Systemic sclerosis (SSc) is a serious disease of unknown cause, characterized by excessive accumulation of collagen and other connective tissue components in the skin and internal organs. An animal model to study the molecular mechanisms of SSc would be extremely useful. We have recently initiated studies of such a model, the Tight Skin 2 or the Tsk2 mouse. During the previous period of funding we have conclusively demonstrated that Tsk2 is a different mutation than Tsk1; we have located the mutation on the proximal arm of chromosome l; and we have narrowed the interval in which the mutation lies from over 50 cM to 2.1 cM. We have shown that the Tsk2 mouse displays marked thickening of the dermis and excessive accumulation of dermal collagen. We found that collagen protein synthesis and type I, III, V and VI collagen mRNA levels were markedly elevated in either Tsk2 mouse skin or dermal fibroblasts. The elevated expression of type I and III collagen genes was due to increased transcription of the corresponding genes. These results demonstrated that the Tsk2 mutant mouse displays connective tissue abnormalities, which resemble those present in the skin of both SSc patients and Tsk1 mice. The discovery of an additional Tsk mutation will allow us to pursue a second avenue of approach to understanding the mechanisms controlling collagen gene expression at the molecular level and should greatly increase our chances of eventually finding an effective treatment for SSc. The overall goal of this renewal application is to identify the Tsk2 gene. To accomplish this goal we will pursue the following specific aims: (1) To further narrow the region on chromosome 1 on which Tsk2 is known to reside by ;continuing to type and map the N2 offspring from the intersubspecific backcross of [(Mus castaneus x C57BL/6-+/Tsk2)F1 x Mus castaneus] mice; (2) to continue to identify screen candidate genes which are known to reside in or near the region of interest; (3) to establish YAC and BAC contigs encompassing Tsk2 and establish refined proxinal and distal boundaries with recombinant markers; (4) to identify coding regions within the contigs by means of identifying CpG islands, exon trapping and cDNA selecton; and (5) to screen and identify the novel gene sequences. These studies will allow us to identify and clone Tsk2 and to characterize the mutation as a prelude to understanding its function. It is expected that the knowledge gained from these studies will be of direct relevance to the understanding of the pathogenesis of the excessive collagen deposition characteristic of Ssc and will provide a more rational approach to develop possible modes of therapy for this incurable and devastating disease.
