Arthur G. Kristiansen

Osteoprotegrin Knockout Mice Demonstrate Abnormal Remodeling of the Otic Capsule and Progressive Hearing Loss

Objectives: The otic capsule, when compared with other bones in the body, is unique in that it undergoes no significant remodeling of bone after development. We previously demonstrated that osteoprotegerin (OPG), which inhibits formation and function of osteoclasts, is produced at high levels in the inner ear of normal mice and secreted into the perilymph from where it diffuses into the surrounding otic capsule bone through a lacunocanalicular system. To test our hypothesis that the high level of OPG may be important in the inhibition of otic capsule remodeling, we studied the light microscopic histology of the otic capsule in OPG knockout mice for evidence of abnormal remodeling of bone. We also tested the hearing in OPG knockout mice to determine whether OPG and its influence on surrounding bone is important for auditory function.

Osteoprotegerin in the Inner Ear May Inhibit Bone Remodeling in the Otic Capsule

Objectives: To elucidate factors that may be responsible for the inhibition of remodeling of bone within the otic capsule.

Temporal bone histopathologic and genetic studies in Mohr-Tranebjaerg syndrome (DFN-1).

Objective To describe the temporal bone histopathologic and genetic abnormalities in a case of Mohr-Tranebjærg syndrome. Background Mohr-Tranebjæzrg syndrome (DFN-1) is an X-linked, recessive, syndromic hearing loss, characterized by postlingual sensorineural hearing loss with onset in childhood, followed in adult life by progressive dystonia, spasticity, dysphagia, and optic atrophy. The syndrome is caused by mutations in the DDP (deafness/dystonia peptide) gene, which are thought to result in mitochondrial dysfunction with subsequent neurodegeneration. The temporal bone pathologic changes in this syndrome have not been reported. Methods Hearing loss developed in the patient at age 4, blindness at age 48, and dystonia at age 57. Genetic studies on peripheral blood showed a l51delT mutation in his DDP gene. He died at age 66. The right temporal bone was subjected to light microscopy and polymerase chain reaction–based analysis of the DDP gene sequence. Results There was near complete loss of spiral ganglion cells with loss of nearly all peripheral and central processes. Only 1,765 spiral ganglion cells remained (8.5% of mean normal for age). The organ of Corti (including hair cells), stria vascularis, and spiral ligament were preserved. There was also a severe loss of Scarpas ganglion cells with preservation of vestibular hair cells. The population of geniculate and trigeminal ganglion cells appeared normal. Sequence analysis from temporal bone DNA showed the 15ldelT DDP gene mutation. Conclusion Sensorineural hearing loss in Mohr-Tranebjærg syndrome is the result of a postnatal, progressive, severe auditory neuropathy.

Association of otosclerosis with Sp1 binding site polymorphism in COL1A1 gene: Evidence for a shared genetic etiology with osteoporosis

Hypothesis: There is an association between otosclerosis and osteoporosis. Background: Both osteoporosis and otosclerosis are common bone diseases to which relatively large portions of the population are genetically predisposed. Recently, a strong association has been described between osteoporosis and an Sp1 binding site of putative functional significance in the first intron of the COL1A1 gene. Methods: We applied polymerase chain reaction-based restriction enzyme analysis to determine the polymorphic distribution of the Sp1 site in 100 patients with otosclerosis and 108 control subjects. Results: This study showed a significant association between otosclerosis and the COL1A1 first intron Sp1 site. The allelic frequency of the Sp1 site is very similar between otosclerosis and osteoporosis. Conclusion: Some cases of otosclerosis and osteoporosis could share a functionally significant polymorphism in the Sp1 transcription factor binding site in the first intron of the COL1A1 gene.

Similar COL1A1 Expression in Fibroblasts from Some Patients with Clinical Otosclerosis and Those with Type I Osteogenesis Imperfecta

Because of the clinical and histopathologic similarities between otosclerosis and type I osteogenesis imperfecta, we examined COL1A1 messenger RNA (mRNA) expression in cultured fibroblasts from patients with clinical otosclerosis to determine whether abnormalities of expression of COL1A1 were present, as has been reported in type I osteogenesis imperfecta. Type I osteogenesis imperfecta has been found to result from mutations in the COL1A1 gene that result in null expression of the mutant allele. Patients with clinical otosclerosis were genotyped for the presence of an expressed 4 base-pair insertion polymorphism in the 3′ region of the COL1A1 gene. Skin biopsies were performed, and cultured fibroblast cell lines were established from patients who were heterozygous for the polymorphism. Allelic expression was examined by reverse transcription–polymerase chain reaction and silver-stained Polyacrylamide gel electrophoresis. Two of 9 patients with clinical otosclerosis demonstrated null or reduced expression of one COL1A1 allele. The differential expression of the Two COL1A1 alleles in all subjects was also examined by a semiquantitative method using an ABI Prism 7700 Sequence Detection System (Taqman). We did this examination to determine whether milder abnormalities in COL1A1 expression might account for the development of otosclerosis in the 7 clinical cases that did not reveal evidence of null expression by the gel technique. Of the same 2 cases of otosclerosis that demonstrated evidence of null expression by gel electrophoresis, both were found to have significant differences in COL1A1 mRNA expression by the Taqman analysis. The remaining 7 cases revealed equal expression of the Two COL1A1 alleles similar to that seen in controls. These results suggest that mutations in COL1A1 that are similar to those that occur in type I osteogenesis imperfecta may account for a small percentage of cases of otosclerosis, and that the majority of cases of clinical otosclerosis are related to other genetic abnormalities that have yet to be identified.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Molecular Biology of Otosclerosis

