ESHG Posters 16

Expression pattern of the RSK2- or Coffin-Lowry syndrome gene during murine development

M. Vogel, B. Fischer, H. Hameister, H. Kehrer-Sawatzki;
Department of Human Genetics, University of Ulm, GERMANY.

Mutations in the RSK2 gene on Xp22.2 are causing Coffin-Lowry syndrome, which is characterized by severe psychomotor retardation, facial and digital dysmorphism, and progressive skeletal deformations affecting mostly the vertebral column. About 50-60% of the mutations identified so far lead to premature translation termination and are predicted to cause loss of function alleles. Some missense mutations are associated with milder phenotypes. In one family, the R383W mutation is causing non-syndromic X-linked mental retardation without other symptoms. RSK2 is a member of the 90kDa ribosomal-S6-serine/threonine kinase family acting at the distal end of the Ras signalling cascade. After activation by MAPKs, RSK2 translocates to the nucleus and regulates gene expression by phosphorylation of transcription factors such as CREB. RSK2 knock-out mice are 10% smaller compared to wildtype littermates. Additionally, RSK2 KO mice have impaired learning and poor coordination, suggesting that RSK2 seems to have similar roles in mental functioning both in mice and humans. To investigate the spatio-temporal expression spectrum of RSK2 during mouse development, we performed RNA in situ hybridization. In early embryonic development (ED 9.5-10.5) high RSK2 expression was observed exclusively in somites and lateral plate mesoderm from which among other tissues the vertebral column develops. At later embryonic stages (ED 12.5-14.5) enhanced RSK2 mRNA levels are detected in the peripheral nervous system (dorsal root ganglia) and in sensory ganglia of the cranial nerves. In contrast to the more widespread expression in multiple tissues of adult mice, RSK2 shows a highly specific expression spectrum during embryonic development.

A. Moroni¹, P. De Marco¹, E. Merello¹, A. Raso¹, M. Crippa², F. Blasi², A. Cama¹, V. Capra¹;
¹G.Gaslini, Genova, ITALY, ²DIBIT-HSR, Milano, ITALY.

Chiari complex is the most frequent pathology among hindbrain malformations, characterized by caudal cerebellar herniation, sometimes associated with lower brain stem dysmorphism, skull bases and vertebral anomalies. In vertebrates hindbrain, generation of regional diversity is achieved through a segmentation process that, during primary neurulation, leads to the formation of 7 metameric units, called rhombomeres. In this process, Hox genes display a key role in controlling and regulating neuronal migration and in mainteining cellular segmental identity. Among labial hortologous, Hoxb-1 gene is the first one to be activated in CNS and the only one to show an expression domain restricted to rhombomere 4 and in the neural crest cells that from r4 migrate in the second branchial arch. We performed the mutational screening of the homologous HOX-B1 gene in 49 patients and 103 control individuals. Sequencing of abnormal SSCP conformers revealed the existence of three allelic variants characterized by the presence of several in cis associated mutations, all affecting the NH2 terminal region of the gene. a1 haplotype is characterized by the presence of two synonimous transitions (C237T and G450A) and one missence mutation (A309T); a2 variant shows, in addition, a 9-bp tandem duplication (CCCACAGCG) at position +80, while a3 presents three silent substitutions (G114A, C213T and G246A) and one missense mutation (C167T). Since the different distribution of the a1 and a2 haplotypes in controls and patients and the absence of a3 variant in the first population, we hypotize these mutations as predisposing genetic factors for the insorgence of pathology.

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibers and eye coloboma

A. Barbieri¹, G. Alfano¹, V. Broccoli¹, A. Bulfone¹, V. Marigo¹, P. Bovolenta², A. Ballabio¹, S. Banfi¹;
¹TIGEM, Naples, ITALY, ²Instituto Cajal, CSIC, Madrid, SPAIN.

Vax2 is a homeobox gene whose expression is confined to the ventral portion of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralization of the retina, thus suggesting its role in the molecular pathway underlying eye development. We have generated and characterized a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalization of the expression of mid-late (EphB2 and ephrin-B2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.

A. Rauch¹, M. Hofbeck², R. Cesnjevar³, G. Buheitel⁴, B. Schenker¹, R. Rauch², H. Singer⁴, M. Weyand³;
¹Institute of Human Genetics of the Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, GERMANY, ²Department of Pediatric Cardiology of the University of Tuebingen, Tuebingen, GERMANY, ³Heart Surgery of the Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, GERMANY, ⁴Department of Pediatric Cardiology of the Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, GERMANY.

