Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels

Abstract

Brachydactyly type A1 (BDA1), the first recorded Mendelian autosomal dominant disorder in humans, is characterized by a shortening or absence of the middle phalanges. Heterozygous missense mutations in the Indian Hedgehog (IHH) gene have been identified as a cause of BDA1; however, the biochemical consequences of these mutations are unclear. In this paper, we analyzed three BDA1 mutations (E95K, D100E, and E131K) in the N-terminal fragment of Indian Hedgehog (IhhN). Structural analysis showed that the E95K mutation changes a negatively charged area to a positively charged area in a calcium-binding groove, and that the D100E mutation changes the local tertiary structure. Furthermore, we showed that the E95K and D100E mutations led to a temperature-sensitive and calcium-dependent instability of IhhN, which might contribute to an enhanced intracellular degradation of the mutant proteins via the lysosome. Notably, all three mutations affected Hh binding to the receptor Patched1 (PTC1), reducing its capacity to induce cellular differentiation. We propose that these are common features of the mutations that cause BDA1, affecting the Hh tertiary structure, intracellular fate, binding to the receptor/partners, and binding to extracellular components. The combination of these features alters signaling capacity and range, but the impact is likely to be variable and mutation-dependent. The potential variation in the signaling range is characterized by an enhanced interaction with heparan sulfate for IHH with the E95K mutation, but not the E131K mutation. Taken together, our results suggest that these IHH mutations affect Hh signaling at multiple levels, causing abnormal bone development and abnormal digit formation.

Endogenously Produced Indian Hedgehog Regulates TGFβ-Driven Chondrogenesis of Human Bone Marrow Stromal/Stem Cells

“…IHH is synthesized as a 45 kDa precursor but is subsequently cleaved to an active 19 kDa Nterminal fragment that undergoes palmitoylation, which is required for multimerization and leads to about a 30-fold increase in bioactivity [33]. This N-terminal fragment is also modified by attachment of cholesterol, and cholesterol interactions with heparan sulfate proteoglycans facilitate longrange diffusion [34]. Cholesterol addition is also required for targeting to membrane lipid rafts and multimerization.…”

Genetic advances in craniosynostosis

“…Indian HH is a pro-osteogenic factor that positively regulates intramembranous calvarial ossification by modulating BMP signaling [Lenton et al, 2011]. Interestingly, in a pattern analogous of BMP2 and BMP14 [Plöger et al, 2008], heterozygous missense mutations in Indian HH also result in brachydactyly [Ma et al, 2011]. …”

A novel pathogenic variant of IHH for Brachydactyly type A1

“…Indian hedgehog (IHH) was the first identified gene to be associated with BDA1 [7].The IHH gene, which encodes a member of the Hedgehog family of signaling proteins, is known for its role in endochondral ossification: regulate the balance between growth and ossification of the developing bones [5]. The IHH protein operates through a feedback control mechanism.…”

“…Based on the X-ray crystal structure of SHH, McLellan, et al showed that these residues are located within a calcium binding site, an important domain for mediating interactions with PTCH1, HIP1, CDO and GAS1 [17]. In particular, p.D100E had been shown to affect IHH interaction with PTCH1 and HIP1, reducing its capacity to induce cellular differentiation [16], whereas p.D100E was shown to change the Hh local tertiary structure and intracellular fate [5] [3,[20][21]. Not all DBA1 individuals with these pathogenic variants exhibited short stature, but they were shorter than non-carrier family members [3,[19][20][21], suggesting variants at this mutational hotspot can affect the growth to various degree but with reduced penetrance for short stature.…”