Controversies in RELN/reelin expression in otosclerosis - Springer Link

Eur Arch Otorhinolaryngol (2012) 269:431–440 DOI 10.1007/s00405-011-1653-4

OTOLOGY

Controversies in RELN/reelin expression in otosclerosis Péter Csomor · István Sziklai · Tamás Karosi

Received: 1 February 2011 / Accepted: 20 May 2011 / Published online: 1 June 2011 © Springer-Verlag 2011

Abstract Several studies have reported a potential genetic association between disease-speciWc single nucleotide polymorphism (SNPs) of RELN and otosclerosis and conWrmed RELN expression in human stapes footplates. These are conXicting results, since RELN expression has been attributed exclusively to neural tissues and to odontoblasts. Otosclerosis is a disease of complex bone remodeling disorder, which is limited to the human otic capsule. Genetic predisposition has long been suspected, however, the pathogenesis remained unclear. Ankylotic stapes footplates (n = 85), cortical bone fragments (n = 4), hearing ossicles (n = 2) and human brain tissue specimens (n = 4) were processed to RELN-speciWc RT-PCR and reelin-speciWc immunoXuorescent assay (IFA). The Wrst group of ankylotic stapes footplates (n = 22) showed a consistent positive reaction against reelin by IFA; however, RELN-speciWc mRNA could not be detected in the second, RT-PCR group (n = 63). Brain specimens were characterized by robust expression of reelin (n = 2) and RELN-speciWc mRNA (n = 2). In case of bone-speciWc controls (n = 6), reelin/ RELN expression was excluded obviously. Concerning current observations, RELN gene does not show active expression in adult stapes footplates. Since, the otic capsule surrounds a special neural structure (membranous labyrinth), reelin might play a coordinative role in the early embryonic stage of development. As being a part of the otic capsule, stapes footplate might be characterized by persisting

P. Csomor · I. Sziklai · T. Karosi (&) Department of Otolaryngology and Head and Neck Surgery, Medical and Health Science Center, University of Debrecen, Debrecen, Nagyerdei Krt. 98, 4032, Hungary e-mail: [email protected]; [email protected] URL: www.earpathology.eu

reelin detectability without mRNA expression. Between these conditions, the etiologic role of RELN is questionable in the pathogenesis of otosclerosis. Keywords Disease-association · Otosclerosis · Otosclerosis genes · Pathogenesis · RELN · Reelin

Introduction Otosclerosis is a common hearing disorder of adults due to pathologically increased bone remodeling in the otic capsule [1]. Otosclerosis causes progressive conductive- and sensorineural hearing loss as a consequence of stapes footplate Wxation and pericochlear osteolysis, respectively [1]. In the Northern white population, the prevalence of manifest otosclerosis is about 0.3–0.4% of the whole population, 5–9% of those with hearing loss and 18–22% of those with conductive hearing loss [1]. Otosclerosis-like bone lesions of the otic capsule are much more common. Histological otosclerosis has been reported as 8–11% of prevalence in large unselected autopsy series [1]. Otosclerosis represents approximately 70% of stapes ankylosis cases leading to consecutive hearing loss [2]. As to simplify the deWnition of stapes ankylosis, we should emphasize that otosclerosis results in stapes Wxation, while stapes Wxation can be determined by other diseases than otosclerosis [2]. Since, otosclerosis and non-otosclerotic stapes ankylosis cannot be separated clinically, diVerential diagnosis is still based on histopathological examination of the removed stapes footplates [2]. Despite of the intense research in the latest years, etiopathogenesis of otosclerosis remained unclear. Several hypotheses suggest viral-, autoimmune-, endocrine- and genetic factors as the basis of otosclerosis [3–8]. However, there are several conXicting results [9, 10].

