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Ocular coloboma (OC) is a defect in optic fissure closure and is a common cause of severe congenital visual impairment. Bilateral OC is primarily genetically determined and shows marked locus heterogeneity. Whole-exome sequencing (WES) was used to analyze 12 trios (child affected with OC and both unaffected parents). This identified de novo mutations in 10 different genes in eight probands. Three of these genes encoded proteins associated with actin cytoskeleton dynamics: ACTG1, TWF1, and LCP1. Proband-only WES identified a second unrelated individual with isolated OC carrying the same ACTG1 allele, encoding p.(Pro70Leu). Both individuals have normal neurodevelopment with no extra-ocular signs of Baraitser-Winter syndrome. We found this mutant protein to be incapable of incorporation into F-actin. The LCP1 and TWF1 variants each resulted in only minor disturbance of actin interactions, and no further plausibly causative variants were identified in these genes on resequencing 380 unrelated individuals with OC., (© 2017 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

Arhinia, or absence of the nose, is a rare malformation of unknown etiology that is often accompanied by ocular and reproductive defects. Sequencing of 40 people with arhinia revealed that 84% of probands harbor a missense mutation localized to a constrained region of SMCHD1 encompassing the ATPase domain. SMCHD1 mutations cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) via a trans-acting loss-of-function epigenetic mechanism. We discovered shared mutations and comparable DNA hypomethylation patterning between these distinct disorders. CRISPR/Cas9-mediated alteration of smchd1 in zebrafish yielded arhinia-relevant phenotypes. Transcriptome and protein analyses in arhinia probands and controls showed no differences in SMCHD1 mRNA or protein abundance but revealed regulatory changes in genes and pathways associated with craniofacial patterning. Mutations in SMCHD1 thus contribute to distinct phenotypic spectra, from craniofacial malformation and reproductive disorders to muscular dystrophy, which we speculate to be consistent with oligogenic mechanisms resulting in pleiotropic outcomes.

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3' (telomeric) to PAX6 and one within a gene desert 5' (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.

Objective: PAX6 is a pleiotropic transcription factor essential for the development of several tissues including the eyes, central nervous system, and some endocrine glands. Recently it has also been shown to be important for the maintenance and functioning of corneal and pancreatic tissues in adults. We hypothesized that PAX6 is important for the maintenance of brain integrity in humans, and that adult heterozygotes may have abnormalities of cortical patterning analogous to those found in mouse models., Methods: We used advanced magnetic resonance imaging techniques, including surface-based morphometry and region-of-interest analysis in adult humans heterozygously mutated for PAX6 mutations (n = 19 subjects and n = 21 controls). Using immunohistochemistry, we also studied PAX6 expression in the adult brain tissue of healthy subjects (n = 4) and patients with epilepsy (n = 42), some of whom had focal injuries due to intracranial electrode track placement (n = 17)., Results: There were significant reductions in frontoparietal cortical area after correcting for age and intracranial volume. A greater decline in thickness of the frontoparietal cortex with age, in subjects with PAX6 mutations compared to controls, correlated with age-corrected, accelerated decline in working memory. These results also demonstrate genotypic effects: those subjects with the most severe genotypes have the most widespread differences compared with controls. We also demonstrated significant increases in PAX6-expressing cells in response to acute injury in the adult human brain., Interpretation: These findings suggest a role for PAX6 in the maintenance and consequent functioning of the adult brain, homologous to that found in other tissues. This has significant implications for the understanding and treatment of neurodegenerative diseases.

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS., (© 2014 WILEY PERIODICALS, INC.)

We identified four different missense mutations in the single-exon gene MAB21L2 in eight individuals with bilateral eye malformations from five unrelated families via three independent exome sequencing projects. Three mutational events altered the same amino acid (Arg51), and two were identical de novo mutations (c.151C>T [p.Arg51Cys]) in unrelated children with bilateral anophthalmia, intellectual disability, and rhizomelic skeletal dysplasia. c.152G>A (p.Arg51His) segregated with autosomal-dominant bilateral colobomatous microphthalmia in a large multiplex family. The fourth heterozygous mutation (c.145G>A [p.Glu49Lys]) affected an amino acid within two residues of Arg51 in an adult male with bilateral colobomata. In a fifth family, a homozygous mutation (c.740G>A [p.Arg247Gln]) altering a different region of the protein was identified in two male siblings with bilateral retinal colobomata. In mouse embryos, Mab21l2 showed strong expression in the developing eye, pharyngeal arches, and limb bud. As predicted by structural homology, wild-type MAB21L2 bound single-stranded RNA, whereas this activity was lost in all altered forms of the protein. MAB21L2 had no detectable nucleotidyltransferase activity in vitro, and its function remains unknown. Induced expression of wild-type MAB21L2 in human embryonic kidney 293 cells increased phospho-ERK (pERK1/2) signaling. Compared to the wild-type and p.Arg247Gln proteins, the proteins with the Glu49 and Arg51 variants had increased stability. Abnormal persistence of pERK1/2 signaling in MAB21L2-expressing cells during development is a plausible pathogenic mechanism for the heterozygous mutations. The phenotype associated with the homozygous mutation might be a consequence of complete loss of MAB21L2 RNA binding, although the cellular function of this interaction remains unknown., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.

