Furthermore, in SHFM families 7, 12, and 18, sequencing of a 7,406 bp region encompassing BHLHA9 and Southern blot analysis using five probes and MfeI-, SspI-, and SacI-digested genomic DNA revealed no variation specific to the patients, and methylation analysis for a CpG rich region at the upstream of BHLHA9 delineated massive hypomethylation in all the patients/carriers examined. This evidence concerns the gene BHLHA9 and Split hand-split foot malformation.