In Vitro Functional Study Of IHH Mutations Causing BDA1 & Association Studies With Schizophrenia

We firstly reported at 2001 that the heterozygous missense mutations in IHH result inBrachydactyly type A1 (BDA1), a condition characterized by shortening of digits dueto hypoplasia/aplasia of the middle phalanx and mild dwarfism. Indian hedgehogsignaling is critical for proliferation and differentiation of chondrocytes and essentialfor endochondral bone formation. So far, other or the same heterozygous mutations inIHH have been identified to be responsible for BDA1. But it was still unclear howthese mutations caused the phenotypes. Here we tested the signaling activities of thethree IHH mutations (p.E95K, p.D100E and p.E131K) by using both the full-lengthIHH cDNA and purified N-IHH proteins. Our in vitro analysis demonstrated for thefirst time that these mutations do not result in a complete loss of function. However,the ability of the mutant IHHs to "fully" induce a Hedgehog response is significantlyimpaired when tested in the C3H10T1/2 cells, suggesting these mutations may beaffecting interaction with the receptor PTC1 either directly or indirectly via partners.Our assay for Ptc1 and Gli1 expression following induction with Wt N-IHH isconsistent with the PTC/SMO/GLI pathway in vivo. Thus, the information gainedcould be related to similar outcomes in chondrocytes. We also performed comparativegene expression profile using a microarray approach. We confirmed three genes,Sostdc1, Penk1 and Igfbp5 are indeed targets of IHH signaling by quantitativeRT-PCR. By gel-shift assay, Penk1 and Igfbp5 were confirmed to be new targetsregulated by GLI1 transcription factor. SOSTDC1 is the antagonist of BMPs and anumber of research articles have implicated that both the BMP and IHH signalingpathways cooperated to regulate the skeletogenesis. The role of Penk1 and Igfbp5 inskeletogenesis is not known. The link that we have provided through expressionanalysis of Igfbp5 and Penk1 with a similar expression profile as Gdf5 in thedeveloping joints of mouse digits is perhaps the first indication for a role of thesegenes in digit joint formation, making another link between IHH signaling and jointformation.We also performed the case-control association studies in the Chinese Han populationin three schizophrenia candidate genes, GRIA4, RGS4 and GRID. No evidence wasfound for GRIA4 and RGS4 association with the disease. While in the associationstudy for GRID1, significant associations were revealed in both the single site analysisand haplotype analysis. In the Northern Han samples, all the three SNPs, rs1902666,rs1159140 and rs999383 showed significance to schizophrenia, the P values were0.0062, 0.000018 and 0.00045, respectively.