A giant novel gene undergoing extensive alternative splicing is severed by a Cornelia de Lange-associated translocation breakpoint at 3q26.3

Abstract

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/microduplication. 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.

Fig. 1

a, b FISH mapping of chromosome breakpoints in the t(3;17) cell line. a FISH identifies BAC RP11–827e14 (green) as spanning the 3q26.3 breakpoint. Red A chromosome 17 centromere-specific probe. Indicated chromosomes are as follows: A chromosomes 3 and der(3) t(3;17)(q26.3;q23.1), B chromosome 17, C der(17) t(3;17) (q26.3;q23.1). b FISH identifies cosmid LA173B3 (green) as spanning the 17q23.1 breakpoint. Red A chromosome 17 centro-mere-specific probe. Indicated chromosomes are as follows: D der (3) t(3;17)(q26.3;q23.1), E chromosome 17 and der(17) t(3;17) (q26.3;q23.1). the original colour FISH images can be viewed at http://www.ncl.ac.uk/ihg/cdls

Fig. 2

a–e Genomic structure, exon-intron organisation and transcript analysis of the NAALADL2 gene. a Genomic organisation of human NAALADL2 relative to the flanking genes neuroligin 1 (NLGN1) and the orthologue of mouse Ira1. These three genes and the position of additional loci predicted by computational analysis (see http://www.ncbi.nih.gov/mapview) are illustrated by filled boxes according to genomic size. Horizontal arrows Transcriptional direction, solid downward-pointing arrow position of the 3q26.3 breakpoint. b NAALADL2 exon organisation. Intron sizes are to scale. c Exon structure and alternative splicing events in NAALADL2. Boxes Exons, black fill-in coding sequence, absence of fill-in untranslated sequences. Exons are drawn to scale with the exception of the 3′UTR following exon 32 (drawn to 1/10th scale). The 2.4-kb 3′UTR (exon 32A) and a shorter 1-kb form (exon 32B) are shown with the alternative polyadenylation site producing the short form (found in AL832931) indicated by a filled triangle at the corresponding position in the exon 32 sequence of AL832144 in d. Asterisks Predicted alternative translational starts. The asterisk below exon 14 (see also d, f) denotes the predicted alternative start used when this exon is spliced directly to exon 11 (when exon 14 is spliced instead to either exon 12 or exon 13, the entire exon contributes to the open reading frame). Exon 18 has been observed in three forms: exon 18A (spliced form), 18B (extended form with a stop codon and 3′UTR) and exon 18C (abbreviated form, with a terminating polyA tail [(A)_n], but without a stop codon or 3′UTR). d cDNA clones and 5′RACE products used to determine the exon structure of NAALADL2. Accession numbers are given for cDNA sequences obtained by in silico screens. Clone names are given for cDNAs where the full-length sequence was established by in-house sequencing. The dagger above exon 20 indicates the presence of a stop codon, resulting from a change in reading frame when this exon is spliced directly downstream of exon 18. e cDNA clones from mouse, cow, pig and chick contain sequences aligning with human exons as indicated. The exon organisation is largely conserved at the genomic level in mouse but was unclear in cow, pig and chick at the time of writing

Fig. 3

a, b Northern blot hybridisation identifies multiple NAALADL2 transcripts in adult and fetal tissues. Human adult (a) and fetal (b) Northern blots containing poly (A)⁺ RNA and normalised to β-actin (Clon-tech) were hybridised with a 581-bp probe NAALADL2 cDNA probe. Right Size markers. Sources of tissue RNA are abbreviated as follows: L peripheral blood leucocytes, Lu lung, P placenta, SI small intestine, Li liver, K kidney, S spleen, T thymus, C colon, M skeletal muscle, H heart, and B brain

Fig. 4

a–c Embryonic expression of NAALADL2. Dark-field microscopy images following hybridisation of a ³⁵S-labelled antisense NAALADL2 probe to sagittal embryonic sections representing Carnegie stages 16 (a) and 21 (b, c). Msn mesonephros, Sto stomach, Mtn metanephros, Duo duodenum, Pan pancreas