Molecular and mutation analysis of hereditary multiple exostoses
lVIOLECULAR AND lVIUTATION ANALYSIS OF HEREDITARY lVIUL TIPLE EXOSTOSES
HUI WING SUM
for the degree of Master of Philosophy at the University of Hong Kong
in September 2002
Hereditary multiple exostoses (HME/EXT; MIM133700) is an autosomal
dominant disorder characterized by the presence of multiple benign cartilage-capped
bone tumors. It has been ascribed to mutations in two "tumor suppressor-related"
genes, E)'71 and EXT2, encoding glycosyltransferases required for heparan sulphate proteogJycan (HSPG) synthesis. The molecular basis for tumor formation is not known but a number of hypotheses have been proposed that center on the alteration of HSPG and deregulated endochondral bone formation. These included altered Indian hedgehog (Ihh) signaling or fibroblast growth factor (FGF) function because diffusion of Ihh is thought to be regulated by HSPG, and FGF function requires binding to haparin. Ihh and FGF are important molecules that regulate chondrocyte
differentiation and proliferation in endochondral ossification.
To gain insight into the molecular basis for HME, we analyzed seven proband:; with exostosis. A novel frameshift mutation, a single nucleotide deletion (386-388
delA) in exon 1 of EXT] was identified in a proband with HME. This mutation altered the amino acid sequence from serine at position 130 with a predicted termination at position 134. In another proband with HME, a previously known amino acid substitution (D227N) was identified in EXT2. This mutation alters the charge of the amino acid in a small domain (DVSIP) that is conserved between the EXTl and EXT2, the function of which is not known.
A third mutation, 1173+ 1 g~t FS R360 mutation was identified and shown to be associated with all affected members in a large family with HME. This mutation represents a nucleotide substitution of guanine to thymine at position 1173 at the + 1 position of intron 7. This mutation alters the 5' splice site and the consequence is the splicing out of exon 7, resulting in a shift of the codon reading frame from arginine at position 360 and a predicted premature termination at position 434. Analysis of the molecular consequence of this mutation using chondrocytes isolated from the patient's tumor cartilage demonstrated for the first time that the result is complete degradation of the mutant mRNA, and the affected individuals in this family are haploinsufficient of EXT2. No mutation in either EXT] or EXT2 was detected for the four cases with solitary exostosis.
Interestingly, in vivo analysis of the tumor cartilage from five probands with or without mutations in EXT] or EXT2 showed an absence of detectable HSPG, suggesting that this a common feature. The impact is disorganized cartilage with many cells resembling hypertrophic chondroctytes, extending from the chondroosseous junction to the surfaces of the cartilage. The data is consistent with the hypothesis that altered chondrocyte differentiation and cellular organization lead to
the formation of exostosis, which is the result of abnormal synthesis of HSPG in the tumor cartilage. The mechanism is unclear, but is most likely due to changes in IHH or FGF signaling. This is an area that needs to be addressed directly, perhaps using chondrocytes isolated from the tumor cartilage.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:exostosis molecular aspects mutation biology cartilage cells genetics genetic disorders
Date of Publication:01/01/2003