Following the major stages of morphogenesis illustrated in Fig. 240-2, ocular tissues undergo an extensive phase of maturation to achieve the diverse and highly specialized structures of the adult eye (Fig. 240-1). The anterior part of the optic cup and the presumptive cornea undergo cell proliferation, differentiation and migration to form the anterior segment, comprising such functionally diverse tissues as the posterior layers of the cornea, the iris and the trabecular meshwork. Molecular mechanisms underlying these complex processes are poorly understood. Identification of mutant genes in humans and mice with developmental abnormalities affecting these structures has proved to be the most successful approach to identifying transcription factors that play a key role in anterior segment development.
Categories of Anterior Segment Anomalies: Phenotypic and Genetic Heterogeneity
Anterior segment anomalies have been broadly divided into those thought to result from abnormal development of the cornea: Peters anomaly (MIM 604229), sclerocornea, congenital endothelial dystrophy, and anterior staphyloma; and those thought to result from abnormal development of the angle and the iris: posterior embryotoxon, Axenfeld anomaly, Rieger anomaly, and iridogoniodysgenesis (MIM 137600 and MIM 601631)274 and Waardenburg syndrome. In fact, the distinction between these two classes is frequently blurred and there are reported cases in which the two eyes of one patient, or individuals within a single family, display anomalies from both classes.200,219 The clinical presentation of these eye diseases has been described in detail (e.g., Taylor1). From a genetic point of view it is more helpful to view these disorders as a spectrum of anterior segment dysgeneses in which there is often no clear genotype-phenotype correlation; indeed there is considerable overlap of the phenotypes elicited by mutations at different loci (see Fig. 240-3). The observed genetic heterogeneity underlines the need for flexibility in classifying this whole group of disorders.
Phenotypic and Genetic Heterogeneity
Axenfeld and Rieger anomalies are related developmental disorders of the anterior chamber, associated with iridogoniodysgenesis: abnormalities of the iris stroma, trabecular meshwork, and cornea. They are usually bilateral, mostly with autosomal dominant inheritance. About half of all cases are associated with glaucoma, which has major manifestations in the posterior segment where optic disk cupping causes gradual loss of vision. Axenfeld anomaly is typically characterized by posterior embryotoxon—a prominent, anteriorly positioned Schwalbe line1,24 —with multiple processes between the iris and Schwalbe line. Rieger anomaly includes the features of Axenfeld anomaly with the addition of iris hypoplasia, polycoria (multiple pupils) and/or corectopia (displaced pupil).174 The distinction between Axenfeld anomaly and Rieger anomaly is often blurred, particularly when differently affected individuals are found in one family, so the two are often classified together as a single entity known as Axenfeld-Rieger anomaly.
Rieger syndrome is a dominantly inherited condition275 involving malformations of the eyes, teeth, and skin (MIM 180500; MIM 601499). It is chiefly characterized by Rieger anomaly (see “Axenfeld-Rieger Anomaly” above) with dental hypoplasia (reduction in the size and number of teeth) and often maxillary hypoplasia resulting in a characteristic facial appearance. Failure of umbilical skin involution is frequently seen.1,174 Occasional isolated growth insufficiency and cardiac defects are additional features. Two loci have been linked to this syndrome, one at 4q25276 for which a gene has been identified (see “PITX2: The Rieger Syndrome Gene at 4q25,” below). Rieger syndrome is no exception to the general rule of genetic heterogeneity in disorders involving anterior segment abnormalities. A large Rieger syndrome pedigree was tested for linkage to 4q25 but no association was found. Further analysis of this family provided evidence for a second locus at 13q14 (MIM 601499),277 where a member of the forkhead family, FKHR, maps (see “FKHL7 in Iridogonial Dysgenesis, Glaucoma, and Axenfeld-Rieger Anomaly” below).
Iridogoniodysgenesis (IGD) is defined by abnormalities of the iridocorneal angle and the anterior stromal layer of the iris. IGD patients suffer from raised intraocular pressure from an early age, resulting in juvenile glaucoma. IGD may occur as an isolated entity (iridogoniodysgenesis anomaly, IGDA) or with systemic defects (iridogoniodysgenesis syndrome, IGDS). IGDA or IRID1 (MIM 601631) has been mapped to the 6p25 region278,279 (see “FKHL7 in Iridogonial Dysgenesis, Glaucoma, and Axenfeld-Rieger Anomaly” below). IGDS or IRID2 (MIM 137600) has been mapped to 4q25.280 The map location and phenotypic similarity with Rieger syndrome suggested the possibility that this family was a variant Rieger syndrome case.
Peters anomaly is a congenital ocular abnormality involving the presence of a central corneal opacity with defects in the underlying corneal endothelium.274 There may be adhesions between the corneal defect and either the iris collarette or the lens. Commonly associated ocular anomalies include glaucoma and cataract.
Most cases of Peters anomaly appear to be sporadic, but a few well-documented familial cases, usually with autosomal dominant inheritance, are also found. Affected relatives of an individual with Peters anomaly may display other anterior segment disorders such as Rieger anomaly or sclerocornea.200 Such intrafamilial heterogeneity again emphasizes the difficulties in classifying anterior segment disorders. In two such cases, PAX6 mutations were identified (see “Missense Mutations in the Human PAX6 Gene Associated with Peters Anomaly and Other Phenotypes,” above), but, as discussed, most Peters anomaly cases show no PAX6 mutation.221 A PITX2 splice mutation has been reported in one case with Peters anomaly in one eye.281
Some cases of Peters anomaly are associated with systemic abnormalities. One well-characterized association is with short-limb dwarfism, called Peters Plus syndrome (MIM 261540). The absence of vertical transmission of Peters Plus syndrome, coupled with the incidence of consanguinity, suggests recessive inheritance.282
Anterior Segment Mesenchymal Dysgenesis.
