Detection of Otosclerosis-Specific Measles Virus Receptor (Cd46) Protein Isoforms

Genetic predisposition of otosclerosis has long been suspected, but unclarified. Unique coexpression pattern of measles virus receptor (CD46) splicing isoforms in the human otic capsule is assumed, since otosclerosis is a measles virus-associated organ-specific disease. In order to identify CD46 involved in the pathogenesis of otosclerosis, we used representative groups of histologically diagnosed otosclerotic, nonotosclerotic, and normal stapes footplates (n = 109). Consecutive histopathological examinations and CD46-specific Western blot analysis were performed. Normal and nonotosclerotic stapes footplates showed consistent expression of the conventional c, d, e, f, and l CD46 isoforms. In contrast, four novel isoforms (os1–4) translated as intact proteins were additionally detected in each otosclerotic specimen. The study herein presented provides evidence for the otosclerosis-associated expression pattern of CD46. This finding might explain the organ-specific, virus-associated and autoimmune-inflammatory pathogenesis of otosclerosis. Regarding our current knowledge, this is the first report that confirms the presence of four new disease-specific protein variants of CD46.


Introduction
Otosclerosis is a complex inflammatory bone remodeling disorder of the human otic capsule that leads to progressive conductive and/or sensorineural hearing loss as a consequence of stapes footplate fixation and cochlear bone resorption with endosteal involvement [1]. In the Caucasian population, the prevalence of clinical otosclerosis is 0.3-0.4% of the general population, 5-9% of those with hearing loss, and 18-22% of those with conductive hearing loss [1,2]. Silent otosclerotic foci are more common: histological otosclerosis without clinical symptoms has been reported as 8-11% in large unselected autopsy series [2]. Otosclerotic foci are limited to the temporal bone, and no lesions have been found outside of the ear [2][3][4][5]. Otosclerosis takes approximately two thirdS of stapes ankylosis cases leading to consecutive conductive hearing loss [6]. Differential diagnosis is still based on postoperative histological analysis of the removed stapes footplates [3,4,7].
However, several hypotheses suggest viral, autoimmune, and endocrine factors in the genesis of disease, etiopathogenesis of otosclerosis remained unexplained [3,[8][9][10]. Genetic factors must play a significant role in the etiopathogenesis of otosclerosis, although the precise mode of inheritance is still uncertain [3,5,10]. The potential etiologic role of measles virus in the pathogenesis of otosclerosis was suggested in the past twenty-five years [8,9]. The otosclerotic otic capsule is assumed to exhibit a unique paramyxovirus favoring receptor expression pattern, which could be the basis of the pathogenesis of otosclerosis and also the reduced humoral immune response against measles virus-derived antigens [3,6,7].
One of the human cellular receptors for measles virus is the CD46 molecule, also known as membrane cofactor protein (MCP) [11]. CD46 has a cofactor activity for inactivation of complement components C3b and C4b by serum factor I, which protects the host cell from damage by the complement system [11]. Signals mediated by CD46 have a great influence on T-cell activation [11,12]. Beyond these functions, CD46 plays a role in the pathogenesis of various inflammatory disorders. Its therapeutic potential in inflammatory diseases has also been suggested. Elevated serum levels of CD46 have been reported in systemic lupus erythematosus (SLE) [13]. Recombinant soluble CD46 has been introduced to animal models of various inflammatory diseases [14]. For example, CD46 treatment inhibited acute cardiac transplant rejection [14]. Accordingly, targeted therapies using recombinant CD46 may be useful in autoimmune-inflammatory conditions [3,14]. The mRNA of CD46 is translated from a single gene linked to chromosome 1q32; however, it is posttranslationally modified by alternative splicing resulting in 14 known splicing variants and corresponding protein isoforms [6,11,12]. Different numbers of CD46 isoforms are coexpressed by all nucleated human cells in various patterns [11]. However, specific functions have not been associated to isoform coexpression yet [6,11]. In 2008, Karosi et al. described four novel otosclerosis-associated splicing variants of CD46 mRNA; however, no additional reports have arisen about the translated and corresponding protein isoforms associated to the etiopathogenesis of otosclerosis [6].
The present study investigates the coexpression pattern of CD46 protein isoforms in histologically proven otosclerotic, nonotosclerotic, and normal stapes footplates in order to establish organ-specific, otosclerosis-associated alternative splicing of the measles virus receptor CD46.

Patients and Controls.
Altogether 109 stapes footplates (male = 39, female = 70) were analyzed. Out of these samples, 92 were ankylotic and removed by stapedectomy and were immediately fixed in 10% (w/v) formaldehyde. The mean age of patients was 41.7 years (range: 21-72 years). Stapes footplate specimens were selected histologically from a larger pool ( = 419) of ankylotic stapes samples with the aim of obtaining representative groups of otosclerotic and nonotosclerotic stapes footplates. Partially removed stapes footplates were not included in the study because the anterior or posterior poles containing the bone lesions fixing the stapes were retained in the oval window niche. However, fragmented and reconstructed footplates were not excluded. The diagnosis of ossicular chain fixation was based on clinical, audiometric, and tympanometric findings. Air-bone gap at 1000 Hz was at least 30 dB. Preoperative tympanometry revealed type-As tympanograms in 67.4% and type-A tympanograms in 32.6% of stapes fixation cases. High resolution CT scan was performed in 18 cases that revealed thickening of the stapes footplate in 11 cases with no apparent signs of hypodensity due to otosclerotic foci in the otic capsule or in the stapes footplate. Seventeen cadaver stapes specimens with negative otopathological history were removed by dissection of temporal bones and were employed as negative controls ( = 17, male = 8, female = 9). These were removed within 20 hours after death. The mean age of negative controls was 53.5 years (range: 49-69 years). Stapes footplates were collected between January 2008 and March 2009 at the Universities of Debrecen, Pécs and Szeged, and at the Department of Otolaryngology, Bajcsy-Zsilinszky Hospital, Budapest, Hungary. We obtained Hungarian Scientific Research Ethical Committee (ETT-TUKEB/2008) and Institutional Ethical Committee (DE OEC-EB/2008/12) approvals. The study was carried out according to the Declaration of Helsinki.