Clinical evaluation and mutation analysis was performed in 51 consecutive probands with severe eye malformations - anophthalmia and/or severe microphthalmia - seen in a single specialist ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions of SOX2, OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and genome-wide array-based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder. Two families with SOX2 mutations demonstrated a parent and child both with significant but highly variable eye malformations. Heterozygous loss-of-function mutations in SOX2 and OTX2 are the most common genetic pathology associated with severe eye malformations and bi-allelic loss-of-function in STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations.

Purpose: To describe the clinical findings of a patient with an early onset retinal dystrophy and a novel mutation in OTX2, and to compare these findings with previously reported cases., Methods: Using direct sequencing, we screened 142 patients, who had either Leber congenital amaurosis (LCA) or early onset retinal dystrophy (EORD), for mutations in OTX2. All patients received a detailed ophthalmic examination including electroretinography and retinal imaging., Results: Only one mutation in OTX2 was identified. A novel heterozygous p.S138X stop mutation was identified in a seven-year-old male who had an infantile onset retinal dystrophy. The mutation was not present in either parent or in 181 blood donor samples. There was a history of failure to thrive in infancy, poor feeding, and growth hormone deficiency. Poor vision and nyctalopia was present from the first year. Funduscopy revealed a hyperpigmented peripapillary ring with a fine granular pigmentation of the RPE throughout the fundus. The scotopic bright flash ERG a-wave was subnormal and the waveform electronegative, in keeping with dysfunction both at the level of the photoreceptor and post-phototransduction. Visual function has been stable to date., Conclusions: Mutations in OTX2 have been reported in association with major developmental malformations of the eye, with retinal dystrophies such as LCA, and with pituitary dysfunction and seizure activity in some cases. This case adds further support for a role of OTX2 both in retinal development and pituitary function, and highlights a novel retinal dystrophy phenotype seen in association with mutations in OTX2.

Purpose: Heterozygous mutations of the PAX6 gene cause a variety of ocular malformations, the best known being aniridia (absence of the iris). Mutation analyses and detailed clinical evaluations were performed in 43 individuals with aniridia or closely related ocular anomalies, to investigate whether phenotype correlates with mutation type., Methods: Case notes and medical records were reviewed and patients were reexamined when necessary. Denaturing high-performance liquid chromatography (DHPLC) analysis and sequencing of the PAX6 coding region was performed in individuals whose mutation was unknown., Results: The most common PAX6 mutations identified were premature termination mutations, amino acid substitutions, and C-terminal extensions. Six novel mutations are reported. Mutations that inactivate one copy of the gene typically caused a severe phenotype including foveal hypoplasia, marked iris anomalies, and severe visual impairment. Missense mutations, all affecting invariant amino acids in the paired domain, caused milder phenotypes in this cohort, with a lower incidence of foveal hypoplasia and less severe visual loss. C-terminal extension mutations caused relatively severe anomalies and marked reduction in vision. Two C-terminal extension cases had a unilateral exudative retinopathy, resembling Coats' disease, which has not previously been reported in association with PAX6 mutation., Conclusions: PAX6 mutations cause panocular malformations that vary considerably in pattern and severity. In our cohort, iris hypoplasia, nystagmus, and foveal hypoplasia were most common, with cataracts, corneal anomalies, and high refractive errors also frequently observed. In this cohort, loss-of-function and C-terminal extension mutations were found to cause more severe phenotypes than missense mutations.