Anterior segment mesenchymal dysgenesis (MIM 107250) is a general term used to describe some families in which no single specific anomaly predominates. The eye phenotype involves all tissues of the anterior chamber, implying abnormalities in all three waves of neural crest migration or of the ectodermally derived structures of the optic cup, lens, and cornea.209
Following linkage studies and candidate gene approaches, several transcription factor genes have been identified that harbor disease-causing mutations in an overlapping spectrum of anterior segment anomalies.
WS is an autosomal dominant disorder characterized by pigment defects of the eye, hair, and skin and by hearing loss. The eye phenotype is hypopigmented or heterochromatic irides. Mutations in the transcription factor gene MITF result in a subset of WS2 patients, termed Waardenburg syndrome type 2. In a later section (see “Human Mutations in MITF Cause Waardenburg Syndrome Type 2” below), we review the relationship between mutations in the MITF gene and WS2, and the essential role of the MITF transcription factor in mammalian eye development, as illustrated by the severe eye defects present in the homozygous Mitf mutant mouse.
FKHl7 in Iridogonial Dysgenesis, Glaucoma, and Axenfeld-Rieger Anomaly
Studies to uncover genes involved in predisposition to early onset glaucoma associated with developmental anomalies of the iris and iridocorneal angle, resulted in identification of a major locus, IRID1 (MIM 601631), at chromosome 6p25. Linkage analyses of families with iridogoniodysgenesis anomaly,278,279 Axenfeld-Rieger anomaly,205 familial glaucoma iridogoniodysplasia,283 and familial glaucoma with goniodysgenesis206 have all implicated involvement of a locus at 6p25.
Chromosomal rearrangements involving 6p25 in patients with anterior segment malformations provided further evidence for the role of IRID1 in normal eye development.217 Candidate genes within the IRID1 region were identified through cloning of the chromosomal breakpoint from an individual carrying a balanced t(6;13) translocation associated with multiple congenital anomalies including glaucoma. Two genes were found nearby, one of which, FKHL7 (previously known as FREAC-3), is a member of a family of transcriptional regulators related to the Drosophila developmental gene forkhead. Four families predominantly affected by Axenfeld-Rieger anomaly were found to have point mutations in FKHL7.217 In an independent study, FKHL7 was analyzed in a large patient panel and found to be mutated in three patients classified as having Axenfeld-Rieger anomaly.206 Although there is considerable phenotypic variability,217 FKHL7 mutations appear to occur mainly in patients or families with a primary diagnosis of Axenfeld-Rieger anomaly, and the presence of an abnormal Schwalbe line may be an important indicator suggesting an underlying FKHL7 mutation. Deletion of the FKHL7 gene is also likely to account for the anterior segment phenotype of patients with monosomy of this 6p region.284 As with PAX6, all available evidence suggests that the ocular anomalies are the consequence of FKHL7 haploinsufficiency. The FKHL7 phenotype has now been extended to a case of glaucoma with heart anomalies and deafness, reflecting even more closely the expression pattern of this gene.285
Extensive analysis of FKHL7 failed to reveal any mutation in four other IRID1-linked families, all of which show phenotypes more characteristic of iridogoniodysgenesis anomaly than Axenfeld-Rieger anomaly.206 In one of these families, FKHL7 involvement was excluded through segregation analysis of an informative polymorphism. Thus, it seems likely that IRID1 is a complex locus and that there is at least one other gene in this region which, when mutated, can predispose to iridogonial dysgenesis/glaucoma. A second forkhead-like gene was recently identified in this chromosomal region286 and in the homologous mouse region.287 In addition a deletion case of anterior segment anomaly where FKHL7 is not included in the deletion has been reported.288
FKHl7 Gene and Protein Structure; Conservation.
FKHL7 is a member of the winged-helix/forkhead-like gene family (see Fig. 240-10). This extensive family is implicated in developmental control of many tissues, often in response to signaling by members of the TGF/BMP family. FKHL7 transcript sizes are 3.0 and 3.9 kb in most of the expressing tissues, with an additional 3.4 kb mRNA present in kidney.217 Length variation may be produced by polyadenylation site variation. A single coding exon, showing 89 percent nucleotide and 92 percent amino acid identity with the mouse ortholog Foxc1/ Mf1, comprises the genomic gene. The coding region occupies 1659 bp, encoding 553 amino acids.206 The conserved DNA-binding forkhead domain, that places FKHL7 into its gene family, extends from amino acid 69 to amino acid 178. This domain is generally described as a winged-helix domain, a variant of the helix-turn-helix motif with two additional loops (the wings) on the C-terminal side of the main HTH region. Monomeric DNA binding is effected by the recognition alpha-helix intruding into the major groove of the DNA, while the loops provide additional backbone contacts.289
Mutations in FKHL7/Foxc1 in Axenfeld-Rieger anomaly. Mutations in the FKHL7 gene presented on a schematic of the FKHL7 protein, including the forkhead domain (FKHD). Above the proteins schematic are deletions; below are missense or nonsense mutations. The mutation associated with the congenital hydrocephalus (ch) mouse is underlined. The starred mutation is a deletion of an alanine in an alanine tract that was found in a patient with primary open-angle glaucoma.147,206,217,285
More distant extragenic elements are probably involved in control of FKHL7 expression, because the translocation breakpoint in a primary congenital glaucoma patient (with additional multiple congenital anomalies) carrying a balanced t(6;13)(p25.3;q22.3) translocation (missing only 11 bp of chromosome 6 sequence) lies about 25 kb outside the FKHL7 transcription unit.217 As several FKHL7 point mutations have been identified in anterior segment anomaly cases,217 FKHL7 is clearly implicated in this phenotype. Its loss of function is presumably elicited by position effect mechanisms similar to those observed for PAX6.214,216 However, the intragenic disruption of the neighboring gene, GMDS, with strong homology to GDP mannose 4,6-dehydratase, or a gene deletion on chromosome 13, may account for the additional more severe anomalies in this translocation patient.217
Regulation of Downstream Target Genes.
