FISH mapping of de novo apparently balanced chromosome rearrangements identifies characteristics associated with phenotypic abnormality.

We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

Developmental malformations of the eye: the role of PAX6, SOX2 and OTX2.

Eye development initiates as an evagination of the early neural plate, before the closure of the neural tube. Structural malformations of the eye such as anophthalmia and microphthalmia arise very early in development. It is not surprising therefore that three of the genes currently identified to play a significant role in these developmental eye anomalies are also major players in brain development and regionalization. However, as has been emerging for a high proportion of transcriptional regulators studied, these genes have evolved to play multiple roles throughout development, and perhaps even in adult tissue maintenance. This complex spatiotemporal expression pattern requires elaborate regulatory systems which we are beginning to unravel. A major component of these complex regulatory networks is a series of cis-acting elements, highly conserved through evolution, which spread large distances from the coding region of each gene. We describe how cross regulation for PAX6, SOX2 and perhaps OTX2 has now been uncovered, pointing to the mechanisms that can fine-tune the expression of such essential developmental components. These interactions also help us understand why there is significant phenotypic overlap between mutations at these three loci.

Cognitive functioning in humans with mutations of the PAX6 gene.

Fourteen patients with PAX6 gene mutations and previously identified MRI abnormalities were administered tests of cognitive functioning. No deficits were found. A subgroup with agenesis of the anterior commissure performed significantly more poorly on measures of working memory than those without this abnormality, suggesting the anterior commissure may play a role in cognitive processing in addition to an earlier identified role in sensory development and processing.

National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology.

We report an epidemiological and genetic study attempting complete ascertainment of subjects with microphthalmia, anophthalmia, and coloboma (MAC) born in Scotland during a 16 year period beginning on 1 January 1981. A total of 198 cases were confirmed giving a minimum live birth prevalence of 19 per 100 000. One hundred and twenty-two MAC cases (61.6%) from 115 different families were clinically examined and detailed pregnancy, medical, and family histories obtained. A simple, rational, and apparently robust classification of the eye phenotype was developed based on the presence or absence of a defect in closure of the optic (choroidal) fissure. A total of 85/122 (69.7%) of cases had optic fissure closure defects (OFCD), 12/122 (9.8%) had non-OFCD, and 25/122 (20.5%) had defects that were unclassifiable owing to the severity of the corneal or anterior chamber abnormality. Segregation analysis assuming single and multiple incomplete ascertainment, respectively, returned a sib recurrence risk of 6% and 10% in the whole group and 8.1% and 13.3% in the OFCD subgroup. Significant recurrence risks were found in both unilateral and bilateral disease. In four families, one parent had an OFCD, two of which were new diagnoses in asymptomatic subjects. All recurrences in first degree relatives occurred in the OFCD group with a single first cousin recurrence seen in the non-OFCD group. A total of 84/122 of the MAC cases were screened for mutations in the coding regions of PAX6, CHX10, and SIX3. No pathogenic mutations were identified in the OFCD cases. A single PAX6 homeodomain missense mutation was identified in a subject with partial aniridia that had been initially misclassified as coloboma.

Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6.

The transcription factor PAX6 plays a critical, evolutionarily conserved role in eye, brain and olfactory development. Homozygous loss of PAX6 function affects all expressing tissues and is neonatally lethal; heterozygous null mutations cause aniridia in humans and the Small eye (Sey) phenotype in mice. Several upstream and intragenic PAX6 control elements have been defined, generally through transgenesis. However, aniridia cases with chromosomal rearrangements far downstream of an intact PAX6 gene suggested a requirement for additional cis-acting control for correct gene expression. The likely location of such elements is pinpointed through YAC transgenic studies. A 420 kb yeast artificial chromosome (YAC) clone, extending well beyond the most distant patient breakpoint, was previously shown to rescue homozygous Small eye lethality and correct the heterozygous eye phenotype. We now show that a 310 kb YAC clone, terminating just 5' of the breakpoint, fails to influence the Sey phenotypes. Using evolutionary sequence comparison, DNaseI hypersensitivity analysis and transgenic reporter studies, we have identified a region, >150 kb distal to the major PAX6 promoter P1, containing regulatory elements. Components of this downstream regulatory region drive reporter expression in distinct partial PAX6 patterns, indicating that the functional PAX6 gene domain extends far beyond the transcription unit.

PAX6 mutation in a family with aniridia, congenital ptosis, and mental retardation.

Congenital aniridia is due to deletions and point mutations in the PAX6 gene. We describe here a case of a mother and her two sons with a syndrome comprising congenital aniridia, ptosis, and slight mental retardation. The sons also show behavioral changes. The possibility of deletion around the PAX6 locus was excluded by polymorphism studies and fluorescence in situ hybridization analysis. Mutation screening of the PAX6 gene revealed the presence of a transversion C719A, resulting in the substitution of arginine for serine at residue 119. We suggest that this missense mutation is responsible both for aniridia and ptosis, and possibly also for the observed cognitive dysfunction in this family.

PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans.

PAX6 is widely expressed in the central nervous system. Heterozygous PAX6 mutations in human aniridia cause defects that would seem to be confined to the eye. Magnetic resonance imaging (MRI) and smell testing reveal the absence or hypoplasia of the anterior commissure and reduced olfaction in a large proportion of aniridia cases, which shows that PAX6 haploinsuffiency causes more widespread human neuro developmental anomalies.

Hemolytic uremic syndrome associated with Denys-Drash syndrome.

The Denys-Drash syndrome is defined by the occurrence of combinations of pseudohermaphroditism, nephrotic syndrome with diffuse mesangial sclerosis, Wilms' tumor, and constitutional mutations in the WT1 suppressor gene. Most patients develop end-stage renal failure. Atypical hemolytic uremic syndrome (HUS) is defined by onset of acute hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and renal failure in the absence of a gastrointestinal prodromal illness of bloody diarrhea. The purpose of this report is to describe the occurrence of features of atypical HUS and Denys-Drash syndrome in two African-American boys aged 13 and 16 months. Each had nephrotic syndrome, diffuse mesangial sclerosis, and WT1 point mutations. Both had grade III hypospadias and undescended testes. They had normal serum creatinine concentrations and hematology a month before presenting with HUS. Stool cultures for Escherichia coli O157:H7 were negative. Each patient has been transplanted with cadaver kidneys without recurrence of HUS.

Phenotypic variability and asymmetry of Rieger syndrome associated with PITX2 mutations.

PURPOSE: Rieger syndrome is an autosomal dominant condition characterized by a variable combination of anterior segment dysgenesis, dental anomalies, and umbilical hernia. To date, reports have shown mutations within the PITX2 gene associated with Rieger syndrome, iridogoniodysgenesis, and iris hypoplasia. The purposes of this study were to determine the range of expression and intrafamilial variability of PITX2 mutations in patients with anterior segment dysgenesis. METHODS: Seventy-six patients with different forms of anterior segment dysgenesis were classified clinically. DNA was obtained and screened by means of polymerase chain reaction (PCR)-single-stranded conformation polymorphism (SSCP) and heteroduplex analysis followed by direct sequencing. RESULTS: Eight of 76 patients had mutations within the PITX2 gene. Anterior segment phenotypes show wide variability and include a phenocopy of aniridia and Peters', Rieger, and Axenfeld anomalies. Mutations include premature terminations and splice-site and homeobox mutations, confirming that haploinsufficiency the likely pathogenic mechanism in the majority of cases. CONCLUSIONS: There is significant phenotypic variability in patients with PITX2 mutations, both within and between families. Developmental glaucoma is common. The umbilical and dental abnormalities are highly penetrant, define those at risk of carrying mutations in this gene, and guide mutation analysis. In addition, there is a range of other extraocular manifestations.

Psychiatric disorder and cognitive function in a family with an inherited novel mutation of the developmental control gene PAX6.

The PAX family of developmental control genes are known to play important roles in the early patterning of the central nervous system. One member of this family, PAX6, is involved in eye development in invertebrates as well as in mouse and man, but is also widely expressed in the developing forebrain. Humans with a mutation in this gene have abnormalities of eye development, and the results presented here suggest, for the first time, that this mutation may also be associated with subtle abnormalities of frontal lobe function in the family studied. We carried out genotyping of individuals within a single family, with and without the characteristic eye abnormalities of PAX6 mutation, and only those individuals with the mutation showed significant abnormalities on tests of frontal lobe function. These individuals also had higher rates of psychiatric disorder. PAX6 is highly conserved between mouse and man, and although the neuroanatomical phenotype associated with PAX6 heterozygosity has only been studied in mice, the resultant cellular disorganization seen in mice is likely to be present in the human forebrain. Although these mice have no obvious behavioural phenotype, the results presented here suggest that humans with the equivalent mutation display a neurobehavioural phenotype.

Role of Pax6 in development of the cerebellar system.

Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here we show that Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6(Sey)/Pax6(Sey)) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6(Sey)/Pax6(Sey) granule cells. Our results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3.

Multiple roles for the Wilms' tumor suppressor, WT1.

