Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations

A HOXD13 p.Q317K mutation causes a severe limb phenotype and alters the DNA binding specificity. (A) Photographs and radiographs of patient with HOXD13^Q317K mutation. Missing digits (oligodactyly) and severe shortening of the digits (brachydactyly), as well as absent distal phalanges and nails are apparent. Radiographs show missing and deformed metacarpals, metatarsals, and rudimentary phalangeal bones. (B) The HOXD13^Q317 mutations alter the binding motif at the 5′ end. The primary motifs found by de novo motif analysis for HOXD13^wt (top), HOXD3^Q317R (middle), and HOXD13^Q317K (bottom). Both Q317 mutant proteins show an altered 5′ end of the sequence as compared with HOXD13^wt. (C) In vitro binding of wild-type and mutant HOXD13 homeodomains to the HOXD13 motif. EMSA of purified wild-type and mutant HOXD13 homeodomains was performed using fluorescent dye-labeled double-stranded oligonucleotides containing a binding sequence for HOXD13 (CCAATAAAA). Oligonucleotides were incubated with purified wild-type and mutant HOXD13 homeodomains. Unlabeled competitor oligonucleotides reduced binding to the labeled oligonucleotide, whereas a mutant competitor did not, indicating sequence-specific binding. The HOXD13^wt motif is bound by HOXD13^wt and (weaker) HOXD13^Q317R but not by HOXD13^Q317K.