Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS

Abstract

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.

Figure 1

Sequences of double-stranded oligonucleotide probes used for EMSA.

Figure 2

(A) Pedigree of family PD1 and haplotypes at 7q36 using microsatellite markers. The region in affected subjects segregating with the phenotype is boxed. Clinically examined individuals are shown by a horizontal line over the symbol. (B) Phenotypic appearance of affected subjects in the family PD1 with PPD triphalangial thumb. (a and b) The hands and feet of III-12, (c) clinical appearance of hands of male subject III-6, (d) radiograph of III-6 demonstrating duplicated thumbs with three phalanges.

Figure 3

(a) Representative chromatogram of the heterozygous mutation (463 T>G) in ZRS. (b) Wild type. (c) Restriction analysis using MseI restriction enzyme in all the sampled family members (normal and affected); the 268-bp mutant allele is indicated by an arrow. (d) EMSA of the conserved element demonstrating binding of Caco-2 cell line nuclear extract to both wild-type (T-allele) and mutant allele (G-allele). Unlabeled probes were added to demonstrate specific binding to both alleles. Specific binding is indicated by arrow.