Proteins of the bone morphogenetic protein (BMP) family are known to have a role in ocular and skeletal development; however, because of their widespread expression and functional redundancy, less progress has been made identifying the roles of individual BMPs in human disease. We identified seven heterozygous mutations in growth differentiation factor 6 (GDF6), a member of the BMP family, in patients with both ocular and vertebral anomalies, characterized their effects with a SOX9-reporter assay and western analysis, and demonstrated comparable phenotypes in model organisms with reduced Gdf6 function. We observed a spectrum of ocular and skeletal anomalies in morphant zebrafish, the latter encompassing defective tail formation and altered expression of somite markers noggin1 and noggin2. Gdf6(+/-) mice exhibited variable ocular phenotypes compatible with phenotypes observed in patients and zebrafish. Key differences evident between patients and animal models included pleiotropic effects, variable expressivity and incomplete penetrance. These data establish the important role of this determinant in ocular and vertebral development, demonstrate the complex genetic inheritance of these phenotypes, and further understanding of BMP function and its contributions to human disease.

PPAX6 is an important transcription factor which plays an essential role in brain morphogenesis and eye development. It is related to migration of neuroblasts to the cerebral cortex and deep telencephalic nuclei, and the specification of cellular and regional identity. Disturbances of brain development in two sib fetuses whose parents were aniridic (both sporadic cases) are reported. Molecular analysis in both parents has shown different mutations in PAX6 gene and a compound heterozygosity for two PAX6 mutations in both fetuses. Neuropathologically both cases showed severe brain malformations with increased germinal proliferation, gross disturbances of migration and organization of the CNS.

Cited2 is a transcriptional modulator with pivotal roles in different biological processes. Cited2-deficient mouse embryos manifested two major defects in the developing eye. An abnormal corneal-lenticular stalk was characteristic of Cited2(-/-) developing eyes, a feature reminiscent of Peters' anomaly, which can be rescued by increased Pax6 gene dosage in Cited2(-/-) embryonic eyes. In addition, the hyaloid vascular system showed hyaloid hypercellularity consisting of aberrant vasculature, which might be correlated with increased VEGF expression in the lens. Deletion of Hif1a (which encodes HIF-1alpha) in Cited2(-/-) lens specifically eliminated the excessive accumulation of cellular mass and aberrant vasculature in the developing vitreous without affecting the corneal-lenticular stalk phenotype. These in vivo data demonstrate for the first time dual functions for Cited2: one upstream of, or together with, Pax6 in lens morphogenesis; and another in the normal formation of the hyaloid vasculature through its negative modulation of HIF-1 signaling. Taken together, our study provides novel mechanistic revelation for lens morphogenesis and hyaloid vasculature formation and hence might offer new insights into the etiology of Peters' anomaly and ocular hypervascularity.

The nature-nurture argument surrounding the mechanisms of disease causation cannot be resolved, as the roles of genes and environment are inextricably entwined. Environmental fluctuation is clearly a major modifier of phenotype, as well as a promoter of evolutionary change. Both types of variability can be mediated by the stress response pathway, with the Hsp90 chaperone family as key components. Hsp90 has been hailed as a capacitor for evolutionary change, because partial inhibition of its functions can uncover cryptic mutations, leading to unexpected phenotypes that, although generally deleterious, will under rare new environmental conditions provide improved survival to the carrier of that variant. There is, therefore, a strong environmentally elicited link between the capacity to reveal hidden variation as human disease phenotype and as novel morphological forms for evolutionary selection.

A series of 125 patients referred primarily with aniridia classified as either sporadic (74), familial (24), or in association with WAGR syndrome (14) or other malformations (13) was analysed for mutations, initially by karyotyping and targeted FISH analysis of chromosome 11p13. These methods identified mutations in a significant proportion of patients, 34/125 (27%). Two cases had chromosome rearrangements involving 11p13, 16 cases had visible deletions, and 16 cases had cryptic deletions identified by 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., ((c) 2008 Wiley-Liss, Inc.)