Very little is known about the genes under the direct regulatory control of FKHL7. In vitro binding selection experiments on random oligonucleotide target sequences have defined a shared core sequence of (RTAAAYA) for all FREAC family genes.289 At the same time, domain-swap experiments implied that gene-specific binding differences at flanking sites result from sequence differences in neighboring protein subregions. More detailed analysis of the forkhead protein-nucleic acid interaction suggested that protein binding leads to an 80° to 90° DNA bending.289
Specific downstream targets have not yet been defined. Analysis of the Mf1 mutant mouse (see “A Mouse Model for FKHL7-Associated Glaucoma,” below)290 suggests that components of cell-cell adhesion systems may be regulated by this forkhead gene. The epithelial tight-junction protein zona occludens, ZO-1, is almost completely absent in the developing cornea of homozygous mutant mice, but is perhaps unlikely to be a direct target of FKHL7 regulation, as there is some residual expression. In contrast, the expression of Pitx2 is unaltered in Mf1−/− mice, implying strongly that this gene which is mutated in some anterior segment anomalies, is not directly regulated by FKHL7.
During mouse development (E7.5 to E9.5), cells of two distinct origins express FKHL7/Mf1:291 neural crest cells and mesoderm cells, both paraxial and lateral. When assessed by northern blot studies using fetal and adult mouse polyA RNAs, the expression pattern is seen to be broad.147,217,289,290 Early studies suggested the absence of adult expression. However, subsequent analysis, including study of the lacZ knockout mice, which share the phenotypic spectrum of the natural congenital hydrocephalus mutant (see “A Mouse Model for FKHL7-Associated Glaucoma,” below), reveals wide-ranging expression. High-level expression is seen in the eye and the kidney,217 particularly in mesodermal derivatives such as the periocular mesenchyme.147,206
Several disease-associated intragenic mutations in FKHL7 have been identified; they are depicted in Fig. 240-10. An 11-bp early deletion, leading to predicted protein termination N-terminal to the winged helix domain, was identified in two brothers with glaucoma, one with Rieger anomaly, the other with iris hypoplasia; their father, carrying the same mutation had only isolated posterior embryotoxon.217 Three missense mutations, all within the third helix of the winged helix domain were identified in three further small families: S131L in a mother and daughter with Rieger anomaly and glaucoma; I126M in a father and son with Axenfeld anomaly; and in a three-generation family, F112S was associated with Axenfeld anomaly in two affected individuals and Rieger anomaly in two others.217 Independent analysis in families and sporadic cases with iridogonial dysgenesis or Axenfeld-Rieger anomaly206 also revealed three disease-causing mutations (a 10-bp early truncating deletion, S82T, and I87M) all in Axenfeld-Rieger anomaly cases. Additional polymorphism, for numbers of repeated glycine (codon GGC) residues at two different sites C-terminal to the winged helix domain, were also observed in this study. The mouse ch mutation (see “A Mouse Model for FKHL7-Associated Glaucoma,” below and reference147) was shown to be a premature truncation at Q123X, in the winged helix domain. It remains to be seen whether FKHL7 mutations C-terminal to the winged helix domain also cause anterior segment anomalies. It is clear, however, that a number of phenotypically similar cases that map to this chromosomal region have not revealed FKHL7 mutations and, in some cases, FKHL7 seems to have been excluded as a candidate gene (see FKHL7 “Gene Identification” above). However, in these cases the possibility of long range derangement of FKHL7 expression must be excluded, as there is evidence that such mechanisms can lead to disease.206,285
A preliminary report has described an FKHL7 frameshift mutation in a family with Rieger anomaly, sensorineural hearing loss, and atrial septal defect.285 The same report also mentions an individual with primary open angle glaucoma (POAG) showing in frame deletion of three alanine residues in a poly-alanine tract in the C-terminal region—a variant not seen in 384 controls.
A Mouse Model for FKHl7-Associated Glaucoma.
Naturally occurring and targeted mutations of Mf1, the original name for the mouse FKHL7ortholog, now renamed Foxc1, are responsible for the classical congenital hydrocephalus (ch) phenotype in mice.147 Like Pax6, Mf1 mutations exhibit clear dosage effects: homozygous ch −/− mice have hydrocephalus, ocular anomalies, and multiple skeletal defects, while heterozygotes have anterior segment anomalies including defects of the trabecular meshwork and Schlemm's canal.206 The ch +/− mouse promises to be a valuable animal model for glaucoma, and provides a fascinating example of a classical mouse mutant which was thought to be recessive but is now known to be semidominant following reexamination in the light of FKHL7 identification as a human disease gene. Recent detailed phenotypic analysis290 revealed that in mice, the major eye anomaly is associated with failure of the cornea to separate from the lens, which is the result of the aberrant development of the mesenchymally derived inner corneal endothelial layer.
The much more severe, neonatally lethal, phenotype of homozygous congenital hydrocephalus mice is not generally seen in the human population. The spectrum of anomalies seen in these mice, does however, illustrate more clearly the expression pattern of the gene.147
PITX2: The Rieger Syndrome Gene at 4q25
The Rieger syndrome gene was identified using a classical approach combining linkage analysis in well-characterized families and definition of chromosomal abnormalities in rare individuals with cytogenetic rearrangements. Linkage studies of three Rieger syndrome families revealed a tight association with markers mapping to 4q25.276 The critical region was further defined by physical mapping, which utilized two Rieger syndrome-associated translocation breakpoints shown to be 50 kb apart, spanned by a cosmid contig. One of these cosmids was shown to include PITX2, a gene possessing a paired-type homeobox, one of three closely-related pituitary homeobox genes, related to that of the Drosophila developmental gene bicoid, with a lysine residue at amino acid 50 in the homeodomain.207
The breakpoints, positioned about 15 kb upstream and 50 kb downstream of the 18-kb genomic gene, apparently cause the phenotype by position effects.216 An additional translocation break 90 kb upstream of the gene has also been reported, suggesting a requirement for intact long-range cis-control of PITX2 expression.218
PITX2 was also isolated several times independently of its role in Rieger syndrome: as a key downstream modulator of left-right asymmetry (reviewed in references292 and 293); as a homeodomain-bearing pituitary developmental regulator;149 and as a target of trithorax regulation modulated in human acute leukemia.294
PITX2 Gene and Protein Structure, Conservation.
