The combination of genetic linkage, mutation localization and DNA sequencing have greatly increased our understanding of the molecular basis of heritable connective tissue disorders affecting type I collagen. The molecular defects range from a single base changes and multi-exon deletions to abnormalities of RNA splicing. The type II collagen gene has many similarities to type I. Certain heritable chondrodystrophies have biochemical or genetic evidence implicating a mutation of the type II collagen gene. However, the methods of mutation identification that have worked for type I collagen will not be readily applicable for type II due to the limited amount of tissue readily available that expresses this gene. The purpose of this gant is to develop a strategy to identify the mutation in a family with a linked type II collagen chondrodystrophy . Before carrying out this process on humans, the methods will be developed on type II collagen mRNA from embryonic chick sternal cartilage. cDNAs to the type II collagen mRNA will be made using a primer just 3' to the helical domain for reverse transcription. The polymerase chain reaction (PCR) will be used to amplify overlapping fragments of approximately 500 base pairs that encompass the helical domain of the mRNA. Each fragment will be applied to acrylamide and denaturing gradient gel electrophoresis to identify the one containing the mutation. The former will detect segments with an insertion or deletion while the later can resolve fragments that differ by one or more base pairs. The fragments containing the potential mutation will be cloned and the DNA from each allelic segment sequenced. Once successful results are obtained from the chick tissue, the strategy will be modified to normal human cartilage tissue. cDNA/PCR techniques will be performed on RNA obtained directly from the cartilage tissue or from cultured chondrocytes. After demonstrating that our methods of mutation identification is feasible on human biopsy tissue, we will apply it to an individual whose chondrodystrophy has been genetically linked to the type II collagen gene. After informed human consent is obtained band the cartilage biopsy is carried out, we will apply the cDNA/PCR method to the RNA obtained from the biopsy sample. We will confirm that the base change found by this method truly underlies the condition by utilizing the PCR to genomic DNA of other family members to determine if the base change is only present in affected individuals. The knowledge gained from this study will have general application to the mutation of the type II collagen genes that affect craniofacial structures by defective cartilage development. The strategy should be applicable to defining mutations of any gene that is expressed in cartilage and provide an avenue for understanding the molecular basis of this extremely heterogeneous group of heritable disorders.