123

432

Genetic background of otosclerosis has long been suspected over the last decades, without obvious target genes or mutations to show up [11]. Several studies have reported genetic associations in populations with conductive hearing loss due to stapes ankylosis without histopathological analysis of the removed stapes footplates [12, 13]. These studies have extrapolated the observations of otosclerotic stapes ankylosis irrespective of non-otosclerotic Wxations [1, 14, 15]. Most of genetic studies on families with stapes Wxation and on large unselected populations have suggested an autosomal dominant inheritance with incomplete penetrance of approximately 40–45% [11, 13]. Linkage studies have demonstrated the presence of eight otosclerosis-speciWc loci (OTSC1-8) located on chromosomes 15q, 7q, 6p, 16q, 3q, 6q and 9p, respectively [11–15]. These loci were mapped and linked to otosclerosis; however, no causative genes and proteins have been identiWed [11–15]. It has been discussed that COL1A1 (Collagen 1A1), BMP2, BMP4 (Bone Morphogenetic Protein 2 and 4), TGFB1 (Transforming Growth Factor-Beta 1) and RELN (Reelin) genes may also contribute to the development of otosclerosis [3, 4, 16–18]. In practice, statistical associations with these genes may account for only a small fraction of the relative risk for otosclerosis or other types of stapes Wxation [3, 18]. The genetic associations reported earlier cannot explain female dominancy, adult onset, organ-speciWcity and the inXammatory bone remodeling disorder, all features that are characteristic of otosclerosis [1]. The association between the SNPs (single nucleotide polymorphism) of RELN gene and otosclerosis appears as the most interesting genetic observation, since the protein, reelin, is exclusively expressed by neural tissues [18–20]. The RELN gene is located to the chromosome 7q22.1 and contains 64 active exons [21]. RELN encodes an extreme large and excreted extracellular matrix protein named as reelin [21, 22]. Reelin is considered to be a special decoy receptor that regulates the primary neuronal and neuroglial interactions, which are critical steps for cell positioning and also for Wnal neuronal migration during human brain development [19, 21, 22]. Reelin has a serine-protease activity that is important for the modulation of cell adhesion [22]. Binding to lipoprotein receptors VLDLR (Very Low Density Lipoprotein Receptor) and ApoER2 (Apolipoprotein-E Receptor 2) induces modulation of Tau phosphorylation and tyrosine phosphorylation of Dab1, which proteins play a central role in the microtubule formation [22]. Two splicing variants of RELN mRNA (variants 1 [NM_005045.3] and 2, [NM_173054.2]) encoding distinct reelin isoforms (isoforms a [NP_005036] and b [NP_774959]) have been identiWed for the RELN gene [21]. Other transcript variants have also been described and reported, but their full-length nature has not been determined [21]. The reelin protein has been suspected to be involved in the pathogenesis of

123

Eur Arch Otorhinolaryngol (2012) 269:431–440

schizophrenia, autism, bipolar disorder, major depression and in neuronal migration defects associated with temporal lobe epilepsy [23–26]. Expression of reelin has been reported to be signiWcantly decreased in bipolar disorder and in schizophrenia; however, these observations remain unclear as several studies showed that psychotropic medication itself aVects the RELN mRNA expression [22, 25]. With regard to our current knowledge, reelin is expressed exclusively by neural tissues (ganglia, brain, and cerebellum) and no signiWcant expression has been found in other non-neural tissue specimens [19, 20, 22]. Reelin is also expressed by odontoblasts, which are premature cells at the anatomical borders of dental pulp [27]. The role of RELN polymorphism has arisen in the last years in the pathogenesis of otosclerosis [18]. Interestingly, Van Camp and his research group have found a signiWcant genetic association between the SNP polymorphism of RELN gene and the pathogenesis of otosclerosis [18]. Schrauwen et al. [18] have performed a genome-wide association study with 555,000 SNPs to screen the relative risk for otosclerosis in Belgian-Dutch and French populations. They have found two SNPs highly associated with otosclerosis that were mapped to the 7q22.1 chromosome and spanned introns 1–4 of RELN gene [18]. Interestingly, authors have also conWrmed the expression of RELN mRNA and protein reelin in the inner ear and in human stapes footplate samples [18]. Later, Schrauwen et al. have discussed very similar observations about the SNPs and allelic heterogenity of RELN gene associated with otosclerosis in multiple European populations [28]. The main power of this study is the relative large number of samples (1.141) obtained from patients with stapes Wxation [28]. In conjunction with previous results, Khalfallah et al. [29] have found the same associations in a smaller Tunisian population with clinical otosclerosis. They have also described a novel statistical SNP association located to the 11q13.1 region with unknown signiWcance [29]. Furthermore, authors have observed a signiWcant interaction with gender for rs3914132 SNP of RELN suggesting an inXuence of sex on the association of RELN with otosclerosis [29]. This study investigates the expression levels and patterns of the RELN mRNA and the reelin protein in human ankylotic stapes footplates, in hearing ossicles and in cortical bone fragments as compared to those of human brain tissue, in order to elucidate the eVective role of RELN-association in the pathogenesis of otosclerosis.