The human gene, transcribed from telomeric to centromeric direction, consists of four exons that encode a 2125-bp transcript.207 The 813-bp open reading frame spans from exon 2 to 4, with the homeodomain encoded mostly by exon 3 but terminating in exon 4. The predicted protein product comprises 271 amino acids, with the homeodomain extending from residues 39 to 98. Alternative splicing with additional intron-exon complexity has been recognized in the mouse, producing a larger 317-amino-acid product. A third isoform, also differing from other products in the N-terminal region, is produced by alternative promoter usage.149 Greater than 99 percent amino acid sequence identity is observed between the human and mouse proteins. In addition, 97 percent nucleotide sequence identity, with probable functional significance, is seen over a 270-bp region in the 3′ untranslated region of the mRNA. Orthologs from a number of vertebrate species have been identified, including the prevertebrate cephalochordate Branchiostoma. In addition, homologs are also found in Drosophila and C. elegans. The availability of these model systems should make functional analysis easier.
Regulation of Downstream Genes.
The PITX2 downstream target genes are not yet defined. Identifying physiologically relevant targets of DNA-binding developmental regulators is one of the toughest goals to fulfill. There is a suggestion that the early acting homeobox gene Hesx1 may be a target because in homozygous Pitx2 knockout mice, Hesx1 expression is abolished in the early E12.5 pituitary.149 In terms of its function in laterality determination, Pitx2 acts downstream of other asymmetry genes such as Shh, nodal, and lefty, iv, and inv, but there is no evidence for direct target relationships.
The PITX2 protein can bind the defined target sequence for Drosophila bicoid. Functional analysis of wild-type and mutant PITX2 proteins using electrophoretic mobility shift assays, protein binding, and transient transfection assays revealed impaired activity and instability of mutant proteins carrying amino acid substitutions found in Rieger syndrome patients.296
In keeping with the Rieger syndrome phenotype, PITX2 expression was originally observed by whole-mount in situ hybridization in 11-day mouse embryos in the mesenchyme around the eye, Rathke's pouch, in the dental lamina, limb mesenchyme, the dorsal mesentery, and the vitelline and umbilical vessels.207 Further analysis is summarized by Yost,293 where the asymmetric Pitx2 expression is discussed, describing broadly that Pitx2 expression is bilateral in the cephalic mesoderm and pituitary precursors, but on the left side of lateral-plate mesoderm structures, and of the cardiac tube and gut. Expression patterns are altered in mouse laterality mutants. Conversely, ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and isomerizes body rotation in developing chick and Xenopus where these experiments can be performed (reviewed in reference 292).
The first intragenic point mutations in PITX2 were described in two sporadic and four familial cases in which all three cardinal signs of Rieger syndrome were present in at least one family member. The mutations (Fig. 240-11) were L54Q, W133X, and two splice-site mutations in the families, and two further missense mutations, T68P and R91P for the sporadic cases. Subsequently, a PITX2 missense mutation, R70H, was identified in a family with iridogonial dysgenesis syndrome (iris hypoplasia, iridocorneal angle defects, and glaucoma, as well as maxillary hypoplasia, dental anomalies, inguinal hernia, and hypospadias).208 Autosomal dominant iris hypoplasia with glaucoma was associated in one family with an R46W missense change.295
Mutations in PITX2 are associated with Rieger syndrome, iridogoniodysgenesis syndrome, and Peters anomaly.207,208,281,295 Mutations in the PITX2 gene presented on a schematic of the PITX2 protein, including the homeodomain (HD) and OAR domain. Splice-site mutations are shown above, while missense and nonsense mutations are shown below the schematic. All the mutations are associated with Rieger syndrome except *, which is associated with iridogoniodysgenesis syndrome, and the underlined mutation, which is mutated in an individual with Peters anomaly.
A PITX2 mutation has also been described in association with Peters anomaly.281 The child in this case had systemic features of Rieger's syndrome and typical features of Rieger's anomaly in the left eye; the right eye, however, showed the characteristic central corneal opacification of Peters anomaly (see “Peters Anomaly” above). A similar combination of ocular phenotypes (Rieger-like in one eye, Peters-like in the other) has been described in association with a PAX6 mutation.199 Clearly, careful evaluation of systemic defects can help in assessing the likelihood of PAX6 or PITX2 involvement.
Mouse Model for PITX2 Loss of Function.
Several groups have generated mice with Pitx2 intragenic deletions by gene targeting in ES cells.297–299 Homozygous total loss-of-function animals die in utero at E13.5 to E14.5, probably as a result of abnormal heart development, but the normal rightward looping of the heart tube and gut is unaltered, although there is right isomerization of the lung. Careful further examination of these null mutants at or before E13.5 reveals optic nerve coloboma, absence of ocular muscles, failure of ventral body wall closure, maxillary defects, and tooth anomalies, as well as arrest of pituitary development. A less severe phenotype, with death of the homozygotes at E18.5, is observed where a partial loss-of-function allele was created by insertion of the LoxP site-flanked selectable marker into an intron which gave rise to an allele with altered splicing characteristics.297 Examination of the heterozygotes from this line, and from the null allele, revealed some of the features typical of Rieger syndrome: corectopia and iris anomalies, as well as malocclusion of teeth.297
PITX3 in Anterior Segment Anomalies
The mouse homolog, Pitx3, was identified through screening a mouse cDNA library with a Pitx2 homeobox probe, and through degenerate RT-PCR.139,300 Initial studies revealed early expression in the developing eye, particularly in the lens which was interesting in view of the chromosomal localization of Pitx3 on mouse chromosome 19, in the region of the mouse locus aphakia associated with maldevelopment of the lens and reduced eye size in homozygotes. The human homolog Pitx3 was subsequently isolated and mapped to chromosome 10q25, the conserved synteny region for the mouse chromosome 19 locus.140 No human eye disease phenotype had been previously assigned to this region, necessitating a less focused search for possible disease associations.140
PITX3 Gene, Protein Structure and Conservation.
