albinism refers to a group of genetic conditions that are characterized by reduced melanin levels leading to hypopigmentation of the eyes, hair, and skin, and importantly, all affect vision. Simple or nonsyndromic forms of albinism affect melanosomes only, whereas complex or syndromic forms of albinism include systemic features caused by disturbances in both melanosomes and other intracellular organelles. While the eponymous syndrome names are useful clinically, genetic classification is recommended. For further information, there are excellent web resources on the disease (National Organization for Albinism and Hypopigmentation at www.albinism.com; Gene Reviews at www. genereviews.com), the laboratories that perform genetic testing (www.genetests.com), and the albinism gene mutation database at the International Albinism Center located at the University of Minnesota (http://albinismdb.med. umn.edu/).

1.Taylor WO. Edridge-Green lecture, 1978. Visual disabilities of oculocutaneous albinism and their alleviation. Trans Ophthalmol Soc UK 1978;98:423–445.

8.Guillery RW. Visual pathways in albinos. Sci Am 1974;230:44–54.

12.von dem Hagen EA, Hoffmann MB, Morland AB. Identifying human

albinism: a comparison of

VEP and fMRI. Invest Ophthalmol Vis Sci 2008;49:238–

249.

13.Sturm RA, Teasdale RD, Box NF. Human pigmentation genes: identification, structure and consequences of polymorphic variation. Gene 2001; 277:49–62.

17.Hutton SM, Spritz RA. A comprehensive genetic study of autosomal recessive ocular albinism in Caucasian patients. Invest Ophthalmol Vis Sci 2008;49:868– 872.

23.Seo JH, Yu YS, Kim JH, et al. Correlation of visual acuity with foveal hypoplasia grading by optical coherence tomography in albinism. Ophthalmology 2007;114: 1547–1551.

28.von dem Hagen EA, Houston GC, Hoffmann MB, et al. Pigmentation predicts the shift in the line of decussation in humans with albinism. Eur J Neurosci 2007;25:503–511.

32.Rachel RA, Dolen G, Hayes NL, et al. Spatiotemporal features of early neuronogenesis differ in wild-type and

albino mouse retina. J Neurosci 2002;22:4249–4263.

33.van Genderen MM, Riemslag FC, Schuil J, et al. Chiasmal misrouting and foveal hypoplasia without albinism. Br J Ophthalmol 2006;90:1098–1102.

41.Toyofuku K, Wada I, Valencia JC, et al. Oculocutaneous albinism types 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wild-type proteins. Faseb J 2001;15: 2149–2161.

44.Manga P, Boissy RE, Pifko-Hirst S, et al. Mislocalization of melanosomal proteins in melanocytes from mice with oculocutaneous albinism type 2. Exp Eye Res 2001;72:695–710.

46.Puri N, Gardner JM, Brilliant MH. Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes. J Invest Dermatol 2000;115:607–613.

Key references

49.Costin GE, Valencia JC, Vieira WD, et al. Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4. J Cell Sci 2003;116:3203–3212.

50.King RA, Oetting WS, Summers CG, et al. Abnormalities of pigmentation. In: Rimoin DL, Conner JM, Pyeritz RE, et al. (eds) Emery and Rimoin’s Principles and Practice of Medical Genetics, vol. 3, 5th edn. Philadelphia: Elsevier, 2007:3380– 3427.

53.Bassi MT, Bergen AA, Bitoun P, et al. Diverse prevalence of large deletions within the OA1 gene in ocular albinism type 1 patients from Europe and North America. Hum Genet 2001;108:51–54.

