ORIGINAL ARTICLE Distribution of Type IV Collagen in the Cochlea in Alport Syndrome Andreas F. Zehnder, MD; Joe C. Adams, PhD; Peter A. Santi, PhD; Arthur G. Kristiansen, MA; Chitsuda Wacharasindhu, MD; Sabine Mann; Raghu Kalluri, PhD; Martin C. Gregory, MD; Clifford E. Kashtan, MD; Saumil N. Merchant, MD Objective: To determine the distribution of ␣1, ␣3, and brane, in the basement membrane of cochlear blood ves- ␣5 chains of type IV collagen in the cochlea in Alport sels and Schwann cells, and within the spiral limbus. In syndrome. the control ear, we also observed strong staining for ␣3 and ␣5 chains in the basement membrane overlying the Design: Case-control study. basilar membrane and within the spiral ligament. In both cases with Alport syndrome, no immunostaining was ob- Patients: Two patients with sensorineural hearing loss served for ␣3or␣5 chains within the cochlea, whereas due to Alport syndrome. Both patients had known mu- ␣1 staining was present in locations similar to that seen tations in the COL4A5 gene. in the control ear. Main Outcome Measures: Immunostaining was used Conclusions: The results indicate that isotype switch- to study the distribution of type IV collagen (␣1, ␣3, and ing does not occur within the cochlea in Alport syn- ␣5 chains) within the cochlea. Immunostaining was also drome. The results are also consistent with the hypoth- performed in the cochlear tissues of an unaffected indi- esis that the sensorineural hearing loss in Alport syndrome vidual used as a control. may be due to alterations in cochlear micromechanics and/or dysfunction of the spiral ligament. Results: In the control ear, ␣1 staining was observed in the basement membrane overlying the basilar mem- Arch Otolaryngol Head Neck Surg. 2005;131:1007-1013 LPORT SYNDROME, AN IN- In Alport syndrome, the glomerular BM herited progressive renal within the kidney shows a characteristic disease with sensorineu- ultrastructural abnormality consisting of Author Affiliations: Ear, Nose, ral hearing loss (SNHL) diffuse thickening and splitting of the 2 and Throat Department, and ocular lesions, is lamina densa. During normal develop- University Hospital Basel, Basel, Acaused by mutations in the genes (COL4) ment, ␣1 and ␣2 chains predominate in Switzerland (Dr Zehnder); that code for the ␣3, ␣4, or ␣5 chains of the glomerular BM. As glomerular matu- Department of Otolaryngology, type IV collagen.1-3 The disease is geneti- ration progresses, the ␣3, ␣4, and ␣5 Massachusetts Eye and Ear cally heterogeneous with X-linked, auto- chains become the predominant type IV Infirmary, Boston (Drs Zehnder, somal recessive and autosomal dominant collagen chains in glomerular BM, a pro- Adams, Wacharasindhu, and forms. Most cases (approximately 85%) of cess referred to as isotype switching.2,3,5-7 In Merchant, Mr Kristiansen, and Alport syndrome are X-linked and are due Ms Mann); Department of most males with Alport syndrome, this iso- to mutations in the COL4A5 gene located type switch does not occur, as a result of Otology and Laryngology ␣ (Drs Adams and Merchant), on Xq22 that codes for the 5 chain of type which the ␣3, ␣4, and ␣5 chains are usu- Harvard Medical School, and IV collagen. More than 300 mutations of ally absent, as determined by immuno- Center for Matrix Biology, the COL4A5 gene producing X-linked Al- staining. The resulting glomerular BM in Department of Medicine, Beth port syndrome have been reported. Alport syndrome is believed to be defec- Israel Deaconess Medical Type IV collagen is the major structural tive in structure and function, causing Center (Dr Kalluri), Boston; component of basement membrane (BM) hematuria early in life and progressing to Departments of Otolaryngology ␣ and it consists of 3 chains, which make a proteinuria and renal failure. and Neuroscience (Dr Santi) triple-helical molecule.2-4 The trimer com- and Division of Pediatric ␣ ␣ ␣ ␣ In contrast to renal studies, investiga- posed of 1 and 2 chains ( 12- 21)is tions of the human inner ear in Alport syn- Nephrology, Department of present ubiquitously in BMs throughout the Pediatrics (Dr Kashtan), drome have lagged behind because of sev- body. On the other hand, trimers com- University of Minnesota, ␣ ␣ ␣ ␣ ␣ ␣ eral factors: Minneapolis; and Department posed of 3, 4, and 5( 31- 41- 51)or ␣ ␣ ␣ ␣ of Medicine, University of Utah 5 and 6( 52- 61) chains are restricted 1. It is not feasible to take a biopsy Medical Center, Salt Lake City in expression to specific BMs of the kid- sample from the cochlea during life with- (Dr Gregory). ney, inner ear, eye, and other organs. out causing severe SNHL. (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 131, NOV 2005 WWW.ARCHOTO.COM 1007 ©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 2. There is a paucity of human postmortem tempo- good speech discrimination. He died at age 57 years from end- ral bone material because temporal bones are not re- stage renal disease, and both temporal bones were removed 4 moved or studied as part of routine autopsies. hours after death. The left temporal bone was examined by light and electron microscopy, and findings were reported previ- 3. When temporal bones are removed and studied af- 8 ter death, tissue alterations are inevitably induced by post- ously. The right temporal bone was fixed in 10% neutral buff- ered formalin, decalcified using EDTA, trimmed so that only mortem autolysis. the inner ear remained, and was then embedded in paraffin. 