Like PITX2, the PITX3 gene consists of four exons, the first of which is predicted to be entirely noncoding. Intron positioning is identical to that of PITX2 and PITX1, with the homeodomain spanning from exon 3 to exon 4. A region of very high GC content (80 percent) is found upstream of the first methionine. There is strong amino acid similarity among PITX family members throughout the protein, but in the homeodomain mouse PITX3 shows 100 percent and 97 percent identity with mouse PITX2 and PITX1, respectively.139 A highly conserved 14-amino-acid motif, the OAR domain, is present C-terminal of the homeodomain in PITX3, as well as in other paired-type homeobox proteins.
In mouse, the 3′UTR of Pitx3 overlaps with the 3′UTR of the Elovl3 gene (formerly Cig30), which is located on the opposite strand (Tyrdik et al, 1999). Elovl3 is involved in the biosynthesis of very long chain fatty acids (Tyrdik et al, 2000). The consequence of the complementary 3′UTR region for normal gene function is unknown. However in mice homozygous for the aphakia mutation, where the level of Pitx3 transcription is reduced to 5% of wildtype, Elovl3 transcription is reduced by about half (Rieger et al, 2001).
In situ hybridization studies139 provided detailed information on eye expression at various stages in development. Strong expression was seen in the equatorial (bow) regions of the lens at E11, and throughout the lens by E15. The eye muscles and eyelid also express Pitx3 at E15. Subsequently, more detailed analysis140 revealed strong expression in the midbrain region, tongue, and incisor primordia, and in the condensing mesenchyme around the sternum and vertebrae, and in the head muscles. Expression in the dopaminergic neurons of the mesencephalic region was also documented in mice.300 Wholemount in situ hybridization studies in Xenopus embryos have confirmed that the developing pituitary and lens are major sites of Pitx3 expression (Pommereit et al, 2001).
PITX3 Mutations Underlie the Classical Mouse Mutation aphakia.
Aphakia (ak) is a classical recessive mouse mutation with a phenotype of bilateral microphthalmia resulting from failure of normal lens development (Doolittle et al, 1997). At E10 in homozygotes, the early lens vesicle is disorganized, and by E10.5-E11 lens development arrests completely at the lens stalk stage. The abnormal lens vesicle then degenerates. The structures of the anterior chamber fail to form, and the optic vesicle, which does not develop to its normal size, becomes filled with abnormally folded retinal tissue (Doolittle et al, 1997; Rieger et al, 2001; Semina et al, 2000).
The ak locus was mapped to chromosome 19 in the vicinity of Pitx3 (Reiger et al, 2001; Semina et al, 2000). In one study, a 650bp deletion was found 2.5 kb upstream of the Pitx3 transcriptional start site. Levels of Pitx3 expression were dramatically reduced in ak homozygotes, and it was concluded that the upstream region must be critical for the normal regulation of the gene (Semina et al, 2000). However, a second study showed that the ak mutation actually consists of two separate deletions, one upstream as previously described and one of 1.4 kb encompassing the promoter of Pitx3, exon 1, and part of intron 1 (Rieger et al, 2001).
The ak mutation is now referred to as Pitx3 ak (http://www.informatics.jax.org/searches/allele_report.cgi?_Marker_key=34563). Mice heterozygous for the Pitx3 ak mutation have been described as normal (Doolittle et al, 1997), but more detailed examination of the anterior chamber of heterozygotes is warranted given that human heterozygotes have anterior segment anomalies (See "Human Mutations in PITX3" below).
Regulation of Downstream Genes.
There is good evidence that the Pitx3 protein is a direct regulator of Th, the gene encoding tyrosine hydroxylase, in midbrain dopaminergic neurones (Lebel et al, 2001). Pitx3 is expressed in the precursors of midbrain neurons from E11, with Th expression apparent by E11.5. Tyrosine hydroxylase is essential for dopamine biosynthesis. In transient transfection assays, Pitx3 was able to stimulate reporter gene expression by binding to a single high-affinity site in the Th promoter (Lebel et al, 2001). No ocular targets of Pitx3 are known at present.
Human Mutations in PITX3.
Four families with various developmental eye anomalies were analyzed for linkage with the PITX3 chromosomal region. One anterior segment mesenchymal dysgenesis (ASMD) family showed tight linkage to polymorphic markers flanking PITX3. ASMD cases have malformations involving each of the three mesenchymal neural crest migrations, making PITX3 a likely candidate gene based on expression pattern. In addition, mindful of the phenotype/genotype heterogeneity observed in developmental eye disease, 80 samples from familial cases with Rieger syndrome, isolated anterior segment malformations, Axenfeld and Rieger anomalies with other associated systemic abnormalities and some familial cases of congenital cataracts, were screened for PITX3 mutations. So far, mutations have been documented in two cases (Fig. 240-12). First, a 17-bp insertion, into a region where an 11-bp repeat is duplicated, has been identified in a family with ASMD syndrome, leading to quite a long region of out-of-frame change before termination.140 The second mutation, a S13N missense change in an autosomal dominant congenital cataract (ADCC) mother and son, was documented as a de novo change when the mother's parents were shown to have only wild-type sequence.140
Mutations in PITX3 are associated with anterior segment mesenchymal dysgenesis.140 Mutations in the PITX3 gene presented on a schematic of the PITX3 protein, including the homeodomain (HD) and OAR domain. The N13S missense mutation is associated with autosomal-dominant congenital cataracts. The 17-nucleotide insertion is present in a family with ASMD and cataracts.
All the anterior segment anomalies described here are caused by loss of function of one copy of a gene (haploinsufficiency). In each case, that gene has many other crucial functions in development, implied by the extensive nonocular expression pattern and demonstrated by the homozygous loss of function phenotype in animal models (e.g., neuronal development, PAX6; left-right asymmetry, PITX2; brain and skeletal development, FKHL7).
