Molecular genetics of Cornelia de Lange Syndrome

Abstract

Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by peculiar facial features, growth and mental retardation, and a variety of other abnormalities affecting multiple organ systems. The molecular pathology underlying the disease phenotypes is not known. In the past, 3q26.3 was considered as a candidate region. However, no patient-specific mutations in the giant gene, NAALADL2, truncated by the 3q26.3 breakpoint were found. Thus, in this study, we tried to find the disease gene in loci other than 3q26.3 mainly by FISH mapping in patients with t(5; 13), t(14; 21) de novo balanced translocation. Of all the three regions (5p13,13g12,14q32) we studied, the novel gene that crosses the 5p translocation breakpoint was found to be the most likely candidate gene for CdIS. Subsequent finer FISH mapping using fosmid clones as probes confirmed the novel gene was truncated by the translocation breakpoint in intron 1. Standard point mutational screening of the newly found gene detected patient-specific mutations. The gene was named, NIPBL, which is the major determinant for this rare syndrome (Tonkin et al., 2004). Analysis of the NIPBL gene in further patients with CdIS revealed a wide variety of pathogenic mutations. More than half of the mutations resulted in premature termination. Loss of function causing haploinsufficiency is supported by patients with heterozygous deletion involving the whole NIPBL gene. No genotype-phenotype correlation was observed. The exact functions of delangin are not known. Tissue in situ studies of NIPBL, using riboprobes in early human embryos, showed expression in developing brain, limbs, muscle, bone, renal tubules and lung bronchioles. In our preliminary data, the expression of NIPBL is mainly in the active proliferating tissue in early mammalian development. However, the tissue expression pattern in later mammalian development is not known. The predicted protein sequence of the NIPBL product, delangin, is 2804 amino acids for the long isoform and 2697 amino acids for the short isoform. In the carboxy-terminal region there is a clustering of HEAT repeats and these motifs (also called adherin) are highly conserved in the carboxy-terminal region of delangin homologues, Nipped-B of Drosophila, Scc2 of Saccharomyces cerevisiae and Mis4 of Schizosaccharomyces pombe, which are required for mitotic sister chromatid cohesion. Delangin is also related to Rad9 of Coprinus cinereus, which is required for DNA repair and meiotic chromosome paring. The Scc2 and Mis4 yeast, and Drosophila Nipped-B adherin homologues of human delangin are required for the Cohesin protein complex that mediates sister chromatid cohesin to associate with chromosomes. The major role in yeast and Xenopus Scc2 is to interact with Scc4 to help in loading the cohesin ring onto chromatin in the early S phase during cell replication. We also identified the sequence of the human Scc4 homologue, which we call human Mau-2. Tissue in situ hybridization data using a probe to dectect human Mau-2 showed a similar expression pattern to NIPBL in human embryos. However, no hMau-2 mutations were detected in CdLS patients in our mutation screen. In Drosophila, Nipped-B was also found to participate in remote activation of the cut and Ultrabithorax genes. Reducing the Nipped-B dosage reduces activation of the wild-type cut gene by the remote wing margin enhancer and causes the wing margin defect in Drosophila. Furthermore, Mau-2, the C. elegans homologue of yeast Scc4, which interacts with Scc2 to help in the establishment of the cohesion loading onto the chromatin, was found to guide axonal migrations in the CNS. However, the exact functions of both NIPBL and hMau-2 need to be further elucidated.