Objective: To assess auditory processing, hearing difficulties, and brain magnetic resonance (MR) imaging abnormalities in children with panocular developmental aniridia due to PAX6 mutations., Design: Case-control study., Setting: Great Ormond Street Hospital and Institute of Child Health., Participants: Eleven case subjects with PAX6 mutations and 11 age-matched and sex-matched healthy control subjects., Interventions: All subjects completed a structured hearing questionnaire, baseline audiometry, and central auditory tests (dichotic speech tests, frequency and duration pattern tests, and gaps-in-noise test). Case subjects underwent brain MR imaging with volumetry, and the results were compared with those of age-matched and sex-matched healthy control subjects randomly selected from the Radiology and Physics Unit database., Main Outcome Measures: Brain MR imaging, central auditory test results, and questionnaire scores., Results: The corpus callosum area was significantly smaller on brain volumetry in the cases compared with the controls. The anterior commissure was small in 7 cases and was normal in 3 cases on visual inspection of brain MR images (conducted in 10 of 11 cases). 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 localizing sound and understanding speech in noise than the controls., Conclusions: Despite normal audiograms, children with PAX6 mutations may experience auditory interhemispheric transfer deficits and have difficulty localizing sound and understanding speech in noise. In view of their additional visual difficulties, thorough audiological evaluation of these children is indicated to initiate appropriate management.

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.

In developed countries, malformations of the eye are among the most common causes of serious visual impairment in newborns. The identification of pathogenic mutations in autosomal and X-linked transcription factors has advanced our understanding of the critical stages in human eye development and has begun to explain some unusual inheritance characteristics of these disorders. The functional characterisation of these genes in model organisms has prompted reinvestigation of affected individuals to identify previously unrecognized but consistent extra-ocular malformations. This dialogue between clinical genetics and basic developmental biology provides a paradigm to enhance our understanding of many critical developmental processes in human embryogenesis.

Transcriptional control is a major mechanism for regulating gene expression. The complex machinery required to effect this control is still emerging from functional and evolutionary analysis of genomic architecture. In addition to the promoter, many other regulatory elements are required for spatiotemporally and quantitatively correct gene expression. Enhancer and repressor elements may reside in introns or up- and downstream of the transcription unit. For some genes with highly complex expression patterns--often those that function as key developmental control genes--the cis-regulatory domain can extend long distances outside the transcription unit. Some of the earliest hints of this came from disease-associated chromosomal breaks positioned well outside the relevant gene. With the availability of wide-ranging genome sequence comparisons, strong conservation of many noncoding regions became obvious. Functional studies have shown many of these conserved sites to be transcriptional regulatory elements that sometimes reside inside unrelated neighboring genes. Such sequence-conserved elements generally harbor sites for tissue-specific DNA-binding proteins. Developmentally variable chromatin conformation can control protein access to these sites and can regulate transcription. Disruption of these finely tuned mechanisms can cause disease. Some regulatory element mutations will be associated with phenotypes distinct from any identified for coding-region mutations.

Purpose: To investigate whether 173 patients with microphthalmia, anophthalmia, and coloboma have mutations in the eye-development gene SIX6., Methods: The two exons of the SIX6 gene were amplified by PCR from patients' genomic DNA and directly sequenced to search for mutations. The PCR products of 75 patients were also analyzed by denaturing high-performance liquid chromatography (DHPLC)., Results: Six SIX6 polymorphisms were identified in the patient panel. Three of these polymorphisms change the encoded amino acid. However, all six polymorphisms were also identified in unaffected individuals. There was no statistically significant difference in genotypes between patients and control subjects., Conclusions: No evidence was found that SIX6 mutations underlie human congenital structural eye malformations.

Cerebral development is governed by genetic and environmental factors. Animal models provide much of our knowledge about genetic influences in the brain but it is not clear how similar or relevant these are to the human brain. The investigation of human subjects with mutations in genes known to be expressed in the developing brain is an alternative approach to improving our understanding of the role of genetic factors in brain development. We investigated 24 subjects with known mutations in the PAX6 gene (including representatives of all the known mutations of the gene) which are associated with characteristic ocular abnormalities in humans and animals. We have quantified MRI data using several techniques to assess qualities of cerebral structure which are difficult to interpret visually. Abnormalities were identified using voxel-based morphometry, statistical morphometrics, and measurement of corpus callosum cross-sectional area when comparing data from subjects with PAX6 abnormality and 72 age-and sex-matched control subjects. These abnormalities include grey matter changes in the cerebellum and occipital poles and white matter loss in the corpus callosum, alteration of sulcal orientation in the occipital lobe, and alteration to overall neuronal connectivity. These abnormalities complement and exceed the changes seen in the mouse models, and those seen on visual inspection alone of the human MRI data. Structural differences were also identified between the two largest genotype mutation subgroups.