Clinical background

Aniridia refers to a bilateral malformation of the eye in which the most prominent clinical finding is variable to near-total absence of the iris.1,2 The word aniridia is a misnomer since the iris is not totally absent (Figure 61.1) and there are in fact a number of other accompanying ocular abnormalities that result from the underlying genetic defect in one of the master ocular developmental genes, PAX6. Even in the more severe cases, a stump of tissue is invariably present at the base of the iris, and gonioscopy may be required for its adequate visualization. Careful ocular examination will reveal other abnormalities that include persistent strands of the fetal pupillary membrane, congenital lens opacities that could be cortical, anterior polar, or of other types, ectopia lentis or subluxation of the crystalline lens as a result of poor zonular development, developmental glaucoma, a superficial keratopathy referred to as corneal pannus, persistence of the retina over pars plana, and foveal hypoplasia that is almost universal and leads to decreased visual acuity and nystagmus.3

Congenital poor visual function in aniridia results from macular, foveal, and optic nerve hypoplasia.4 Acquired causes of visual loss in aniridia include the development or progression of cataracts, optic nerve damage from glaucoma,5,6 corneal opacification, and anisometropic or strabismic amblyopia. The keratopathy/corneal pannus of aniridia appears late in the first decade of life and is presumably due to insufficient/absent limbal stem cells that depend on the presence of normal PAX6 complement for their development and maintenance.7 Nystagmus is most likely due to congenital poor visual acuity, although underlying central nervous causes may exist that have not been adequately investigated. There are at least two families in which some members have classical aniridia and other members have atypical iris defects ranging from radial clefts or atypical colobomas and relatively good vision.8,9 We have recently described four patients with aniridia, preserved vision, little or no foveal hypoplasia and no detectable mutations in PAX6.10 Vascular anomalies of the iris have also been described.9 Aniridia can also occur in association with malformations of the globe such as Peters’ anomaly or congenital anterior staphyloma or with microcornea and subluxated lenses.

Aniridia

Elias I Traboulsi

Aniridia is associated with systemic abnormalities when it occurs in the context of the well-defined contiguous gene syndrome of “Wilms’ tumor–aniridia–genitourinary abnor- malities–retardation” (WAGR) or Miller syndrome (Box 61.1). This type of aniridia is of the nonheritable variety and is always associated with a deletion of band 13 on the short arm of chromosome 11.11,12

Aniridia can also occur in the context of multisystem malformation syndromes and chromosomal abnormalities such as a ring chromosome 6,13 pericentric inversion of chromosome 9,14 the syndrome of multiple ocular malformations, and mental retardation described by Walker and Dyson15 and Hamming et al,16 and the syndrome of aniridia and absence of the patella.17 When iris hypoplasia is not severe, aniridia may be confused with conditions such as Rieger anomaly (Figure 61.2), ectopia lentis et pupillae, atypical coloboma of the iris, or essential iris atrophy (Chandler syndrome).

PAX6 is widely expressed in the central nervous system. Sisodiya et al18 performed magnetic resonance imaging (MRI) and smell testing in patients with aniridia and showed absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of cases, demonstrating that PAX6 haploinsuffiency causes more widespread human neurodevelopmental anomalies18; Mitchell et al19 demonstrated widespread structural abnormalities of the brain, including absence of the pineal gland and unilateral polymicrogyria on MRI of 24 patients heterozygous for defined PAX6 mutations. Thompson et al20 studied 14 patients with PAX6 gene mutations and MRI abnormalities for defects in cognitive functioning. None were found except in a subgroup of patients with agenesis of the anterior commissure who performed significantly more poorly on measures of working memory than those without this abnormality. In another study, brain MRI, central auditory testing, and a questionnaire were administered to a group of 11 children with aniridia.21 The corpus callosum area was significantly smaller on brain volumetry in patients compared with controls. The anterior commissure was small in 7 cases and was normal in 3 cases on visual inspection of brain MR images. Audiograms showed no abnormalities in any of the children. Central auditory test results were normal in all the controls and were abnormal in all the cases, except for 1 case with a pattern of abnormalities consistent with reduced auditory interhemispheric transfer. The cases had greater difficulty

Figure 61.1  Classic aniridia phenotype with almost total absence of iris tissue. Only a small rim of iris is present. The edge of the lens is visible.

