CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-phenotype correlation.

Meckel-Gruber syndrome (MKS) is a lethal fetal disorder characterized by diffuse renal cystic dysplasia, polydactyly, a brain malformation that is usually occipital encephalocele, and/or vermian agenesis, with intrahepatic biliary duct proliferation. Joubert syndrome (JBS) is a viable neurological disorder with a characteristic "molar tooth sign" (MTS) on axial images reflecting cerebellar vermian hypoplasia/dysplasia. Both conditions are classified as ciliopathies with an autosomal recessive mode of inheritance. Allelism of MKS and JBS has been reported for TMEM67/MKS3, CEP290/MKS4, and RPGRIP1L/MKS5. Recently, one homozygous splice mutation with a founder effect was reported in the CC2D2A gene in Finnish fetuses with MKS, defining the 6th locus for MKS. Shortly thereafter, CC2D2A mutations were also reported in JBS. The analysis of the CC2D2A gene in our series of MKS fetuses, identified 14 novel truncating mutations in 11 cases. These results confirm the involvement of CC2D2A in MKS and reveal a major contribution of CC2D2A to the disease. We also identified three missense CC2D2A mutations in two JBS cases. Therefore, and in accordance with the data reported regarding RPGRIP1L, our results indicate phenotype-genotype correlations, as missense and presumably hypomorphic mutations lead to JBS while all null alleles lead to MKS.

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.

Expression pattern of NOGO and NgR genes during human development.

Nogo protein has been identified as the component of central nervous system (CNS) myelin that limits axonal regeneration. We investigated the expression of the genes encoding Nogo and its receptor, NgR, between weeks eight and 23 of human embryonic development, by quantitative radioactive in situ hybridization. At 8 weeks, we detected NOGO and NgR transcripts in developing neuronal and non-neuronal structures. We focused on two different structures: the brain and the dental germs. During this period of development, NOGO and NgR transcripts colocalized in the cortical and ventricular zones of the brain, with expression strongest for these two genes in the postmitotic cells of the cortical plate. In developing dental germs, NgR was more strongly expressed than NOGO at 16 and 21 weeks. NOGO and NgR were expressed in zones of epithelium-mesenchyme interaction, which induce the differentiation of ameloblasts/odontoblasts. These genes were expressed most strongly in differentiated cells.

Expression of the RSK2 gene during early human development.

The 90 kDa ribosomal S6 serine/threonine kinase 2 gene (RSK2, U08316) has been recently identified as a disease-causing gene in an X-linked disorder, the Coffin-Lowry Syndrome (MIM 303600) characterized by severe mental retardation, facial dysmorphisms and progressive skeletal malformations. To investigate its possible role in cerebral cortex development, we performed RNA in situ hybridization at three stages of human development: day 32 (Carnegie 15), 9 weeks (Carnegie 23) and 13 weeks. RSK2 expression is detected in the embryonic anterior and posterior telencephalon (hippocampus anlagen), mesencephalon, rhombencephalon and cerebellum. RSK2 gene expression is also observed in dorsal root ganglia, cranial nerve ganglia, and sensory epithelium of the inner ear, liver, lung and jaw anlagen. This pattern of expression may be involved in cognitive impairment and facial dysmorphisms found in Coffin-Lowry Syndrome.

A new syndrome of congenital generalized osteosclerosis and bilateral polymicrogyria.

We report a 29-week male fetus with healthy consanguineous parents. He showed a severe sclerosing bone disorder affecting all skeletal elements, resulting in insufficient modeling, generalized densification, and fragility of the skeleton. This skeletal dysplasia was associated with an abnormal craniofacial development (hypertelorism, severe microretrognathia, cleft palate, absent epiglottis, reduced number, and mineralization of teeth buds) and abnormal terminal phalanges. Neuropathologic examination showed bilateral fronto-parietal cerebral polymicrogyria. This syndrome appears to represent a new variant of congenital sclerotic bone disorder of unknown origin. Autosomal recessive inheritance is possible.