Wilms' tumor is a childhood kidney tumor that is a striking example of the way that cancer may arise through development gone awry. A proportion of these tumors develop as a result of the loss of function mutations in the Wilms' tumor suppressor gene, WT1. Inherited mutations in the WT1 gene can lead to childhood kidney cancer, severe gonadal dysplasia, and life-threatening hypertension. Knockouts show that the gene is essential for the early stages of kidney and gonad formation. These tissues are completely absent in null mice. The WT1 gene encodes numerous protein isoforms, all of which share four zinc fingers. There is a large body of evidence supporting the notion that WT1 is a transcription factor, particularly a transcriptional repressor. Recently, however, we obtained evidence that WT1 colocalizes and is physically associated with splice factors. What is more, one alternative splice isoform of WT1 containing three amino acids, Lys-Thr-Ser (KTS; inserted between zinc fingers 3 and 4) is preferentially associated with splice factors, whereas the other alternative splice version, lacking these three amino acids, preferentially associates with the transcriptional apparatus. Both genetic and evolutionary considerations suggest that these two different forms of the protein have different functions. We will discuss recent evidence to further implicate WT1 in splicing. Our results raise the possibility that regulation of splicing is a crucial factor in the development of the genitourinary system, and that tumors may arise through aberrant splicing. To pursue the regulation and function of WT1 in whole animals, we have been introducing the human gene and large flanking regions cloned in yeast artificial chromosomes directly into mice. These studies have allowed us to dissect the function of WT1 at late as well as at early stages in organogenesis and to identify new sites and surprising new potential functions for the gene.

Missense mutations in the most ancient residues of the PAX6 paired domain underlie a spectrum of human congenital eye malformations.

Mutations of the human PAX6 gene underlie aniridia (congenital absence of the iris), a rare dominant malformation of the eye. The spectrum of PAX6 mutations in aniridia patients is highly biased, with 92% of all reported mutations leading to premature truncation of the protein (nonsense, splicing, insertions and deletions) and just 2% leading to substitution of one amino acid by another (missense). The extraordinary conservation of the PAX6 protein at the amino acid level amongst vertebrates predicts that pathological missense mutations should in fact be common even though they are hardly ever seen in aniridia patients. This indicates that there is a heavy ascertainment bias in the selection of patients for PAX6 mutation analysis and that the 'missing' PAX6 missense mutations frequently may underlie phenotypes distinct from textbook aniridia. Here we present four novel PAX6 missense mutations, two in association with atypical phenotypes: ectopia pupillae (displaced pupils) and congenital nystagmus (searching gaze), and two in association with more recognizable aniridia phenotypes. Strikingly, all four mutations are located within the PAX6 paired domain and affect amino acids which are highly conserved in all known paired domain proteins. Our results support the hypothesis that the under-representation of missense mutations is caused by ascertainment bias and suggest that a substantial burden of PAX6 -related disease remains to be uncovered.

EYA4, a novel vertebrate gene related to Drosophila eyes absent.

We have isolated a family of four vertebrate genes homologous to eyes absent (eya), a key regulator of ocular development in Drosophila. Here we present the detailed characterization of the EYA4 gene in human and mouse. EYA4 encodes a 640 amino acid protein containing a highly conserved C-terminal domain of 271 amino acids which in Drosophila eya is known to mediate developmentally important protein-protein interactions. Human EYA4 maps to 6q23 and mouse Eya4 maps to the predicted homology region near the centromere of chromosome 10. In the developing mouse embryo, Eya4 is expressed primarily in the craniofacial mesenchyme, the dermamyotome and the limb. On the basis of map position and expression pattern, EYA4 is a candidate for oculo-dento-digital (ODD) syndrome, but no EYA4 mutations were found in a panel of ODD patients.

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: dramatic compaction and conservation of synteny with human chromosome 11p13.

The pufferfish Fugu rubripes has a genome approximately 7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305-306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

In utero nephropathy, Denys-Drash syndrome and Potter phenotype.

We report an unusual case of Denys-Drash syndrome presenting in a newborn infant with end-stage renal failure of antenatal origin and Potter phenotype. DNA analysis showed a novel missense change in arginine 394 of zinc finger 3 of the WT1 gene. This mutation may lead to an earlier and more severe presentation of Denys-Drash syndrome. It may be of interest to look for this mutation in other Potter phenotype cases.

Position effect in human genetic disease.

The spatially, temporally and quantitatively correct expression of a gene requires the presence not only of intact coding sequence, free of adverse nucleotide changes, but also correctly functioning regulatory control. With the identification of an increasing number of disease-related genes, the molecular defect in many cases has been defined. It is becoming clear that it is not always the transcription unit that bears the defect: there are a number of cases where the regulation of gene expression has been compromised. Cases associated with chromosomal rearrangement outside the transcription and promoter regions are categorized as position effects. A number of different mechanisms may explain their aetiology. Here, we examine the human disorders where such position effects are implicated. Further study of such cases may lead to important insights into mechanisms of gene regulation and transcriptional control.

A new set of primers for mutation analysis of the human PAX6 gene.

Mutations in the human PAX6 gene are an important cause of dominantly inherited congenital malformations of the eye, including aniridia, Peters' anomaly, keratitis, and isolated foveal hypoplasia. To satisfy the need for efficient detection of PAX6 mutations, we have developed a new set of oligonucleotides for genomic SSCP based on the recently completed genomic sequence of the entire human PAX6 gene. We also describe PAX6 mutations in eight aniridia patients, five of which are novel.