Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B

Abstract

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

Figure 1

Characteristic features of CdLS. (a) Full face view of a child with CdLS showing characteristic facial features, including arched eyebrows with synophrys, long eyelashes, ptosis, depressed nasal bridge with anteverted nares, long philtrum with thin upper lip and micrognathia. (b,c) Variability of upper limb abnormalities in CdLS. (b) Oligodactyly defect with absence of ulna and most digital structures (note also hirsutism of back). (c) Distal reduction defect with missing and fused digits.

Figure 2

dentification of NIPBL as the gene underlying CdLS. (a) Critical region on chromosome 5p13, with microsatellite markers and their distances (in Mb) from the p-terminal arm of chromosome 5 indicated above the diagram. Arrows mark the refined critical region after high-resolution analysis and identification of obligate recombination events. BAC clones used for FISH analysis are indicated by their RP11 addresses. FISH analysis further narrowed the critical region, as shown by arrows below the diagram. The expanded view identifies genes in the defined critical region (from the July 2003 build of the University of California Santa Cruz genome browser). (b) FISH analysis using BACs from the linkage-defined chromosome 5p13 critical region. The chromosome 5p telomeric control probe is labeled in green and the 5q telomeric control probe is labeled in red. In the left panel, BAC RP11-252F20 is labeled in green. Both signals from BAC RP11-252F20 are on chromosome 5 p (arrows), indicating that it is proximal to the translocation breakpoint. In the middle panel, BAC RP11-14I21, which contains NIPBL, is labeled in green. There is signal on both of the chromosome 5p arms as well as on chromosome 13q (arrows), indicating that the probe was split on the translocated chromosome. In the right panel, BAC RP11-317I23 is labeled in red. One signal is present on the normal chromosome 5p arm and the other is present on 13q, indicating that this probe is distal to the translocation breakpoint.

Figure 3

Expression of NIPBL in the developing mouse. (a–c) Embryonic day (E) 9.5 embryos, whole-mount in situ hybridization. (d–i) E10.5 embryos, vibratome sections (200 µm) of embryos processed for whole-mount in situ hybridization. (a,d,g) Sense control. (b,e,h) Mouse homolog of NIPBL. (c,f,i) Fgf8 (positive control). (b) The mouse homolog of NIPBL is expressed throughout the embryo, especially in the limb buds and branchial arches (arrow, fore limb bud; arrowhead, first branchial arch). (c) Fgf8 expression marks a portion of the surface ectoderm of the same structures (arrow and arrowhead as in b). (e,f) At E10.5, sections through the forelimb bud show that expression of the mouse homolog of NIPBL is concentrated in the mesenchyme (asterisk in e marks ventral limb bud mesenchyme; dorsal mesenchyme is also stained), whereas Fgf8 expression marks the apical ectodermal ridge (arrow in f). No significant differences in intensity between fore- and hindlimb buds were observed (data not shown). Coronal sections at the level of the head show expression of the mouse homolog of NIPBL in the mesenchyme of both the lateral (L) and medial (M) nasal processes (h). In contrast, Fgf8 hybridization marks the ectoderm surrounding the developing nasal pit. Scale bars: a, 0.5 mm (for a–c); g, 0.5 mm (for d–i).