Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects 1,2 . We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-Blike orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.The multisystem nature of the CdLS phenotype suggests that it is caused by a microdeletion or microduplication affecting several genes or by a single gene that regulates various target genes. A high-density BAC microarray comparative genome hybridization screen found no evidence for a consistent pattern of microdeletion or microduplication 3 . Because CdLS is rare and most cases are sporadic, genome-wide linkage screens are problematic. As an alternative, we analyzed chromosomal breakpoints associated with CdLS, focusing first on three classical cases with de novo balanced translocations, including the previously described translocations t(3;17)(q26.3;q23.1) 4 and t(14;21)(q32;q11) 5 . We first analyzed the 3q26.3 breakpoint because of NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome Arrows indicate the normal chromosome 5 and the der(5) t(5;13)(p13.1;q12.1) and der(13) t(5;13)(p13.1;q12.1) chromosomes. In occasional metaphases a weak G248P84262B4 signal can be detected on the der(5) chromosome as well as a strong signal on the der(13). The combined data suggest that the most likely location for the breakpoint is close to the proximal end of the region of overlap for inserts of G248P84262B4 and G248P8840C10 (Fig. 2a).

Cornelia de Lange syndrome (CdLS) is a rare developmental malformation syndrome characterised by mental handicap, growth retardation, distinctive facial features and limb reduction defects. The vast majority of CdLS cases are sporadic. We carried out a high density bacterial artificial chromosome (BAC) microarray comparative genome hybridisation screen but no evidence was found for a consistent pattern of microdeletion/micro-duplication. As an alternative, we focused on identifying chromosomal regions spanning associated translocation breakpoints. We prioritised the distal 3q region because of the occurrence, in a classical CdLS patient, of a de novo balanced translocation with a breakpoint at 3q26.3 and of reports of phenotypic overlap between cases of mild CdLS and individuals trisomic for the 3q26-q27 region. We show that the 3q26.3 breakpoint severs a previously uncharacterised giant gene, NAALADL2, containing at least 32 exons spanning 1.37 Mb. Northern blot analysis identified up to six different transcripts in the 1-10 kb range with strongest expression in kidney and placenta; embryonic expression was largely confined to duodenal and stomach endoderm, mesonephros, metanephros and pancreas. Transcript analysis identified extensive alternative splicing leading to multiple 5′ and 3′ untranslated regions and variable coding sequences. Multiple protein isoforms were defined by different N-terminal regions (with at least four alternative initiating methionine codons), and by differential protein truncation/use of alternative C-terminal sequences attributable to alternative splicing/polyadenylation. Outside the N-terminal regions, the predicted proteins showed significant homology to N-acetylated alpha-linked acidic dipeptidase and transferrin receptors. Mutation screening of NAALADL2 in a panel of CdLS patient DNA samples failed to identify patient-specific mutations. We discuss the possibility that the 3q26.3 translocation could nevertheless contribute to pathogenesis. Tonkin et al.