A recurring theme is the extreme phenotypic variability that occurs in these disorders, sometimes even between the two genetically identical eyes of a single individual carrying one mutation. This variability probably reflects the dependence of normal eye development on the precise dosage of these genes, and the fact that a molecular defect in the early stages of anterior segment development may not have an entirely predictable and reproducible outcome given the enormous complexity of the subsequent tissue interactions that must take place. In a single individual, the phenotypic outcome may depend on the precise number of transcripts from a gene at some critical stage. Between individuals, the activity of other genes may ameliorate or exacerbate the shortfall in the mutated gene. From a genetic perspective, remember that there is generally no simple genotype-phenotype correlation. From a clinical perspective, knowledge of the underlying mutation may be useful in deciding what clinical strategies to take; there may be increased risks associated with some surgical interventions due to the reduced regenerative power of tissues carrying certain mutations.
The phenotypic overlap between patients with mutations in PAX6, PITX3, PITX2, and FKHL7 must point to functional interrelationships between these proteins. The ectodermally derived (PAX6-expressing) structures of the optic cup, the lens, and the presumptive cornea must communicate through cell-surface markers and secreted signaling proteins with the incoming neural crest-derived mesenchyme (PITX2-, PITX3-, and FKHL7-expressing). The stage is set for a more detailed understanding of how these genes act to coordinate the multiple tissue interactions of the anterior segment.
Waardenburg syndrome (WS) is a phenotype characterized principally by auditory and pigmentary abnormalities. The characteristic eye pigmentation defects of WS (Table 240-7, Fig. 240-13) are hypopigmented or heterochromatic irides and occasionally, hypopigmented fundi.301,302 The major nonocular phenotypes of WS include defects of pigmentation of the skin or hair (particularly a white forelock) and hearing loss. The underlying cause of these diverse phenotypes is a disruption in the development, migration, or differentiation of melanocytes and neural crest cells. Four types of WS have been distinguished by clinical and molecular criteria and found to result from mutations in at least five different genes (Table 240-7).302–304 Additional WS genes remain to be identified because mutations in the five known WS genes do not account for all patients. WS is discussed comprehensively in Chap. 244. Only a brief overview is presented here, with particular attention to the associated eye defects.
Photograph of the iris of a patient with a hypopigmented iris and thin iris stroma. This patient was not demonstrated as having WS2 or an MITF mutation, but the phenotype of the iris and white forelock suggest WS2. The arrowhead identifies the hypopigmented radial section of the left iris. See Chap. 244 for photographs of patients with identified Waardenburg syndrome. (Photograph kindly provided by Isabelle Russell-Eggitt, Great Ormond Street Hospital for Children, London, England.)
Waardenburg syndrome type 1 (WS1) is a dominantly inherited condition due to mutations in the transcription factor gene PAX3.302 Affected patients show the canonical pigmentary defects of WS in addition to a craniofacial defect, dystopia canthorum (outward displacement of the inner canthi of the eyes). WS2, caused by mutations in MITF, is also dominant, but can be distinguished from WS1 by the absence of dystopia canthorum. WS3 patients resemble those with WS1 except that, by definition, WS3 patients also have limb defects. Like WS1, WS3 is also the result of mutations in PAX3 although both recessive and dominant inheritance of WS3 have been identified. WS4 patients have classic WS pigmentary defects and no dystopia canthorum, but their phenotype includes Hirschsprung disease. When WS4 is dominantly inherited, mutations have been found in the putative transcription factor SOX10. In contrast, patients with recessive WS4 have been discovered to have defects in either the gene encoding the Endothelin 3 (EDN3) ligand, or one of its receptors, EDNRB. Mouse models for all four Waardenburg syndromes have been identified (WS1 and 3: Splotch mouse (Pax3);305 WS2: microphthalmia mouse (Mitf);116,306 WS4: Dom mouse (Sox10),307 lethal-spotting mouse (Edn3),302 and piebald-lethal mouse (Ednrb)302).
The differences in the WS phenotypes may be due to the cells affected.302 WS2 appears to result from defects in melanocytes of neural crest origin fated to become auditory and pigmentary cells. However, WS1, 2, and 3 appear to be the consequence of abnormalities in melanocytes as well as other neural crest cells. Neural crest cells are involved in the development of various tissues including facial bones and limb muscles, accounting for the more complex phenotypes of WS1, 2, and 3.
Waardenburg Syndrome Type 2A.
Although mutations in the MITF gene are associated with WS2, they are responsible for only 15 percent of cases, and consequently at least one other gene, presently unidentified, is associated with WS2;302,308 WS2 patients with MITF mutations are classified as having WS2A. In this chapter, we examine in particular the role of the MITF gene (associated with one form of WS, type 2A) in eye development, because MITF is the only one of the WS-associated genes known to be expressed in the developing eye.116,118 The MITF gene encodes a basic helix-loop-helix, leucine-zipper transcription factor, which is of fundamental importance to mammalian eye development, because homozygous mutations in the mouse Mitf gene are associated with severe developmental eye defects. These defects extend well-beyond the abnormalities in iris pigmentation (hypopigmented or heterochromatic irides) found in patients with all types of WS, including type 2. The iris phenotypes are thought to originate in abnormalities in the neural crest cells that migrate into the iris, rather than in cells that originate in the developing eye per se. The homozygous Mitf mouse eye abnormalities include a small eye and retinal degeneration due to hyperproliferation of the (RPE) cells.
In 1993, mutations in the mouse Mitf gene were found to be associated with a classic semidominant mouse mutant, microphthalmia (mi).116,306 This discovery led to the cloning of the human MITF gene from a melanocyte cDNA library. The mouse Mitf gene is located on chromosome 6, 40 cM from the centromere.309 Human MITF maps to chromosome 3p14.1-p12.3.310 Two WS2 families were also mapped to proximal 3p, in the region of MITF,311 suggesting MITF as a WS2 candidate gene. This candidacy was supported by the similarity of the auditory and pigmentary phenotype of WS2 patients and the microphthalmic (mi) mouse. WS2 patients were found to carry MITF mutations (see “Human Mutations in MITF Cause Waardenburg Syndrome Type 2,” below).308 Spontaneous Mitf mutations have also been identified in hamster,312,313 quail,314 and rats.313
MITF Protein Structure and Conservation.
