The human PAX6 gene is mutated in two patients with aniridia.

Aniridia is an inherited ocular disorder of variable expressivity characterized by iris hypoplasia. A candidate aniridia gene, AN, which is the human homologue of the mouse Pax-6 gene, has recently been isolated by positional cloning from the WAGR region of 11p13. Here we describe mutations in this gene in two cases of sporadic aniridia, one detected at the DNA level and one at the RNA level, both of which are predicted to affect protein function. Mutations in Pax-6 have been described previously in Small eye, the proposed mouse model for aniridia. We present new phenotypic evidence for the validity of this mouse model.

Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.

Mutation or deletion of the PAX6 gene underlies many cases of aniridia. Three lines of evidence now converge to implicate PAX6 more widely in anterior segment malformations including Peters' anomaly. First, a child with Peters' anomaly is deleted for one copy of PAX6. Second, affected members of a family with dominantly inherited anterior segment malformations, including Peters' anomaly are heterozygous for an R26G mutation in the PAX6 paired box. Third, a proportion of Sey/+ Smalleye mice, heterozygous for a nonsense mutation in murine Pax-6, have an ocular phenotype resembling Peters' anomaly. We therefore propose that a variety of anterior segment anomalies may be associated with PAX6 mutations.

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.

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.

Modulation of DNA binding specificity by alternative splicing of the Wilms tumor wt1 gene transcript.

The technique of whole-genome polymerase chain reaction was used to study the DNA binding properties of the product of the wt1 gene. The zinc finger region of this gene is alternatively spliced such that the major transcript encodes a protein with three extra amino acids between the third and fourth fingers. The minor form of the protein binds specifically to DNA. It is now shown that the major form of wt1 messenger RNA encodes a protein that binds to DNA with a specificity that differs from that of the minor form. Therefore, alternative splicing within the DNA binding domain of a transcription factor can generate proteins with distinct DNA binding specificities and probably different physiological targets.

Transcription factors in disease.

Mutations affecting several predominantly tissue-specific transcriptional regulators have recently been associated with disease phenotypes. Although the mutational spectrum is variable, many of the reported cases involve clear loss-of-function mutations-such as Waardenburg syndrome type 1, aniridia and Rubinstein-Taybi syndrome-suggesting that the genetic mechanism involved in disease is haplo-insufficiency. The high degree of dosage sensitivity often appears to affect only a subset of the tissues that express the gene. Position effects with cytogenetic rearrangements well outside the coding region have been implicated for four of the genes discussed: POU3F4, SOX9, PAX6, and GL13.

Pax6: more than meets the eye.

The paired-box motif, originally defined in Drosophila segmentation genes is conserved in the Pax family of vertebrate developmental genes. Mutations that reduce Pax6 dosage cause dominantly inherited eye malformations in man and mouse. Remarkably, it has now been found that Drosophila has a homologue of Pax6, which also plays a key role in eye development.

Wilms' tumour: reconciling genetics and biology.

Wilms' tumour, a paediatric malignancy of the kidney, is a striking example of the relationship between aberrant development and cancer. Several different genetic loci have been implicated in the aetiology of the tumour; genomic imprinting also plays a role. One Wilms' tumour predisposition gene (WT1), encoding a zinc finger protein, is expressed in a limited set of tissues, including developing nephrons and gonads. The biology and genetics of Wilms' tumour underline the developmental relationship between kidneys and gonads.

Molecular and physical arrangements of human DNA in HRAS1-selected, chromosome-mediated transfectants.

We used mitotic chromosomes isolated from a human EJ bladder carcinoma cell line for morphological transformation of mouse C127 cells. These chromosome-mediated transformants were analyzed for cotransfer of markers syntenic with c-Ha-ras-1 on human chromosome 11. We also used cloned, dispersed human DNA repeats, in a general mapping strategy, to quantitate the amounts and molecular state of human DNA transferred along with the activated c-Ha-ras-1 gene. In situ hybridization was used to visualize the physical state of the transfected human chromatin. The combined use of these various techniques revealed the occurrence of both chromosomal and DNA rearrangements. However, our analysis also demonstrated that, in general, very substantial lengths of DNA are transferred intact. Closely linked markers are likely to cosegregate. Therefore, these transformants should be invaluable sources for the complete molecular cloning of isolated fragments of the short arm of human chromosome 11.

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.

The evolution of WT1 sequence and expression pattern in the vertebrates.