Box 61.1  Clinical findings in aniridia

•Aniridia is an autosomal-dominant panocular malformative disorder in which the most prominent clinical abnormality is absence of iris tissue

•Poor vision in aniridia results mostly from foveal hypoplasia

•Aniridia results from mutations in the PAX6 gene on 11p13

•The WAGR syndrome of Wilms’ tumor, aniridia, genitourinary malformations and retardation results from deletions of chromosome 11p13

B

Figure 61.2  (A, B) Variable degrees of absence of iris tissue in aniridia. The phenotype can simulate Rieger anomaly.

localizing sound and understanding speech in noise than the controls. These findings indicate that, despite normal audiograms, children with PAX6 mutations may experience auditory interhemispheric transfer deficits and have difficulty localizing sound and understanding speech in noise.

Aniridia with cerebellar ataxia and mental retardation is a very rare condition inherited in an autosomal-recessive fashion and known as Gillespie syndrome.22,23 Gillespie syndrome is not caused by mutations in PAX6.24

Most recently glucose intolerance and diabetes mellitus have been described in some patients with aniridia and are possibly due to the importance of PAX6 in pancreatic development and function.25–27 PAX6 plays an indispensable role in islet cell development. Yasuda et al performed oral glucose tolerance tests in patients with PAX6 mutations and found glucose intolerance characterized by impaired insulin secretion.26

Etiology and distribution

Aniridia occurs in 1/50 000 live births. Shaw et al1 estimated the prevalence of aniridia in the lower peninsula of Michigan in 1960 to be about 1 in 64 000. Approximately two-thirds

to three-fourths of patients have at least one other affected family member; the remainder are sporadic.

In one large series of 125 patients, 74 cases were sporadic, 24 were familial, and 14 had the WAGR syndrome, or other malformations.28 Two cases had chromosome rearrangements involving 11p13, 16 cases had visible deletions, and 16 cases had cryptic deletions identified by fluorescent in situ hybridization (FISH). The frequency of cryptic deletions in familial aniridia was 27% and in sporadic isolated aniridia was 22%. Of the 14 cases referred with WAGR syndrome, 10 (71%) had chromosomal deletions, 2 cryptic, and 8 visible. Of the 13 cases with aniridia and other malformations, 5 (38%) had a chromosomal rearrangement or deletion. In 37 cases with no karyotypic or cryptic chromosome abnormality, sequence analysis of the PAX6 gene was performed. Mutations were identified in 33 cases: 22 with sporadic aniridia, 10 with familial aniridia, and 1 with aniridia and other non-WAGR syndrome-associated anomalies. Overall, 67 of 71 cases (94%) undergoing full mutation analysis had a mutation in the PAX6 genomic region.

Aniridia is caused in most cases by mutations in PAX6, a homeobox transcription factor on 11p13.29,30 There appears to be a correlation between the type of mutation and the clinical phenotype. A classic severe phenotype results from mutations that lead to stop codons and protein haploinsufficiency.31 Missense mutations cause aniridia as well as other

phenotypes, including cataracts, Peters’ anomaly, other types of anterior-segment dysgenesis, and occasionally a clinical picture predominated by keratopathy (Figure 61.3).32 Rarely, the iris is so well preserved (Figure 61.4) that the phenotype is one of isolated foveal hypoplasia (Figure 61.5).33 Some patients with mutations in PAX6 have microphthalmia.

Prognosis, prevention, and treatment

Once the clinical diagnosis of aniridia is made, it becomes imperative to determine whether the patient has a mutation inside the PAX6 gene or whether he/she carries a deletion that involves the adjacent Wilms’ tumor gene WT1. Clinical molecular genetic testing is available and will identify a mutation in more than 75% of cases. Karyotyping has been superseded by microarray analysis, a test that will detect small deletions or chromosomal rearrangements of 11p13 where the PAX6 gene is located. FISH analysis can also be used to detect submicroscopic deletions of 11p13. As a general rule, familial cases have intragenic mutations, while sporadic cases may be due to either PAX6 mutations or to