The wide range of clinical variability in patients with 22q11.2 deletions has been demonstrated in numerous studies. Nevertheless, it is still an open question if major genetic factors contribute to clinical expression. Therefore one aim of this study was to investigate, if patients with 22q11.2 deletion and conotruncal heart defects show a “second hit” somatic 22q11.2 deletion in tissue from the conotruncus, heart vessels or thymus. The second aim was to analyse patients with conotruncal heart defects without 22q11.2 deletion in blood cells for somatic deletion mosaicism. Parents of 19 patients with conotruncal heart defects (IAA, TAC, pulmonary atresia with VSD) agreed to collect and study somatic tissue from heart surgery in their children. 5 of these 19 patients had 22q11 deletions shown by FISH analysis on metaphase spreads from peripheral lymphocytes with 10 DNA probes from the DGS1 region. DNA was prepared from thymus and/or heart vessels and/or conotruncus tissue and peripheral lymphocytes in each patient and analysed with 18 microsatellite markers from the DGS1 region for allelic loss. Results did not show any allelic loss, thus there was no evidence for a somatic 22q11.2 deletion. Therefore somatic 22q11.2 deletions apparently do not play a major role in conotruncal heart defects in patients with or without germ line 22q11.2 deletion.

M. Zollo¹, V. Aglio¹, P. Carotenuto¹, M. Cocchia¹, V. Avantaggiato¹, A. Andre'¹, A. Faedo², A. Ballabio¹, A. Bulfone²;
¹Telethon Institute of Genetics and Medicine, Napoli, ITALY, ²Stem Cells Research Institute (SCRI), Dibit-HSR, Milano, ITALY.

Our approach combines gene array expression technology and murine subtractive cDNA library to isolate unique and specific genes preferentially expressed in the embryonic telencephalon. We have randomly sequenced 3600 cDNA clones (ESTs) from a cDNA subtractive library. A set of unique transcripts (1026) have been identified, selected and arrayed on glass coated slides. A series of experiments based on the potential of cell lines (P19, neuro2A, PC12) to be induced to differentiation into specific neuronal cell subtypes by Retinoic Acid (RA) or Neural Growth factor (NGF), are undergoing. This will permit the isolation of genes that are differentially expressed before and after neuronal "in vitro"differentiation. The results will be confirmed by Real time PCR assays. A detailed sequence analysis of the 372 identified cDNA clones was performed using public domain DataBases (such as dbEST, Unigene, Homologene, Locus Link and OMIM), to verify the quality of the library, to identify the human homologs, and to map and correlate them to neurological disorders. In particular 20% of the selected clones have no public database match to date, and 23% correspond to genes with unknown function. To determine the spatio-temporal expression profile of 110 cDNAs, we have performed in-situ mRNA hybridization on mouse embryos (sagittal and coronal sections of E14.5 embryos and whole-mount E10.5 embryos) and adult brains. Moreover, experiments are undergoing for testing the value of this cDNA array in order to unravel the molecular defects of the developing brain of mice models mutated in the Tbr1 and Lis1 genes.

Expression of SMADIP1 during early human development correlates with the phenotype of a syndromic form of Hirschsprung disease

Y. Espinosa-Parrilla, T. Attié-Bitach, J. Augé, A. Munnich, S. Lyonnet, M. Vekemans, J. Amiel;
Département de Génétique et INSERM U-393, Hôpital Necker-Enfants Malades, Paris, FRANCE.

The smad binding protein 1 gene (SMADIP1, MIM 605802) has been identified as causing a polytopic embryonic defect (MIM 235730) including midline anomalies (agenesis of the corpus callosum, congenital cardiac defect, hypospadias), facial dysmorphism, mental retardation and enteric nervous system malformation (Hirschsprung disease, HSCR). We recently screened the SMADIP1 locus in a series of 19 unrelated patients with this phenotype and identified de novo large-scale SMADIP1 deletions or truncating mutations in 8 cases. To further investigate the role of SMADIP1 during embryogenesis, we performed RNA in situ hybridization at early stages of human development. According with HSCR and facial dysmorphism in patients, SMADIP1 is expressed in the enteric nervous system and other neural crest derived cells (peripheric nervous system, facial neurectoderm and cranial nerve ganglia). SMADIP1 mRNAs are also detected in the central nervous system as soon as day 33 (carnegie 15). In agreement with other clinical features (hypospadias, strabismus, limbs and kidney anomalies, and hypotonia) SMADIP1 is further expressed in genital tubercle, developing eye, limbs, kidney and muscles. Although congenital cardiac defects are frequently observed, no SMADIP1 expression is detected in the developing heart. However, this expression pattern correlates with the spectrum of malformations observed in patients and confirms the pleiotropic role of SMADIP1 during human development.