A New Split Hand/Foot Malformation with Long Bone Deficiency Familial Case

Abstract

Split hand/foot malformation with long bone deficiency (SHFLD) is a congenital limb anomaly where hands and/or feet cleft and syndactyly are associated with long bone defects, usually involving the tibia. Previously published data reported that 17p13.3 chromosomal duplication, including the BHLHA9 gene, has been associated with the distinct entity, termed SHFLD3 (OMIM 612576), inherited as an autosomal dominant trait. Here, we present a family with three members affected by SHFLD harboring BHLHA9 duplication. We exploited in vitro differentiation system to promote proband's skin fibroblasts toward osteoblastic lineage, and we observed a slight but consistent delay in the mineralization pattern. This result possibly suggests an impairment of the osteogenic process in the affected members.

Keywords: 17p13.3 microduplication; SHFLD3; osteogenic differentiation; split-hand/foot malformation.

Fig. 1

Clinical features of SHFLD3 patients. (A) Two generations pedigree of the Italian family. Filled black symbols indicate members affected by SHFLD3. (B) Representative images of the affected mother on the newborn period (7 days of birth) (on the left) and pictures of her hands (top) and her radiographs(bottom) (on the right). (C, D) Representative photos of the upper and lower limb anomalies of the affected siblings.

Fig. 2

Molecular findings of SHFLD3 patients and in vitro osteogenic differentiation. (A) Relative DNA amount was measured by quantitative polymerase chain reaction in three controls and in all family members. Each assay was made in triplicates. (B) Osteogenic differentiation was revealed by the formation of extracellular mineralized matrix, as shown by von Kossa staining. Control (left), fibroblast cell line derived from a healthy subject; I-2 (right), fibroblast cell line derived from the proband (I-2); noninduced (top), fibroblast cell lines maintained with standard medium; induced (bottom), fibroblast cell lines cultured in osteogenic differentiation medium. For all acquisitions the ×10 magnification was used. (C) Quantification of possible differences in osteogenic potential measured in control-derived compared with I-2-derived osteoblast precursors (black and gray bars, respectively) using ImageJ software. Von Kossa stained areas (µm²) and number of counted spots, representing the calcium deposits, were used as parameters for the measurement (see text for details). The quantification was achieved from two independent experiments and the mean and standard deviation are shown.