474

chromosomal deletions that may include adjacent genes and cause the WAGR syndrome. In any patient with aniridia and a negative family history, the risk of developing Wilms’ tumor is 20%. About 1 in 70 patients with Wilms’ tumor have aniridia. The presence of other systemic abnormalities in a sporadic case of aniridia should raise the suspicion of a chromosome 11p deletion or rearrangement. If access to genetic testing is not possible, careful, repeated examination and imaging of the renal system should be performed. Ultrasound examination of the kidneys is done at 6-month intervals supplemented with intravenous pyelography, computed tomography, or MRI to evaluate further any suspicious finding.

Other family members should be examined for the presence of mild degrees of iris hypoplasia as this may indicate dominant inheritance with variable expressivity and circumvent the worries about the potential occurrence of Wilms’ tumor.

The management of ocular problems in patients with aniridia can be very challenging (Box 61.2). Visual acuity is less than or about 20/200 in most patients, but may be as good as 20/20 in patients with aniridia and preserved ocular

function.10 The main cause of acquired visual loss in aniridia is glaucoma, and patients are screened for its presence at regular intervals.5 The glaucoma in aniridia typically develops in late childhood or in adulthood; however, it may be present in the first year of life. Aniridic glaucoma may be due to trabeculodysgenesis, but a more likely mechanism is occlusion of the filtering angle by an up-pulling of the iris stump. Goniotomy or trabeculotomy may be successful in controlling or preventing aniridic glaucoma34; however, filtering surgery or cyclocryotherapy may be required. Medical therapy should be tried in older individuals with aniridic glaucoma. Cataracts, which develop in most aniridic patients, are extracted if they produce significant further decrease in visual acuity. Some patients have congenital anterior polar cataracts while others have acquired cataracts that usually develop in early adulthood. Ectopia lentis is occasionally found in aniridic eyes and should be looked for before a lensectomy is performed. Finally, penetrating keratoplasty may be required in some instances if progressive keratopathy leads to corneal opacification and to further loss of vision.

Pathology

There are very few histopathologic studies of the eye in aniridia, some of which are from eyes blind with advanced glaucoma.35 In typical cases, the iris is rudimentary and there is an absence of dilator and pupillary sphincter muscles. Grant and Walton6 reported their observations of the angle in a large number of patients with aniridia. They noted that the peripheral stump of the iris gradually extended anteriorly to cover the filtration portion of the angle as patients got older. They also noted that this correlated with worsening of glaucoma. The lens may be subluxated from underdeveloped zonular apparatus, and the ciliary processes may be small.

Etiology

Mutations in PAX6 account for a majority of cases of aniridia (Box 61.3). There is evidence of genetic heterogeneity and some cases with aniridia do not have detectable mutations in PAX6.10

Using positional cloning and DNA samples from patients with aniridia and deletions involving the 11p13 aniridia locus, Ton and coworkers cloned a cDNA which they presumed to be complementary to AN2.29 This gene was found

Box 61.3  Mutations in PAX6 cause aniridia

•More than 300 mutations in PAX6 have been reported to date, most of which result in protein truncation and haploinsufficiency

•Rare cases of Peters’ anomaly and cataracts have resulted from PAX6 mutations

•Insect and animal models of aniridia have been discovered and result from mutations in Pax6 in the mouse and rat for example, and in eyeless in Drosophila. These models have been very important in studying the function of the gene