Seventy-seven patients with aniridia, referred for cytogenetic analysis predominantly to assess Wilms tumor risk, were studied by fluorescence in situ hybridization (FISH), through use of a panel of cosmids encompassing the aniridia-associated PAX6 gene, the Wilms tumor predisposition gene WT1, and flanking markers, in distal chromosome 11p13. Thirty patients were found to be chromosomally abnormal. Cytogenetically visible interstitial deletions involving 11p13 were found in 13 patients, 11 of which included WT1. A further 13 patients had cryptic deletions detectable only by FISH, 3 of which included WT1. Six of these, with deletions <500 kb, share a similar proximal breakpoint within a cosmid containing the last 10 exons of PAX6 and part of the neighboring gene, ELP4. Two of these six patients were mosaic for the deletion. The remaining four had chromosomal rearrangements: an unbalanced translocation, t(11;13), with a deletion including the WAGR (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) region, and three balanced rearrangements with what appear to be position effect breakpoints 3' of PAX6: (a) a t(7;11) with the 11p13 breakpoint approximately 30 kb downstream of PAX6, (b) a dir ins(12;11) with a breakpoint >50 kb from PAX6, and (c) an inv(11)(p13q13) with a breakpoint >75 kb downstream of PAX6. The proportion and spectrum of chromosome anomalies in familial (4/14, or 28.5%) and sporadic (26/63, or 41%) cases are not significantly different. An unexpectedly high frequency of chromosomal rearrangements is associated with both sporadic and familial aniridia in this cohort.

PAX6 function was first identified through aniridia-associated null mutations. Since then, this transcription factor, with a paired domain and a homeodomain, has become a paradigm, illustrating functional conservation in developmental pathways. The Small eye mouse and Drosophila eyeless have served as major model systems in defining the multistage roles for Pax6 in eye and olfactory system development throughout evolution. The overt phenotypic consequences of heterozygous human and mouse Pax6 mutations were initially confined to the eye, with some interesting genotype-phenotype correlations being noted. Recently, structural and functional abnormalities in the olfactory system have been identified. Alterations in brain structure have also been documented, in line with the wider forebrain and cerebellar expression of Pax6. The broad PAX6 expression pattern is controlled by a number of long-range control elements, and is reflected in the severe homozygote phenotype. Upstream regulators and a multitude of downstream targets of PAX6 have been identified, and its varied tissue-specific functions are emerging.

The human eye anomaly aniridia is normally caused by intragenic mutations of PAX6. Several cases of aniridia are, however, associated with chromosomal rearrangements that leave the PAX6 gene intact. We have identified and characterized a novel gene, PAXNEB (C11orf19), downstream (telomeric) of PAX6. Sequence analysis, including interspecies comparisons, show this gene to consist of 10 exons, with an unusually large final intron spanning 134 kb in human and 18 kb in Fugu. This intron is disrupted by each chromosomal rearrangement. The 2-kb PAXNEB transcript, encoding a 424-amino acid protein, is expressed in all cell lines tested. The homologous mouse cDNA is broadly expressed in mouse embryos. PAXNEB is highly conserved from mammals to fish, with some regions of the protein showing conservation to invertebrates, yeast, and plants. The possible role of PAXNEB in aniridia was assessed. Using a transgenic mouse model, we show that the aniridia phenotype of the chromosomal rearrangement cases is not due to the heterozygous loss of PAXNEB function.

Human congenital cataract and ocular anterior segment dysgenesis both demonstrate extensive genetic and phenotypic heterogeneity. We identified a family where ocular developmental abnormalities (cataract, anterior segment dysgenesis and microphthalmia) co-segregated with a translocation, t(5;16)(p15.3;q23.2), in both balanced and unbalanced forms. We hypothesized that this altered the expression of a gene of developmental significance in the human lens and ocular anterior segment. Cloning the 16q23.2 breakpoint demonstrated that it transected the genomic-control domain of MAF, a basic region leucine zipper (bZIP) transcription factor, first identified as an oncogene, which is expressed in vertebrate lens development and regulates the expression of the eye lens crystallins. The homozygous null mutant Maf mouse embryo demonstrates defective lens formation and microphthalmia. Through mutation screening of a panel of patients with hereditary congenital cataract we identified a mutation in MAF in a three-generation family with cataract, microcornea and iris coloboma. The mutation results in the substitution of an evolutionarily highly conserved arginine with a proline at residue 288 (R288P) in the basic region of the DNA-binding domain of MAF. Our findings further implicate MAF/Maf in mammalian lens development and highlight the role of the lens in anterior segment development. The 16q23.2 breakpoint transects the common fragile site, FRA16D, providing a molecular demonstration of a germline break in a common fragile site.