Identification of four new PITX2 gene mutations in patients with Axenfeld-Rieger syndrome.

PURPOSE: Axenfeld Rieger syndrome (ARS) is an autosomal dominant inherited disorder affecting development of the ocular anterior chamber, abdomen, teeth and facial structures. The PITX2 gene is a major gene encoding a major transcription factor associated with ARS. METHODS: ARS patients were collected from six unrelated families. Patients and their families were ophthalmologically phenotyped and their blood was collected for DNA extraction. We screened the coding region of human PITX2 gene by direct sequencing. The consequences of the mutations described were investigated by generating crystallographic representations of the amino acid changes. In order to better understand the occurrence of glaucoma in ARS patients, we studied the PITX2 gene expression in human embryonic and fetal ocular tissue sections. RESULTS: We identified four novel PITX2 genetic alterations in four unrelated families with ARS. These mutations included two nonsense mutations (E55X and Y121X), an eight nucleotides insertion (1251 ins CGACTCCT) and a substitution (F58L), in familial and sporadic cases of ARS. We also showed for the first time that PITX2 is expressed at early stages of the human embryonic and fetal periocular mesenchyme, as well as at later stages of human development in the fetal ciliary body, ciliary processes, irido corneal angle and corneal endothelium. The human fetal eye PITX2 gene expression pattern reported here for the first time provides a strong basis for explaining the frequent occurrence of glaucoma in patients affected by PITX2 gene mutations. CONCLUSIONS: Two mutations identified affect the homeodomain (E55X and F58L). The E55X nonsense mutation is likely to alter dramatically the DNA-binding capabilities of the PITX2 homeodomain. Furthermore, there is a complete loss of the carboxy-terminal part of the PITX2 protein beyond the site of the mutation. The phenylalanine F58 is known to contribute to the hydrophobic network of the homeodomain. The crystallographic representations of the mutation F58L show that this mutation may change the conformation of the helical core. The F58L mutation is very likely to modify the homeodomain conformation and probably alters the DNA binding properties of PITX2. The other mutations (Y121X and the eight-nucleotide insertion (1251 ins CGA CTC CT) CGA CTC CT, at position 224 in PITX2A) result in partial loss of the C-terminal domain of PITX2. Pitx2 synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. Pitx2 activity is regulated by its own C-terminal tail. This region contains a highly conserved 14-amino-acid element involved in protein-protein interactions. The C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2 interactions with other transcription factors, for Pitx2-Pit-1 interaction and Pit-1synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Thus, the partial or complete loss of the C terminus tail can lead to decreased or absent DNA binding activity and trigger severe ARS phenotypes. Our in situ hybridization results obtained on human embryonic and fetal ocular tissue sections constitute the first molecular histological data providing an explanation for the occurrence of precocious glaucoma in human patients affected by ARS caused by PITX2 mutations. Further structural and biochemical studies are needed for understanding the wide spectrum of clinical phenotypes caused by the increasing number of new PITX2 mutations found in ARS affected patients.

VEGF and KDR gene expression during human embryonic and fetal eye development.

PURPOSE: It is important to understand the development of the normal retinal vascular system, because it may provide clues for understanding the mechanisms underlying the neovascularization associated with several retinopathies of infancy and adulthood. However, little is known about normal human ocular vascularization. VEGF is a key growth factor during vascular development and one of its receptors, KDR, plays a pivotal role in endothelial cell proliferation and differentiation. The purpose of this study was to analyze VEGF and KDR gene expression patterns during the development of the human eye during the embryonic and fetal stages. METHODS: The gene expression of VEGF and KDR was analyzed by in situ hybridization in 7-week-old embryos and in 10- and 18-week-old fetuses. In addition, we performed VEGF and KDR immunohistochemistry experiments on 18-week-old fetus tissue sections. RESULTS: These results clearly demonstrated that the levels of VEGF and KDR transcripts are correlated during the normal development of the ocular vasculature in humans. The complementarity between the patterns of VEGF and KDR during the early stages of development suggests that VEGF-KDR interactions play a major role in the formation and regression of the hyaloid vascular system (HVS) and in the development of the choriocapillaris. In later stages (i.e., 18-weeks-old fetuses), the expression of KDR seems to be linked to the development of the retinal vascular system. VEGF and KDR transcripts were unexpectedly detected in some nonvascular tissues-that is, in the cornea and in the retina before the development of the retinal vascular system. CONCLUSIONS: The expression of VEGF and KDR correlates highly with the normal ocular vascularization in humans, but VEGF may also be necessary for nonvascular retinal developmental functions, especially for the coordination of neural retinal development and the preliminary steps of the establishment of the definitive stable retinal vasculature.

The beta3 integrin gene is expressed at high levels in the major haematopoietic and lymphoid organs, vascular system, and skeleton during mouse embryo development.

Integrins are a family of cell surface molecules that mediate the attachment of cells to the extracellular matrix (ECM). These alphabeta heterodimers are involved in many biological processes. We used northern blotting and in situ hybridization to study the pattern of beta3 integrin gene expression during mouse embryogenesis. Northern blotting detected two species of beta3 mRNA from 7 to 17 days post coitum (dpc). These transcripts were abundant in the adult testis, kidney, liver, spleen, and heart. In situ hybridization experiments detected high levels of beta3 in the major haematopoietic and lymphoid organs: yolk sac, liver, and thymus. Moreover, beta3 transcripts were also detected in the vascular system, where beta3 integrin probably plays a key role in angiogenesis and vasculogenesis. We also detected a hybridization signal in the gut, the bronchioles of the lungs, and the bladder wall. beta3 transcripts were also present in the medullary regions of the adrenal glands and in the developing skeleton. Our study shows that beta3 gene expression is not restricted to the liver and gut during mouse development. We also detected beta3 integrin mRNA in the yolk sac, vessels, lung, bladder, and developing bones. Our data suggest that beta3 integrin plays a key role in many important physiological processes like haematopoiesis, angiogenesis, phagocytosis, and bone resorption.

Pleiotropic and diverse expression of ZFHX1B gene transcripts during mouse and human development supports the various clinical manifestations of the "Mowat-Wilson" syndrome.

ZFHX1B encodes Smad-interacting protein 1, a transcriptional corepressor involved in the transforming growth factors beta (TGFbeta) signaling pathway. ZFHX1B mutations cause a complex developmental phenotype characterized by severe mental retardation (MR) and multiple congenital defects. We compared the distribution of ZFHX1B transcripts during mouse and human embryogenesis as well as in adult mice and humans. This showed that this gene is strongly transcribed at an early stage in the developing peripheral and central nervous systems of both mice and humans, in all neuronal regions of the brains of 25-week human fetuses and adult mice, and at varying levels in numerous nonneural tissues. Northern blot analysis suggested that ZFHX1B undergoes tissue-specific alternative splicing in both species. These results strongly suggest that ZFHX1B determines the transcriptional levels of target genes in various tissues through the combinatorial interactions of its isoforms with different Smad proteins. Thus, as well as causing neural defects, ZFHX1B mutations may also cause other malformations.