Etiology

to be the human homolog of the murine Pax6 gene that, when mutated, results in the small-eye (Sey) phenotype. Homozygous Sey/Sey mice are anophthalmic, lack nasal structures, and die shortly after birth.36 Hemizygous Sey/+ mice are microphthalmic and have a range of anteriorsegment abnormalities ranging from colobomas to iris hypoplasia and lenticulo-irido-corneal adhesions. A neuropathological study of Small eye mice showed that there was a delay of premigratory neurons and an impairment of axonal growth and differentiation. This eventually results in a broad spectrum of neuronal migration disorders of the neocortical roof.37 The murine Pax6 gene mapped to a region of chromosome 2 in the mouse which is syntenic to the aniridia locus on chromosome 11 in humans, giving further support that Sey is the murine homolog of the aniridia gene. PAX6 is expressed in the fetal eye, forebrain, cerebellum, and olfactory bulbs.29,38 In the developing eye Sey is expressed first in the optic sulcus and subsequently in the eye vesicle, in the lens, in the differentiating retina, and finally in the cornea. Glaser and coworkers described the complete genomic structure of the human PAX6 gene and discovered mutations in familial and sporadic cases.30 Using mutation analysis of the PAX6 gene, investigators from around the world identified numerous mutations in patients with aniridia (http://pax6.hgu.mrc.ac.uk/).

The human PAX6 gene spans 22 kb and consists of 14 exons (Figure 61.6). It belongs to the Pax family of developmental control factors that possess the paired domain originally identified in the Drosophila melanogaster segmentation gene paired. The encoded PAX6 protein contains two DNAbinding domains: the paired box of 128 amino acids and a paired-type homeobox of 61 amino acids, separated by the linker region. The paired domain has two functional subdomains, the 74-amino-acid N-terminal domain which is relatively conserved among paired domains, and the less well conserved 54-amino-acid C-terminal domain. PAX6 is alternatively spliced and inclusion of exon 5a (PAX6-5a) alters the DNA-binding properties resulting in DNA contact by the C-terminal domain instead of the N-terminal domain. In the C-terminal part of the protein there is a proline, serine, and threonine-rich (PST) domain of 152 amino acids which resembles the activation domain of transcription factors, and has been shown to possess transcriptional activity in vitro. Pax6 is involved in the transcriptional regulation of the crystalline genes and interacts closely with a number of genes essential in embryonic eye development. A striking amino acid identity is observed of PAX-6 proteins between species, especially in the functional domains, with only one amino acid difference between the mouse and the human gene located in the alternatively spliced exon. The expression patterns of Pax-6 are also conserved throughout the vertebrates and transcripts have been detected in the developing eye, the

Figure 61.6  Organizational diagram of the PAX6 gene. PST, proline, serine, and threonine-rich.

brain, spinal cord, and pancreas. It has been shown in the bovine eye that the 5a isoform predominates in the iris in contrast to the lens and retina where PAX6 and PAX6-5a seem to be equally represented, indicating that the PAX6-5a isoform is important for iris development.

In the human PAX6 database there are now more than 300 mutations and over 100 sequence variations (http:// pax6.hgu.mrc.ac.uk/). Most of the mutations cause premature truncation of the protein and relatively few missense mutations have been reported, some with aniridia and others with congenital cataracts, Peters’ anomaly, and foveal

hypoplasia. Autosomal-dominant keratitis has been associated with a splice site mutation (IVS10-2A>T) in PAX6.

Missense mutations are generally associated with milder phenotypes.

Some PAX6 mutations give rise to panocular effects with signs that are less severe or different from classic aniridia. For example, a missense mutation (R26G) caused a heterogeneous syndrome of anterior-segment malformations, including iris hypoplasia and Peters’ anomaly. A nonsense mutation in codon S353 produces a truncated protein in the PST domain and is associated with normal irides, cataracts, and late onset of cone dystrophy.39These two truncated PST domains have partial transcriptional activity, possibly accounting for the milder phenotype. An exon 11 splice acceptor mutation caused autosomal-dominant keratitis in a family.32 Yanagisawa and colleagues40 reported a missense mutation at nucleotide 799, a C to T transition associated with a phenotype dominated by foveal hypoplasia and, according to the authors, no iris defects.