Materials and methods Patients and controls Figure 1 shows the study design. Eighty-Wve ankylotic stapes footplates (n = 85, male = 32, female = 53) removed

Eur Arch Otorhinolaryngol (2012) 269:431–440 Fig. 1 Schematic representation of study design. Partially removed stapes footplates or fragmented stapes superstructures were not analyzed (crossed images)

433

Specimens Ankylotic stapes footplates (n=85) Controls: Cortical bone fragments (n=4) Human brain specimens (n=4) Hearing ossicles Incus (n=1) Malleus (n=1)

Histological examination – Conventional H&E staining and Reelin-specific immunofluorescent assay Ankylotic stapes footplates (n=22) Controls: Cortical bone fragments (n=2) Human brain specimens (n=2) Incus (n=1)

by stapedectomy were analyzed. The mean age of patients was 42.11 years (range 25–63 years). Air-bone gap at 1,000 Hz was at least 30 dB on pure tone audiometry. Stapes Wxation was clinically bilateral in twenty-three patients; however, only one stapes obtained from each patient was analyzed, because over this period, only unilateral stapes surgeries have been performed. Four cortical bone fragments harvested from the external ear canal during the lateral atticotomy (n = 4) and one incus and malleus specimens removed during CWD mastoidectomy were used as tissue-speciWc controls of RELN expression of stapes footplates. Cortical bones were obtained from patients with stapes ankylosis, while hearing ossicles originated from patients with middle ear cholesteatoma. Four cadaver brain tissue samples (n = 4, male = 3, female = 1) were employed as positive controls of RELN expression in the RELN-speciWc RT-PCR and immunoXuorescent assays. Cadaver specimens were removed within 10 h after death. The mean age of positive controls was 55.85 years (range 49–63 years). Stapes footplates and control tissue specimens were collected from February 2010 until October 2010 (University of Debrecen, Medical and Health Science Center, Departments of Otolaryngology and Head and Neck Surgery and Pathology; and ENT Department of Bajcsy-Zsilinszky Hospital, Budapest; Hungary). All patients gave their informed written consents to our study. The Hungarian ScientiWc Research Ethical Committee (ETT-TUKEB/2008-113-547/89) approved this study. The

Reelin-specific RT-PCR Ankylotic stapes footplates (n=63) Controls: Cortical bone fragments (n=2) p ((n=2)) Human brain specimens Malleus (n=1)

study was carried out according to the Declaration of Helsinki. RNA extraction and RELN mRNA-speciWc RT-PCR Cortical bone specimens (n = 2), malleus (n = 1) and human brain tissue samples (n = 2) and the majority of ankylotic stapes footplates (n = 63) were stored in sterile silicone tubes at ¡70°C temperature (Fig. 1). Individual tissue specimens were frozen in liquid nitrogen and pulverized in sterile dry rubber cups. The pulverized tissues were suspended in DEPC (diethyl-pirocarbonate) treated, destilled water. Total RNA was extracted from the homogenized tissue suspensions by using of TRI Reagent™ solution (Sigma-Aldrich, St. Louis, MO, U.S.A.) according to the manufacturer’s instructions. Reverse transcription was performed on 1 g of total RNA in a total volume of 20 l with the High-Capacity cDNA Reverse Transcription Kit™ (Applied Biosystems, Foster City, CA, U.S.A.) with hexagonal random primers according to the manufacturer’s instructions. RELN-speciWc primers were created and selected by online Primer-BLAST™ software (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/) using the sequence of RELN transcript variant 1 mRNA (NM_005045.3, GI: 223718138). RT-primers (Reln-7F 5⬘– 3⬘: GGA CCA TGT GGA GGT CGT CCT AGT A, GC% = 56, Tm = 58.8°C and Reln-7R 5⬘–3⬘: CAC TCG GTC TTG AGA AGG GCT TTC, GC% = 54.17,

123

434

Fig. 2 ImmunoXuorescent demonstration of reelin expression in ankylotic stapes footplates with diVerent histopathologies. a1 Active otosclerosis at the anterior pole of the stapes footplate with hypercellular osteoid substance and several osteolytic lacunae (H.E., left ear). Air-bone gap at 1 kHz was 35 dB. a2 Reelin-speciWc immunoXuorescent staining of the previous section. The otosclerotic focus displays intense, granular reelin-speciWc immunoreaction. b1 Inactive focus of otosclerosis at the anterior pole of the stapes footplate (H.E., right ear). The lesion is characterized by hypocellularity, empty cellular halos and large pseudo-vascular osteolytic lacunae. Air-bone gap at 1 kHz was

