Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia

Abstract

Cleidocranial dysplasia (CCD) is a dominantly inherited disorder characterized by patent fontanelles, wide cranial sutures, hypoplasia of clavicles, short stature, supernumerary teeth, and other skeletal anomalies. We recently demonstrated that mutations in the transcription factor CBFA1, on chromosome 6p21, are associated with CCD. We have now analyzed the CBFA1 gene in 42 unrelated patients with CCD. In 18 patients, mutations were detected in the coding region of the CBFA1 gene, including 8 frameshift, 2 nonsense, and 9 missense mutations, as well as 2 novel polymorphisms. A cluster of missense mutations at arginine 225 (R225) identifies this residue as crucial for CBFA1 function. In vitro green fluorescent protein fusion studies show that R225 mutations interfere with nuclear accumulation of CBFA1 protein. There is no phenotypic difference between patients with deletions or frameshifts and those with other intragenic mutations, suggesting that CCD is generally caused by haploinsufficiency. However, we were able to extend the CCD phenotypic spectrum. A missense mutation identified in one family with supernumerary teeth and a radiologically normal skeleton indicates that mutations in CBFA1 can be associated exclusively with a dental phenotype. In addition, one patient with severe CCD and a frameshift mutation in codon 402 had osteoporosis leading to recurrent bone fractures and scoliosis, providing first evidence that CBFA1 may help maintain adult bone, in addition to its function in bone development.

Figure 1

Summary of mutations identified in CCD patients. The bar depicts the CBFA1 protein. Glutamine-alanine repeat (Q/A), DNA binding runt homology (RUNT), putative NLS, and PST domains are indicated. The TLE corepressor protein interaction motif VWRPY is shown at the carboxy terminus. AD1–3 and RD denote transcriptional activation and repression domains as described by Thirunavukkarasu et al. (1998). Mutations are indicated as follows: circle, missense; octogon, nonsense; triangle, insertion; inverted triangle, deletion; and square, polymorphism.

Figure 2

Radiological findings in patient F (887delC). A, Anteroposterior radiograph of the skull showing multiple Wormian bones in lambdoid and sagittal sutures. B, Chest radiograph demonstrating cone-shaped thorax, right clavicular hypoplasia, and abnormally shaped left clavicle with lateral gap. C, Abnormal pelvis with hypoplastic iliac wings, reduced pelvic diameter necessitating cesarian section, and coxa vara.

Figure 3

Radiological findings in patient C. A, Anteroposterior radiograph of patient C, carrying the mutation 1205insC. The chest is severely deformed because of compression fractures of the thoracic vertebrae and the subsequent development of a kyphoscoliosis. A Harrington rod was implanted for stabilization. Both clavicles are absent. The lumbar vertebrae have an irregular structure, are severely osteopenic, and are partially collapsed. The entire skeleton is extremely osteopenic, giving an almost radiotranslucent appearance especially of the ribs and the thoracic spine. The iliac wings are hypoplastic. B, Radiograph of the left hand and the distal part of the radius and the ulna of patient C. Note severe osteoporosis and greatly reduced trabecular and cortical bone. There is also hypoplasia of the terminal phalanges.

Figure 4

Subcellular localization of mutant CBFA1. Fluorescence (left), visible-light (right), and double-projection (middle) micrographs of CBFA1-GFP fusion proteins expressed in NIH 3T3 cells. Note the different subcellular localization of control GFP (A), wild-type CBFA1 (B), and mutant R225Q CBFA1 (C).

Figure 5

Subcellular localization of runt domain-GFP fusion protein in NIH 3T3 cells. Laser-scan micrographs show the intracellular distribution of control GFP (A), wild-type CBFA1-runt (B), R225Q (C), and R225W (D) mutant CBFA1-runt proteins.