WT1 is a Wilms' tumour predisposition gene, encoding a protein with four C-terminal Kruppel-type zinc fingers, which is also a major regulator of kidney and gonadal development. To pinpoint key regulatory domains involved in development and evolution of the vertebrate genitourinary system, we have isolated WT1 orthologues from all gnathostome classes. Partial nucleotide sequence from chick, alligator, Xenopus laevis and zebrafish reveals extensive conservation. Both the zinc fingers and the transregulatory domain exhibit a high level of similarity in all the species examined. However, of the two alternatively spliced regions only one, the three amino acid KTS insertion between zinc fingers 3 and 4, is found in species other than mammals. The 17 amino acid insertion at the C-terminal end of the transregulatory domain is present only in mammals. Residues with reported human pathological mutations are also unaltered across species, underlining their structural significance. Studies in chick and alligator reveal that the mammalian intermediate mesoderm expression pattern is conserved in birds and reptiles. A wider role in mesodermal differentiation is suggested by expression in some paraxial and lateral mesoderm derivatives.

Equivalent expression of paternally and maternally inherited WT1 alleles in normal fetal tissue and Wilms' tumours.

Observations of non-random maternal 11p allele loss in Wilms' tumour (WT) have implied the possible involvement of an imprinted 11p locus in WT aetiology. A proposed 11p13 Wilms' tumour gene, WT1, has recently been isolated and encodes a zinc finger DNA-binding protein, the 3' untranslated region of which contains a polymorphic dinucleotide repeat (CA repeat) motif. We have exploited this transcribed CA repeat to examine the allelic expression pattern of WT1 and thereby determine whether transcriptional imprinting of this gene occurs. DNA and reverse-transcribed RNA from tumours and normal tissue were subjected to the polymerase chain reaction (PCR) using radiolabelled primers flanking the CA repeat. The gene was seen to be expressed from both of the constitutive alleles in 9-week human fetal kidney, all informative Wilm's tumours and neonatal kidney tissue adjacent to the tumours. In one tumour, known to be heterozygous for a point mutation in zinc finger 2, direct sequencing confirmed that both mutant and wild-type transcripts were being expressed. These results demonstrate that this gene is not subject to transcriptional imprinting in tumours or normal fetal kidney.

Subunit structure of calgranulins A and B obtained from sputum, plasma, granulocytes and cultured epithelial cells.

The calcium-binding proteins calgranulins A and B co-purified with an elastase-specific inhibitor after the affinity and cation-exchange chromatography of the perchloric acid-soluble fraction of pooled sputum collected from patients with chronic obstructive pulmonary disease (Sallenave, J.-M. and Ryle, A.P. (1991) Biol. Chem. Hoppe-Seyler 372, 13-21). The calgranulins were separated from the inhibitor by reverse-phase FPLC. Protein blot analysis of the calgranulin fraction in the absence of reducing agent revealed a band of 25 kDa corresponding to the disulphide-bonded heterodimerization of the two monomer components. Similar results were obtained from the immunoprecipitation and protein blot analysis of plasma, granulocytes and cultured epithelial cells. This implies that the calgranulins exist in the heterodimeric form in secretions in vivo. Their association with pancreatic elastase during the affinity chromatography stage of purification implicates them in the tissue destruction elicited by the inflammatory response in chronic obstructive pulmonary diseases.

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.

Placement and refined mapping of established and new markers on human chromosome 11q using a small panel of somatic cell hybrids.

Constructing detailed chromosome maps relies on the ability to place new markers rapidly and accurately. Here we map two new anonymous DNA markers precisely on chromosome 11q and refine the location of established markers using a panel of just six fragmentation hybrids, with reference to four translocation chromosomes. As several of the hybrids retain multiple segments of chromosome 11, it is possible to quickly assign new markers to subintervals using this modest set of hybrid DNAs.

A monoclonal antibody-based immunoassay for human lactoferrin.

Monoclonal antibodies against human lactoferrin define at least 3 and possibly as many as 6 different epitopes. A sandwich enzyme-linked immunoassay, using monoclonals against different epitopes, has been optimised for the measurement of serum lactoferrin. In 35 samples from healthy adults the mean lactoferrin content of serum from blood clotted overnight was 0.54 +/- 0.26 micrograms/ml.

A simple method for ranking the affinities of monoclonal antibodies.

Measurement of the binding of constant trace amounts of labelled antigen by increasing dilutions of culture supernatant allows the ranking of monoclonal antibodies in the order of their affinity for the antigen. The theoretical basis for this method is discussed and it is illustrated with data from a set of anti-alphafoetoprotein monoclonal antibodies. Hybridomas secreting antibodies of desired affinity for immunoassay, histochemistry or antigen purification can thus be selected at an early stage after fusion.