123


40 dB. b2 Reelin-speciWc immunoXuorescent assay (IFA) of the previous section. The lesion forming cells show weak cytoplasmic immunoreaction. c1 Non-otosclerotic, annular calciWcation of the stapes footplate with hypocellular and bulky poles (H.E., left ear). Air-bone gap at 1 kHz was 30 dB. c2 Reelin-speciWc IFA of the previous section. The osteocytes are characterized by weak reelin expression similar to those in case of inactive otosclerosis. In contrast, chondrocytes of the hyaline cartilage layer of the vestibular surface of the stapes footplate show a quite intense immunoXuorescent reaction (small insert)


435

Table 1 Expression of reelin adhesion molecule in ankylotic stapes footplates with diVerent histopathologies and in tissue-speciWc controls Histological specimens (H.E., n = 27)

Reelin-speciWc IFAa

Stapes (n = 22) Otosclerosis (n = 14, 63.6%) Active (n = 9, 64.3%)

++++

Inactive (n = 5, 35.7%)

++

Non-otosclerotic stapes Wxation (n = 8, 36.3%) Annular calciWcation (n=8, 100%)

++

Control specimens (n = 5) Cortical bone fragments (n = 2)

¡

Incus (n = 1)

¡

Brain (n = 2)

++++

a

Intensity of immunoXuorescent assays (IFA): +, weak annular immunoreactivity; ++, weak homogeneous immunoreactivity; +++, strong homogeneous or weak granular immunoreactivity; ++++, strong conXuent and robust granular reaction; ¡, negative reaction

Tm = 57.5°C) spanning the 3⬘ end of RELN mRNA were employed in the cDNA PCR (Sigma-Aldrich, St. Louis, MO, USA). The estimated product size was 264 base pairs. AmpliWcation reactions were performed in a total volume of 25 l, containing 1£ PCR buVer, 1.5 mM MgCl2, 100 M of each dNTP, 0.2 M of both primers, 2.5 units of AccuTaq LA DNA Polymerase™ (Sigma-Aldrich, St. Louis, MO, USA), and 2 l cDNA. PCR conditions were 2 min at 94°C for initial denaturation, 35 cycles of 15 s at 94°C for denaturation, 30 s at 60°C for annealing, 1 min at 68°C for extension, and a Wnal extension for 5 min at 68°C. Negative control RT-PCR with DNA-free water was included in all experiments. The suitability of extracted RNA samples for RT-PCR detection of RELN mRNA was tested by using h36B4 + (5⬘–3⬘: AGA TGC AGC AGA TCC GCA T) and h36B4 ¡ (5⬘–3⬘: ATA TGA GGC AGG AGT TTC TCC AG) primer set targeting human ribosomal RNA. This human ribosomal RNA-speciWc housekeeping control RT-PCR was performed in each specimens. Reelin-speciWc immunoXuorescent assay The remnants of ankylotic stapes footplates (n = 22), cortical bone fragments (n = 2), incus (n = 1) and two human brain tissue samples (n = 2) were Wxed in 10% (w/v) formaldehyde (Fig. 1). Stapes footplate specimens and control bones were decalciWed by 0.5 M Na-EDTA containing 0.02% (w/v) sodium azide (72 h, 4°C). Human brain samples were not treated by Na-EDTA. Specimens were embedded in 15% (w/v) puriWed gelatine (24 h, 56°C) and reWxed in 4% (w/v) paraformaldehyde (24 h, 20°C). Blocks were cryoprotected in 20% (w/v) saccharose-solution