The microphthalmia basic helix-loop-helix transcription factor contains a leucine-zipper domain, a motif that, in other proteins, mediates protein-protein interactions (Fig. 240-14).317,318 The human and mouse MITF proteins are 94 percent identical overall, with 100 percent identity in the bHLH domain.116,306,310 In addition, the leucines of the leucine zipper, as well as the spacing between them, are perfectly conserved. The MITF polypeptide is similar to the bHLH-ZIP proteins TFEB, TFEC, and TFE3.319 Each of these three related proteins, which together with MITF make up the small MiT protein family, has been shown to heterodimerize both with MITF and with one another, each combination having a different DNA-binding affinity. TFEB and TFE3 are expressed ubiquitously, while TFEC is tissue-restricted in its expression; apart from MITF, no other member of the MiT family has been shown to have a role in development.
Mutations in the human MITF gene presented above a schematic of the MITF protein, a basic helix-loop-helix, leucine-zipper transcription factor. Truncating mutations are shown above; nontruncating mutations are shown below. All the mutations shown are associated with WS2 except *, which is associated with the Tietze-Smith syndrome (see text). The insertion point for the 18-bp alternatively spliced exon is shown above the schematic (arrowhead). A stop codon is abbreviated as an X. (Adapted from Read.302 Used with permission.) Mutations in the mouse Mitf gene presented below a schematic of the MITF protein. Deletions and promoter mutations are shown above, point mutations below. All mutations are alleles associated with the microphthalmic mouse. *The red-eyed white allele is a deletion of upstream sequences resulting in cryptic splicing and a novel N-terminal peptide. **An intragenic deletion. §A small deletion disrupts splicing and removes the alternatively spliced exon in the spotted allele which has no homozygous phenotype, but is associated with a microphthalmic phenotype only when present with another Mitf allele in a compound heterozygote. ¥A small deletion in the basic domain, 3′ to the alternatively spliced exon. ∥One arginine, exact position unknown, is deleted from the basic domain. (Figure compiled using data from Steingrimsson et al.315 and Moore.316 )
In addition to the potential modulation of MITF activity through the formation of various heterodimers, differential splicing produces multiple MITF isoforms. In mouse, there are three alternatives of exon 1, each generating different proteins at the N-terminus,315,320,321 one specifically present in melanocytes, another in the heart, and a third isoform that is specific to the RPE. Another alternative splice event occurs within intron 5, resulting in two isoforms that either include or omit six amino acids immediately N-terminal to the bHLH domain.116 The splice form with the six-amino-acid insert has slightly higher DNA-binding affinity.319 MITF is regulated by MAP kinase phosphorylation, part of the Steel/Kit pathway in melanocytes.322 This phosphorylation event increases the transactivation of the MITF downstream gene, tyrosinase (see Fig. 240-15).
The MITF protein has been positioned in various biochemical and genetic pathways. The MITF protein has been shown in melanoma cell lines to be phosphorylated by the MAP kinase pathway in response to Steel factor. This phosphorylation event increases the ability of MITF to transactivate one of its downstream targets, the major pigmentation gene Tyr. MITF may also transactivate Tyrp1, Dct, and Kit. All four promoters (Tyr, Tyrp1, Dct, and Kit) contain an MITF binding site called the M-box. MITF functions by heterodimerizing with proteins of the TFE family. PAX3 protein binds to an MITF promoter construct in vitro, producing a significant increase in the transcription of the MITF gene in melanoma cells. See text for details and references.
Mitf expression in the developing mouse eye has been characterized extensively.116–118,320 The transcript is first detected at E9.5 in the whole-optic vesicle, but by E10.5 expression is reduced to the presumptive RPE, where it continues to be detected until E16.5. Mitf is also expressed in the developing neural crest, specific cells surrounding the otic vesicle (the presumptive inner ear structure), and hair follicles. This pattern is consistent with the disruption of normal auditory intermediate cells of the stria vascularis and pigment cell formation in WS2.323 Adult mice, and cells lines derived from the corresponding tissues, express Mitf in heart, lung, uterus, skin, mast cells, and melanocytes.116 The phenotype associated with heterozygous WS2 mutations is milder than the homozygous phenotype observed for most recessive mouse mi alleles, where affected tissues include the eye—particularly the retina—the inner ear, coat pigment, mast cells, and osteoclasts.316,324 RPE and forebrain melanocytes are of distinct origin, and the latter appear to be unaffected in individuals heterozygous for MITF mutations (in both humans and mice), while RPE melanocytes are very abnormal in mice homozygous for Mitf mutations.
Regulation of the MITF Gene and Target Genes of the MITF Protein.
MITF is a transcriptional activator that interacts with other proteins to regulate the expression of downstream genes by binding to a conserved 11-bp DNA element: the M box (core sequence: CATGTG).319 The three major pigmentation enzyme genes—Tyrosinase (Tyr), Tyrosinase related peptide 1 (Tyrp1), and Dopachrome tautormerase (Dct), previously known as Tyrosinase related peptide 2 (Tyrp2)—all contain an M-box in their promoters. Although there is no evidence for in vitro binding of MITF to the human Tyrosinase promoter, MITF does activate transcription from this promoter in expression assays.325,326 There is also evidence for MITF regulation of Tyrp1 and Dct, as well as the Kit gene (cytokine receptor).117,327,328 Mitf also interacts with the retinoblastoma and CBP/p300 protein in vitro.329,330
The MITF protein has been positioned in a number of biochemical pathways (Fig. 240-15): as a target of phosphorylation from the Steel/Kit/MAP kinase pathway,322 directly downstream of PAX3,331 and upstream of pigmentation enzyme genes.319 PAX3 up-regulates the expression of MITF in vitro and mutant PAX3 proteins associated with WS1 fail to transactivate MITF.331 The PAX3 transactivation of MITF is consistent with the similar auditory-pigmentary phenotypes observed in WS1 (due to mutations in PAX3) and WS2 (due to mutations in MITF).
