Restriction analysis of otosclerosis-associated CD46 splicing variants.

ABSTRACT

Otosclerosis is a primary bone remodeling disorder of the human otic capsule and is associated with persistent measles virus infection. The human cellular receptor of measles virus is the membrane cofactor protein (MCP, CD46), which has 14 well-described splicing variants. Unique CD46 expression pattern of the otic capsule and the stapes footplate may determine the susceptibility for persistent measles virus infection. A total of 51 surgically removed ankylotic stapes footplates were analyzed by histopathological and molecular biological methods, respectively. Nucleic acids were extracted. Measles virus sequences were detected by nucleoprotein RNA-specific reverse transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNA of CD46 isoforms was amplified by RT-PCR; cDNA amplimers were separated by SDS poly-acrylamide gel electrophoresis and were purified from the gel. Complementary DNA of CD46 isoforms was restricted by endonuclease enzymes having CD46-specific recognition sites. The presence of viral RNA was associated exclusively with the histopathological diagnosis of otosclerosis; the stapes specimens with negative measles virus belonged to non-otosclerotic stapes fixations. All specimens (N = 51) were characterized by the consecutive expression of five CD46 variants (c, d, e, f and one shorter unidentified isoform). Histologically confirmed ostosclerotic specimens (N = 21) were characterized by increased expression levels of variant "f" and the unknown isoform. Increased expression levels of these isoforms and special CD46 expression pattern of the human otic capsule might produce modified or pathological intracellular signalization that could create the possibility of persistent measles virus infection.