(2 h, 4°C) and sectioned into 10 m slides at ¡25°C (MNT-200, Slee, Mainz, Germany). Slides were stored in 0.1 M PBS containing 0.03% (w/v) sodium azide at 4°C. Two consecutive 10 m frozen cut sections were examined as follows: (1) Staining with hematoxylin and eosin (H.E.); and (2) Reelin-speciWc immunoXuorescent assay (IFA). First sections of each individual specimen were processed by a conventional H.E. staining. The second series of sections were washed and protein blocked in 0.2 M PBS containing donkey normal serum (SC-2044, Santa Cruz, CA, USA). Blocked sections were incubated with 1:200 polyclonal goat anti-human reelin primary antibody solution (20°C, 48 h, continuous shaking) (SC-32554, K-15, Santa Cruz, CA, USA). Primary antibody was washed with 0.2 M PBS and sections were incubated with 1:300 donkey antigoat FITC-labeled IgG solution (20°C, 12 h, continuous shaking) (SC-2024, Santa Cruz, CA, USA). Slides were covered by UV transparent mounting medium (Vectashield™, Vector Laboratories, CA, USA). Imaging was performed under UV-light with 510 nm wavelengths and 69– 87 ms exposure time and the photographs were archived in jpeg Wle format (Axioskop2 MOT™, Axiovision 3.0™, Zeiss, Jena, Germany). Histological examinations were blinded for two independent researchers: P.C. analyzed the sections stained by HE, while T.K. interpreted the immunoXuorescent assays. The histopathological activity of otosclerosis was based on the cellularity and the structure of cement lines by conventional HE staining. Since otosclerosis is a multifocal disease, the histopathological activity of a stapedial osteolytic focus is not in full correspondence with the clinical activity of otosclerosis [1, 5]. Active otosclerosis is displayed by wide pseudo-vascular spaces Wlled with increased numbers of large, misshapen, and multinucleated osteoclasts. Cement lines show a woven pattern. Inactive otosclerosis is marked by obliterated vascular spaces and resorption lacunae with decreased numbers of osteoclasts (empty halo-cells).

Results Ankylotic stapes footplates (n = 85), cortical bone fragments (n = 4), hearing ossicles (n = 2) and human brain tissue specimens (n = 4) were analyzed through RELNspeciWc RT-PCR and reelin-speciWc immunoXuorescent assay, respectively (Fig. 1). Reelin-speciWc immunoXuorescent assay (IFA) Histological diagnosis of otosclerosis was established in 14 ankylotic stapes footplates [2] (Fig. 2, Table 1). Among these patients, the otosclerotic foci were seen to be active in nine stapes footplates and inactive in Wve cases (Fig. 2,

123

436

Fig. 3 ImmunoXuorescent demonstration of reelin expression in bone-speciWc controls and in human brain specimens. a1 Incus that represents a normal structure of cement lines and blood vessels (H.E., right ear). a2 The reelin-speciWc immunoreaction of the previous section is negative. b1 Cortical bone fragment harvested from the posterior wall of the external auditory canal (H.E., left ear). b2 Reelin expression is not detectable. c1 Human brain cortex removed from the

123


superior temporal gyrus (H.E.). A complex axonal- and dendritic web can be observed. The inserted picture presents a pyramid cell (neuron) with intense basophilic staining. c2 Reelin-speciWc immunoXuorescent assay of the previous section. Neuroglial elements (astrocytes and oligodendrocytes) show robust reelin expression. The reelin expressing oligodendroglial complex is marked by the empty arrow. The small picture shows a reelin-positive astrocyte (white arrow)

ontr. 2 Neg. Co

Neg. Co ontr. 1

Brain 2

Brain 1

Brain 2

MW

Brain 1

Malleus s

Stapes 5

Stapes 4

437

Stapes 3

Stapes 2

Stapes 1


A RELN-specific RT-PCR RT PCR 264 bp

MW

B Housekeeping control RT-PCR

150 b bp

Fig. 4 Agarose gel electrophoresis of RT-PCR amplimers. a RT-PCR ampliWcation of RELN-derived mRNA in ankylotic stapes footplates in a malleus and in human brain tissue specimens applied as positive controls. RELN mRNA was not detectable in the bone samples (left part of the Wgure), while brain tissues gave strong positive reactions by

264 bp amplimers. RNA-free controls (negative controls 1–2) were negative for RELN mRNA. b Housekeeping control RT-PCR of previous specimens. All samples showed positivity on human ribosomal RNA (150 bp)

Table 2 Expression of RELN mRNA in ankylotic stapes footplates and in tissue-speciWc controls

plates (Fig. 2, Table 1). The hyaline cartilage layer of the vestibular surface was displayed by the most intense reelinspeciWc immunoreaction, while the superstructures did not indicate reelin expression (Fig. 2). In contrast, the cortical bone fragments and the incus applied as bone-speciWc controls were characterized by negative reelin-speciWc IFA results (Fig. 3, Table 1).