Effects of MITF Activity in Vivo.
In tissue culture model systems, MITF is required for the determination of RPE cell fate and for the down-regulation of cell proliferation. Ectopic MITF expression converts NIH 3T3 fibroblasts into cells with features of melanocytes,332 including characteristic morphology and the expression of MITF downstream proteins. RPE cells expressing mutant Mitf undergo increased proliferation, and lose their pigmented phenotype; ectopic Mitf can rescue both of these changes.314 MITF also appears to negatively regulate PAX6 expression in both chick and quail, although it is not known whether this regulation is direct.314,320 The ectopic PAX6 expression may be causal in the loss of an RPE phenotype and gain of neuroretina-like features in RPE cells.
Human Mutations in MITF Cause Waardenburg Syndrome Type 2.
As of 1997, 11 human MITF mutations had been identified in association with WS2302,308,333 (Fig. 240-14). MITF mutations associated with WS2 include truncating mutations predicted to be loss-of-function null alleles (splice mutations, nonsense changes, and frameshift deletions), and nontruncating changes that may or may not be loss-of-function mutations (a three-nucleotide deletion and missense mutations). One MITF mutation was also found in a family with autosomal dominant Tietz-Smith syndrome (MIM 103500), of which the major phenotypes are albinism and complete nerve deafness.334 Given that MITF functions in several biochemical pathways, the remaining, as yet unidentified genes in these pathways may be mutated in WS2 (Fig. 240-15). In contrast to the mouse no recessive disease-causing mutations in the human MITF gene have been identified to date, but this may be due in part to ascertainment bias in human populations that are largely outbred with low mutant gene frequency.
Recently digenic inheritance involving MITF was identified,335 highlighting the role of MITF as a genetic modifier locus. Two families with both WS2 and autosomal recessive ocular albinism (AROA) were shown to (a) harbor a heterozygous mutation in MITF (a one-nucleotide deletion in exon 8) and (b) to be either heterozygous or homozygous for a TYR polymorphism (R402Q) that by itself is nondeleterious. Thus, MITF appears to act as a modifier locus of the TYR phenotype, consistent with MITF being a transcriptional regulator of TYR. These findings illustrate an important principle of medical genetics: transcription factor variants that regulate a polymorphic gene product can increase the clinical heterogeneity of a disease.
Mouse Mutations in MITF Cause Microphthalmia.
To date, 21 alleles of Mitf have been shown to produce the microphthalmic (mi) mouse phenotype118,316 (Fig. 240-14). A diversity of mutations has been shown to cause both semidominant and autosomal recessive disease. Mitf mutations affect the cells in which the protein is developmentally expressed (see “Mitf Expression,” above).117,316 The most severe phenotypic manifestations occur with alleles in which the DNA-binding domain is altered. The range of phenotypic characteristics includes microphthalmia, retinal degeneration, osteoporosis, an inner ear defect with possible hearing loss, pigmentation abnormalities, reduced mast cell numbers, and neural crest cell defects. A major feature of the severe eye phenotype is hyperplasia of the RPE, indicating that Mitf normally represses RPE cell proliferation. The RPE proliferation leads to defects in the choroidal fissure.324 The expression of Tyr and Tyrp1 is undetectable in Mitf mutant RPE cells, which may also contribute to the RPE cell abnormalities.117 Progressive retinal degeneration is seen with some alleles, and is probably due to defects in RPE cell function;118,316 a normal RPE is essential for the maintenance of the mammalian photoreceptor.336
Lessons from the Mouse Model.
The study of the microphthalmic mouse has made several major contributions to the understanding of the role of the MITF gene in human disease. Identification of the mouse Mitf gene116,306 and analysis of the microphthalmic mouse led to the discovery of the association between mutations in human MITF gene and the human disease Waardenburg syndrome type 2A.302,308 Subsequently, analysis of Mitf/MITF mutations in mouse and humans has highlighted major unexpected differences in the genetic mechanisms at this locus in the two species.
A striking example is the contrast in the pattern of inheritance and phenotype resulting from two near-identical nonsense mutations, R263X in mouse and R259X in human, both of which occur just N-terminal to the leucine zipper motif.319,337 The R263X allele is fully recessive in mouse, while the human R259X allele is autosomal dominant. The homozygous R263X mouse (cloudy eyes allele) has microphthalmia, reduced eye pigmentation, and cataracts.316 In contrast, human heterozygotes for the R259X allele have Waardenburg syndrome type 2A, which is not associated with cataracts.
Additional complexity in the effect of the same mutation in the two species is shown by the different heterozygous phenotypes that occur in carriers of the R217Δ allele. In human carriers, the allele causes autosomal dominant Tietz Syndrome, which is similar to WS2A except that the patients also have albinism and complete nerve deafness.333 In contrast, R217Δ is semidominant in mice, their hearing is normal, and they have only a small depigmented skin splotch.116,324
Finally, molecular evidence of mutations in MITF/Mitf protein supports different genetic mechanisms between mouse and human. In the mouse, in vitro studies suggest that these semidominant alleles are dominant negatives that disrupt the basic DNA-binding domain.319 In contrast, human MITF alleles that result in autosomal dominant WS2 are likely to be due to haploinsufficiency.333,337 These examples suggest that humans are either more sensitive than mice to MITF gene dosage effects, or that there are substantial differences in the effects of modifier genes in the two species.
In contrast to the mouse, the Syrian hamster has dominant Mitf mutations that lead to haploinsufficiency.116,312 The hamster phenotype is similar to humans with WS2 pigment defects, and heavy hearing loss is present in heterozygotes. Thus, the hamster may be a better model for WS2 than the mouse. Because no humans with homozygous MITF mutations have been identified, either the result is in embryonic lethality or a phenotype that has not yet been identified.