Otosclerosis is a bone disease of the human otic capsule, which is among the most common causes of acquired hearing loss. The pathologic process is characterized by a wave of abnormal bone remodeling in specific sites of predilection within the endochondral layer of the temporal bone. Although the cause of otosclerosis remains uncertain, there is a clear genetic predisposition with half of all cases occurring in families with more than one affected member. There is also compelling evidence that measles virus may play a role in some cases. Ultimately, how genetic factors and viral infection result in otosclerosis must be explained by effects on the molecular factors that control bone remodeling.

Studies of otic capsule morphology and gene expression in the Mov13 mouse - : An animal model of type I osteogenesis imperfecta

Type I osteogenesis imperfecta (OI) is a disorder of skeletal bones characterized by bone fragility and blue sclera, which can result from mutations in genes encoding for type I collagen – the COL1A1 and COL1A2 genes. Fifty percent of patients with type I OI develop hearing loss and associated histopathological changes in the otic capsule that are indistinguishable from otosclerosis, a major cause of acquired hearing loss. In an attempt to elucidate molecular and cellular mechanisms of hearing loss in type I OI, we have studied the Mov13 mouse, which has served as an animal model of type I OI by virtue of exhibiting variable transcriptional block of the COL1A1 gene. We studied the morphometry of the Mov13 otic capsule and compared expression levels of 60 genes in the otic capsule with those in the tibia and parietal bone of the Mov13 and wild-type mice. The degree of transcriptional block of the COL1A1 gene and its downstream effects differed significantly between the bones examined. We found that expression levels of bone morphogenetic protein 3 and nuclear factor ĸ-B1 best distinguished Mov13 otic capsule from wild-type otic capsule, and that osteoprotegerin, caspase recruitment domain containing protein 1, and partitioning defective protein 3 best distinguished Mov13 otic capsule from Mov13 tibia and parietal bone. Although the Mov13 mouse did not demonstrate evidence of active abnormal otic capsule remodeling as seen in type I OI and otosclerosis, studying gene expression in the Mov13 mouse has provided evidence that osteocytes of the otic capsule differ from osteocytes in other bones.

Deoxyribonucleic acid contamination in archival human temporal bones: a potentially significant problem.

Hypothesis Contamination of archival human temporal bones with extraneous deoxyribonucleic acid may represent a potentially significant problem in the analysis of nucleic acids isolated from archival specimens. Background During the past decade, there has been growing interest in the development of molecular biologic techniques that can be applied to the investigation of pathologic changes in archival human temporal bones. The impetus for the development of these techniques is in part related to the fact that the temporal bone collections represent a repository of archival material compiled over decades, which is not available from living patients. Methods An archival human temporal bone specimen from a male patient with the Mohr-Tranebjærg syndrome (formerly called DFN-1) and a well-characterized mutation was analyzed for the presence of the mutation by a standard method for extraction, isolation, amplification, and sequencing of deoxyribonucleic acid. The experiment was repeated four times. Results The deoxyribonucleic acid sequence from three of four extractions was normal. The known mutation was easily and repeatedly demonstrated in a blood sample from the same individual. Because Mohr-Tranebjærg syndrome is X-linked, there is only one allele, and therefore there is no potential endogenous source to account for the normal sequence that was amplified. Contamination of the tissue sections by extraneous deoxyribonucleic acid presumably occurred during acquisition and processing of the temporal bone. Conclusions Contamination of archival temporal bones with exogenous deoxyribonucleic acid is a significant potential problem that must be considered in the interpretation of the results of deoxyribonucleic acid retrieved from archival sections. The authors recommend collecting blood samples from temporal bone donors in the future to ensure the availability of a reliable source of deoxyribonucleic acid.

Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear.

Tumor necrosis factor (TNF) family cytokines are important mediators of inflammation. Elevated levels of serum TNF‐α are associated with human sensorineural hearing loss via poorly understood mechanisms. We demonstrate, for the first time, expression of TNF‐related apoptosis‐inducing ligand (TRAIL) and its signaling death receptor 5 (DR5) in the murine inner ear and show that exogenous TRAIL can trigger hair cell and neuronal degeneration, which can be partly prevented with DR5‐blocking antibodies.