The Drosophila gene eyeless (ey) is the homolog of PAX6 in humans and Pax6 in the mouse. Eyeless flies have partial or total absence of their compound eyes. Hypomorphic (weak) alleles lead to the reduction or absence of compound eyes but do not affect the ocelli or simple eyes. Null alleles are not available now but presumably affect all eyes and are lethal when homozygous. The proteins encoded by ey, Sey, and AN2 share 94% sequence identity in the paired domain and 90% identity in the homeodomain. Furthermore, there are similarities in the flanking sequences and some of the splice sites in the paired box and in the homeobox are conserved between the fly and mammalian genes, indicating that the genes are orthologous.41

Grove et al42 reported two matings of aniridic patients in a large family. One couple had no children. A second couple had a total of six children: one girl with aniridia lived to 11 months and died of central nervous system problems; three boys died at less than 24 hours of age; there was also one spontaneous abortion at 3 months of gestation and one near-term intrauterine fetal death. Elsas and coworkers8 also reported one mating between aniridics. The couple had four living children: three had aniridia and one was normal; there was a stillborn child with unknown phenotype. Other cases are those reported by Hodgson and Saunders,43 who described the necropsy findings in a stillborn girl whose mother and father had aniridia. There were two previous miscarriages at 10 weeks of gestation. The fetus had absence of the palpebral fissures and eyes. The nasal bones were completely absent and the nasal cavity was small. Both parietal bones had elliptical defects at their posterior medial aspects and overlapped the occipital bone. The adrenals were absent. The skeletal, urogenital, alimentary, cardiovascular,

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Box 61.4  Functions of PAX6

•PAX6 is a master control gene that is expressed in the developing eye and brain

•PAX6 is essential in lens placode formation and other early developmental stages of the eye

•PAX6 interacts with a large number of other transcription factors that are essential in normal ocular development

•PAX6 continues to be expressed in ocular surface cells such as the limbal epithelium, where it plays a vital role in corneal epithelial cell maintenance and regeneration

and respiratory systems appeared normal. The thyroid and thymus were normal. The brain was macerated. Glaser et al reported a family where two mutations of the PAX6 gene segregated independently, causing either aniridia or a syndrome of cataracts and late-onset corneal dystrophy.39 A compound heterozygote for the mutations at codons 103 and 353 had severe craniofacial and central nervous system defects and no eyes. This study demonstrates a dosage effect of the PAX6 gene and its critical role in the development of eye and brain structures.

Pathophysiology

PAX6 is widely expressed in the neurectoderm and the surface ectoderm of the developing eye, and in their derivatives with specific dosage requirements of the transcription factor in these tissues.44 PAX6 activity is essential in the ectoderm for lens placode formation (Box 61.4).

Ashery-Padan and coworkers showed that several independent, fully differentiated neuroretinas developed in a single optic vesicle in the absence of a lens from a mutation in Pax6, demonstrating that the developing lens is not necessary for the differentiation of the neuroretina but is required for the correct placement of a single retina in the eye.45 PAX6 continues to be expressed in the adult retina, lens, and cornea. The progressive nature of the aniridia phenotype with corneal and lens changes over time reflects the maintenance functions of PAX6 in the adult eye.46 PAX6 is also expressed in the olfactory system, from the earliest nasal placode to the mature olfactory bulb and the olfactory epithelium. It is also expressed in the developing telencephalon, thalamus, pituitary, pineal, cerebellum, spinal cord, and pancreas. PAX6 plays a key role in the development of the brain where it affects cell fate, cell proliferation, and patterning. The paired domain is necessary for the regulation of neurogenesis, cell proliferation, and patterning effects of PAX6, whereas the homeodomain plays a lesser role in the brain. Splicing of one or the other exon 5 appears to play a pivotal role in neurogenesis.47

The Drosophila gene eyeless (ey) is homologous to the mouse Small eye (Pax6) gene and to the aniridia gene in humans. By targeted expression of the ey complementary DNA in various imaginal disc primordia of Drosophila, Halder et al induced ectopic eye structures on the wings, legs, and antennae. The ectopic eyes appeared morphologically normal and consisted of groups of fully differentiated