Analysis of two translocation breakpoints and identification of a negative regulatory element in patients with Rieger's syndrome.

BACKGROUND

Rieger's syndrome is an autosomal dominant disorder characterized by eye, tooth, and umbilical anomalies. A gene responsible for Rieger's syndrome, PITX2, has previously been cloned using two patients with balanced translocations, t(4;16) and t(4;11), with breakpoints that lie near the gene, but which do not interrupt it.

METHODS

We sequenced both breakpoint regions on chromosome 4 and screened this area for novel genes. Fluorescence in situ hybridization (FISH) was used to determine if PITX2 was still present on the 4:16 chromosome. Both the chromosome 16 and chromosome 11 breakpoints were cloned and sequenced using panhandle polymerase chain reaction (PHPCR). Transient transfection studies were performed to compare effects on a reporter gene between native chromosome 4 sequence and chromosome 11 sequence.

RESULTS

The region surrounding PITX2 on chromosome 4 is rich in repetitive elements, but no novel genes were identified. FISH demonstrated that PITX2 was intact on the 4:16 translocation chromosome. The PHPCR experiments demonstrated that the translocated regions of chromosomes 16 and 11 were repeat-rich, and transfection studies revealed a slight enhancer effect with the chromosome 4 sequence, and a strong silencer effect when the chromosome 11 sequence was present.

CONCLUSIONS

Given the lack of any novel genes near either breakpoint, changes in potential regulatory elements may be the best model to explain the loss of PITX2 expression in these patients and hence the Rieger's syndrome phenotype.