4. The human temporal bone contains the delicate Serial sections were cut at a thickness of 8 µm. A representa- membranous labyrinth within an extremely dense shell tive (mid modiolar) section was stained with hematoxylin- of bone, which imposes serious technical challenges in eosin (H&E), while other sections through the cochlea were processing temporal bone tissue in a manner that al- immunostained. lows for preservation of morphology and the ability to immunostain for various proteins. CASE 2 In a recent study,8 our research team examined the Patient 2 was a man aged 31 years with X-linked Alport syn- histopathologic features of the cochlea in 9 patients with drome caused by COL4A5 mutation. This individual had a de- Alport syndrome and described abnormalities of the BM letion of exons 43-47 of the COL4A5 gene. He had bilateral SNHL of cells of the organ of Corti, as well as cellular infilling since the age of 9 years and wore amplification for several years. of the tunnel and extracellular spaces of the organ of Corti His last audiogram at age 25 years showed a downsloping 60- in 8 of the 9 cases. This particular study used a combi- to 110-dB SNHL on the right with speech discrimination of 76% nation of light and electron microscopy to study the mor- and a 60- to 90-dB SNHL on the left with speech discrimina- phologic changes in the cochlea. tion of 90%. He died at the age of 31 years from pancreatitis Knowledge of the distribution of various chains of type and complications of renal failure, and both temporal bones IV collagen in the cochlea may help in the understand- were removed 10 hours after death. The right temporal bone ing of the pathogenesis of SNHL at a cellular level. There was used in the present study. The specimen had been stored in 10% formalin for a number of years. The bone was prepared is a single extant study that examined the distribution by decalcification using EDTA and trimming so that only the of type IV collagen in the normal human inner ear. In inner ear remained, followed by embedding in paraffin and sec- 9 ␣ the study by Kleppel et al, expression of 1 chains was tioning at a thickness of 8 µm. A mid modiolar section was observed in the BM overlying the basilar membrane (ie, stained with H&E, while immunostaining was performed on the BM of cells of the organ of Corti), in the BM of coch- other sections. lear blood vessels, and in the spiral limbus and in the BM of Schwann cells. In contrast, expression of ␣3 and ␣4 CASE 3 chains was restricted to the BM overlying the basilar mem- brane. The study did not examine expression of ␣5 chains. Case 3 involved a woman aged 57 years with no otologic symp- To our knowledge, no study has described the distribu- toms (control case). This individual died as a result of a hem- tion of type IV collagen within the cochlea in Alport syn- orrhagic cerebral infarct with pulmonary thromboembolism. drome. Both temporal bones were removed 5.5 hours after death, fixed In the present study, we describe the distribution of in 10% formalin, and processed in a manner identical to that type IV collagen (␣1, ␣3, and ␣5 chains) as determined described for cases 1 and 2. Before proceeding with immunostaining on human tempo- by immunostaining within the inner ear of 2 patients with ral bones, we conducted experiments using temporal bones of SNHL due to Alport syndrome. Both patients had known CBA mice to optimize the protocol for immunostaining. The mutations in the COL4A5 gene resulting in Alport syn- temporal bones of CBA mice were fixed in 10% formalin, de- drome. Results indicate that there is selective distribu- calcified in EDTA, embedded in paraffin, and sectioned in a man- tion of ␣3 and ␣5 chains within the cochlea, including ner similar to the processing used for human tissues. Antibod- within the collagen bundles in the spiral ligament and ies against the ␣1, ␣3, and ␣5 chains of type IV collagen were limbus. Results in the cases with Alport syndrome sug- used to immunostain the animal material to optimize the gest that isotype switching for collagen does not occur protocol.