Genetic heterogeneity in Rubinstein-Taybi syndrome: mutations in both the CBP and EP300 genes cause disease

Abstract

CREB-binding protein and p300 function as transcriptional coactivators in the regulation of gene expression through various signal-transduction pathways. Both are potent histone acetyl transferases. A certain level of CREB-binding protein is essential for normal development, since inactivation of one allele causes Rubinstein-Taybi syndrome (RSTS). There is a direct link between loss of acetyl transferase activity and RSTS, which indicates that the disorder is caused by aberrant chromatin regulation. We screened the entire CREB-binding protein gene (CBP) for mutations in patients with RSTS by using methods that find point mutations and larger rearrangements. In 92 patients, we were able to identify a total of 36 mutations in CBP. By using multiple ligation-dependent probe amplification, we found not only several deletions but also the first reported intragenic duplication in a patient with RSTS. We extended the search for mutations to the EP300 gene and showed that mutations in EP300 also cause this disorder. These are the first mutations identified in EP300 for a congenital disorder.

Figure 1

Conservation of amino acids that are predicted to change by missense mutations. All five mutations we found that are predicted to change the amino acid residues are situated in the highly conserved HAT domain. The changed residues are conserved in man (Homo sapiens [Hs]), mouse (Mus musculus [Mm]), and the fruit fly (Drosophila melanogaster [Dm]). Numbers indicate individual patients.

Figure 2

Mutations in EP300 in patients with RSTS. A, Results for patient 254-1. DGGE analysis of patient 254-1 and the healthy parents shows that only the affected child has the mutation. Subsequent sequence analysis revealed a transition, c.1942 C→T, that predicts the protein change p.Arg648X in this patient. B, Results for patient 256-1. DGGE analysis of family 256 shows a de novo mutation. The allele-specific PCR confirms the exact location of the deletion seen by sequence analysis. The patient has an 8-bp deletion (c.2877_2884 del) with the following sequence (deleted region is in capital letters; nucleotides of allele-specific forward primer are underlined): gcctcctccatctactagtagCACAGAAGtgaat. The forward primer skips the deleted part and anneals with two nucleotides after the deletion. Only DNA from the patient shows a band of 168 bp, whereas, in the lanes for DNA from the healthy parents, only the prominently visible primer dimers can be seen. C, Bar diagram of MLPA results for patient 149-1. MLPA reveals a deletion at the first exon of EP300. The X-axis shows the DNA probes. The probes upstream of the first exon are at positions 787–716 and 5–54 bp before the transcription start site (indicated by −787 and −54, respectively). Ex = exon; Int = intron. The Y-axis represents the dosage of DNA: a dosage of 1 indicates the presence of the normal amount of DNA—that is, both alleles are present—whereas a dosage of ∼0.5 typically indicates a deletion of one allele. The diagram clearly shows that the deletion runs from the upstream region of exon 1 into intron 1 and that exons 2 and 3 are present for both alleles. The exact size of the deletion is unknown.

Figure 3

Photographs of the three patients with EP300 mutations. A, The face, a hand, and a foot of patient 149-1. B, A hand, a close-up of the fingers with fetal pads, and a foot of patient 254-1. C, A hand, a foot, and an X-ray of the foot of patient 256-1. Patient 256-1 has the overall typical appearance of a patient with RSTS, with the exception of the feet. The feet have abnormally short first metatarsal bones, as can be clearly seen in the X-ray photograph. Although this is not a typical feature, it does appear in some other patients with RSTS, as well as in individuals with mutations in CBP.