Screening for Mutations in Cartilage ECM Genes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 126

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Screening for Mutations in Cartilage ECM Genes
Michael D. Briggs
1. Introduction
1.1. Genetic Disorders of Cartilage
Genetic disorders of cartilage (chondrodysplasias) are a clinically and
genetically heterogeneous group of diseases ranging in severity from relatively
mild to severe and lethal forms (1-2) . There are over 100 unique well-charac-
terized chondrodysplasia phenotypes and remarkable progress has been made
in the last few years identifying the underlying genetic basis of many of these
disorders (3) . In most cases, a molecular genetics approach was employed
involving a combination of genetic linkage mapping, positional (candidate)
cloning and DNA sequence analysis (4-9) . By its nature this approach requires
extensive mutation screening in any potential candidate gene, first to deter-
mine if it is the disease gene and then subsequently to identify a range of dis-
ease causing mutations. In chondrodysplasia phenotypes this approach has been
hampered by a difficulty in obtaining appropriate pathological tissue, such as
cartilage, for the isolation of mRNA. This problem is compounded by the com-
plex genomic structure of many genes that encode cartilage structural ECM
molecules. For most cartilage diseases, a combination of these difficulties has
necessitated screening for mutations in a large numbers of exons using a vari-
ety of techniques such as single-stranded conformational polymorphism
(SSCP) (10) , conformational sensitive gel electrophoresis (CSGE) (11) , het-
eroduplex (12) and chemical cleavage mismatch (CCM) analysis (13) .
1.2. Multiple Epiphyseal Dysplasias and Pseudoachondroplasia
Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED)
are clinically similar phenotypes manifesting in varying degrees of dispropor-
tionate short stature and early onset osteoarthritis (2) . Mutations in the gene-