U ' d !,~ L, ' ½ U _~. Expression of mRNA for Type IV CoLLagen czl, (z5 and Chains by CuLtured Dermal Fibrobtasts from Patients with X-Linked ALport Syndrome SATOSHI SASAKI*, BING ZHOU*, WEI WEI FAN*, YOUNGKI KIM*, DAVID F. BARKER t, 30YCE C. DENISON t, CURTIS L. ATKINt, MARTIN C. GREGORY t, 31NG ZHOU*, YOAV SEGAL*, YOSHIKAZU SADO ~, YOSHIFUMI NINOMIYA~, ALFRED F. MICHAEL* and CLIFFORD E. KASHTAN* *Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota, t Departments of Physiology, Medicine and Biochemistry, University of Utah Health Sciences Center, Salt Lake City, Utah, * Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA Division of Immunology, Shigei Medical Research Institute and II Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, Japan Abstract COL4A5 mutations causing X-linked Alport syndrome (XLAS) are frequently associated with absence of the c~3, 0t4, c(5 and ct6 chains of type IV collagen from basement membranes and increased amounts of the 0d(IV) and 0~2(IV) chains in glomerular basement membrane. Although many COL4A5 mutations have been described in XLAS, the mechanisms by which these mutations influence the basement membrane appearance of chains other than o~5(IV) remain poorly understood. In this study, we used dermal fibroblasts from eight normal indi- viduals and nine males with XLAS to test the hypotheses that COL4A5 mutations increase transcription of COL4A1 and suppress transcription of COL4A6. Ribonuclease protection assays revealed that cd(IV), 0~5(IV) and 0~6(IV) transcripts were expressed in cultures of der- mal fibroblasts. The mRNA levels for c~l(IV) in eight of nine patients with XLAS were not increased compared to controls; one patient with a large COL4A5 deletion showed signifi- cant elevation of cd(IV) mRNA levels. No differences in steady-state mRNA levels for Ix6(IV) were found when XLAS fibroblasts were compared with controls, even though little or no c~6(IV) protein was detectable at the dermal-epidermal junction by immunofluores- cence study. This finding suggests that post-transcriptional events account for the absence of c~6(IV) in the Alport dermal-epidermal junction. Key words: Alport syndrome, cultured fibroblasts, mutation, type IV collagen mRNA. Abbreviations used: ARAS, autosomal recessive form of AS; AS, Alport syndrome; DEJ, dermal-epidermal junction; GBM, glomerular basement membrane; RPA, RNase protection Matrix Biology Vol. 17/1998, pp. 279-291 © 1998 by Gustav Fischer Verlag assay; TBM, tubular basement membrane; XLAS, X-linked form of AS