Discussion
Genetic predisposition for otosclerosis has long been disputed over the last decades, without obvious target genes or mutations to show up [3,10,15]. Several studies have reported genetic associations in populations with clinical otosclerosis without histopathological confirmation and have extrapolated the observations to otosclerosis irrespective nonotosclerotic fixations [10,15]. The majority of genetic studies on families with stapes fixation and on large unselected populations has suggested an autosomal dominant mode of inheritance with incomplete penetrance of approximately 40-45% [10,15]. Genetic linkage studies have demonstrated the presence of eight loci (OTSC1-8) located on chromosomes  15q, 7q, 6p, 16q, 3q, 6q, and 9p, respectively [3, 10, 15].  Although these loci have been mapped, no causative genes and proteins have been identified and we have a little idea of the molecular process involved in this disease [3,10,15]. It has been reported that COL1A1, BMP2, BMP4, TGFB1, and RELN genes may also contribute to the development of otosclerosis [16][17][18][19]. Furthermore, prior associations with these genes account for only a small fraction of the relative risk for otosclerosis or other types of stapes fixation [16][17][18][19]. These associations reported earlier cannot explain female dominancy, adult onset, organ specificity, and the inflammatory bone remodeling disorder, all characteristic features of otosclerosis [3,4]. As to previous results, average life span of an active otosclerotic focus is about 5 to 7 years until inactivation; hence a dynamic genetic hypothesis is necessary to explain this "healing" process [20]. The involvement of T-cells, inflammatory cytokines, and other mediators in otosclerosis suggest an autoimmune-inflammatory nature of the disease [3,20].

ISRN Otolaryngology
No animal model exists for the otosclerotic bone remodeling disorder, which is associated to the presence of measles virus in the foci [3,4]. Measles virus is found exclusively in otosclerotic bone in human [8,9]. This called the attention to the CD46 molecule, which is not expressed as virus receptor in other mammalian species, except humans [6,11,12]. Specific diseases have not yet been attributed to CD46 isoform coexpression; however, present study supplies essential information about novel translated isoforms of CD46 emphasizing a potential association between isoform coexpression and otosclerosis.
Various expressions of different splicing variants of CD46 seem to be one of the acceptable explanations for the genetic background of otosclerosis [6]. Newly described CD46 protein isoforms have a shorter or missing transmembrane and uncommon cytoplasmic domains; however, virus-binding domain remains conservative (Figure 3). Transmembrane and soluble isoforms of CD46 protein have been identified in humans and transgenic mice [6,11,12]. In mice, there is an exon spliced alternatively, which allows the encoding of a soluble, cytoplasmic isoform [6,11]. Although soluble isoforms of human CD46 have been found in different body fluids, mRNA encoding soluble forms remain unidentified until now [11]. The present study supplies direct evidence of a human soluble CD46 isoform (os4) produced by alternative splicing (Figures 2-3). These changes should result in functional consequences of signaling that may be responsible for the persisting replication of measles virus. Decreased serum level of anti-measles IgG is characteristic for otosclerosis, which is independent from vaccination or measles virus infection and can be determined by expression of otosclerosis-associated CD46 isoforms [6,7]. Newly described isoforms are supposed to allow virus internalization without TCR-dependent activation of primary CD4 positive T-helper cells and consecutive induction of B-celldependent immunoglobulin production [6,7,11,12]. It is supposed that the most important factor in the pathogenesis of otosclerosis is a consecutive autoimmune reaction due to continuous CD46-presented viral antigen stimulation of natural killer cells and CD8 positive T-lymphocytes [6,11,12]. Several inflammatory cytokines (TNF-alpha, IL-1, and TGFbeta) and bone-specific proteins (osteoprotegerin, BMP) also may play a secondary promoting role in this process [4,17,18,20].
Proteins expressed as different splicing isoforms are able to imitate regular Mendelian inheritance [3,6,10,11]. Familial cases of otosclerosis showing obscure autosomal dominant inheritance with incomplete penetrance might be considered as a common disease caused by unique alternative splicing of measles virus receptors in the otic capsule [10,15]. An unresolved question is whether measles virus can induce the expression of new CD46 splicing variants, or existing novel isoforms lead to the increased virus affinity and smooth virus replication [6,11,12]? The answer may hide in the expression of different regulatory proteins of alternative splicing leading to a special expression pattern and altered functions of CD46 that could explain the organ-specific and virus-associated pathogenesis of otosclerosis [4,6,11,12].
In conclusion, we found a strong association between the expression of novel CD46 protein isoforms and histologically confirmed otosclerosis. Our results are consistent with four new splicing variants of CD46 mRNA being causally related to the viral pathogenesis of otosclerosis [6]. Special expression pattern of CD46 isoforms due to organ-specific alternative splicing may explain genetically determined susceptibility for persisting measles virus infection observed