Specimens (n = 68)

Housekeeping control RT-PCR

RELN mRNAspeciWc RT-PCR

Ankylotic stapes footplates 47 (n = 63)

0

Cortical bone fragments (n = 2)

2

0

Human brain specimens (n = 2)

2

2

RELN mRNA-speciWc RT-PCR

Malleus (n = 1)

1

0

Human cellular RNA was detected in 47 ankylotic stapes footplates and in all tissue-speciWc controls (n = 5) conWrming that nucleic acid extraction process was correct (Fig. 4, Table 2). The sixteen ankylotic stapes footplates with negative cellular control results were excluded from further analysis (Table 2). Human brain specimens applied as positive controls showed obvious positivity on RELN mRNA expression (Fig. 4, Table 2). In contrast to these results, in cases of ankylotic stapes footplates and bone-speciWc controls no considerable RELN expression could be observed (Fig. 4, Table 2).

Table 1). In the eight non-otosclerotic stapes footplates, histological examinations revealed annular, stapediovestibular calciWcation with marked thickening of the stapes footplate [2] (Fig. 2, Table 1). In agreement with previous observations, human brain specimens employed as positive controls showed robust cytoplasmic reelin expression by IFA (Fig. 3, Table 1). Oligodendrocytes and astrocytes showed rather intense immunoreaction standing out the complex structure of neuroglial web (Fig. 3). Unexpectedly, ankylotic stapes footplates were also characterized by an intense reelin expression, which was independent from the histological diagnosis of stapes Wxation (Fig. 2, Table 1). DiVerent expression levels of reelin seemed to be in association with the cellularity of the pathologically remodeled stapes foot-

Discussion Our study was performed with the aim of precise description of tissue-speciWc expression of RELN-derived mRNA and reelin in human ankylotic stapes footplates. The results

123

438


Fig. 5 Hypothetical model attempting to explain the variable RELN/reelin expression in the human otic capsule

Enchondral bone formation of the otic capsule - Paraxial mesoderma - Chordal chondrocranium (4.-10. weeks)

RELN mRNA + Reelin +

Development of brain nerves (VII and VIII) - Ectoderma - Metencephalon (6. week)

Development of the membranous labyrinth (sacculus, utriculus, ampulla, cochlear duct) and ganglia (vestibular and spiral) - Ectoderma - Otocysts (4. week)



RELN mRNA Persisting Reelin + Mature bony and membranous labyrinth - No synapsis formation - No remodeling - Invariable biomechanical characteristics of organ of Corti

shown in this report are of value, since several studies have reported astonishing and novel information about reelin expression in human stapes footplates regarding this association as a potential genetic basis of otosclerosis [18, 28– 30]. With regard to current observations, RELN/reelin expression was unambiguously excluded in incus, malleus and in cortical bone fragments applied as tissue-speciWc controls of stapes specimens. These observations are not surprising, since reelin expression has been reported only in neural- or in neural-associated tissues (brain, ganglia, retina and odontoblasts) [20, 27]. Surprisingly, there was a conXict between RELN mRNA and reelin expression in human ankylotic stapes footplates, since mRNA did not show a detectable expression level, while reelin gave strong immunoreaction by immunoXuorescent assay. Van Camp and his research group have reported several single nucleotide polymorphisms of RELN gene associated with the pathogenesis of otosclerosis [18, 28, 29]. A nonnegligible diVerence with our study is that diagnosis of otosclerosis was made without histopathological analysis of surgically removed stapes footplate [18]. It might be explained by the surgical technique: stapedectomy is not a suitable method to obtain stapes footplate fragments containing the Wxing lesions. It was very important to conWrm the RELN-speciWc mRNA and also the protein expression in human stapes footplate specimens, since without these results genetic association turns into a pointless Wnding and remains a simple statistical observation [18]. In their Wrst paper, Schrauwen et al. [18] have employed RELN mRNAspeciWc primers RELNex61.62F (5⬘–3⬘: TACGCCCATGG

123

AGACTCACT) and RELNex61.62R (5⬘–3⬘: CCGTTGTT GACGCTGTATTC) to detect RELN expression in human stapes footplates (with the courtesy of Ealy M.). We were unable to detect RELN-speciWc mRNA by the virtual online analysis (http://www.ncbi.nlm.nih.gov/tools/primerblast/) of these primer pairs. We do think that several interconnected factors associated with the special features of the human otic capsule might explain these incongruent Wndings about RELN/ reelin expression in human stapes footplates (Fig. 5). As being a unique bone in the human skeleton, otic capsule contains three cranial nerves and their ganglia (n. VII., n. VIII. and n. IX.—Jacobson’s nerve) and several neuroectodermal elements (membranous labyrinth, organ of Corti, spiral ganglion), which are neccessary to be escorted by reelin during the early phase of embryonic development [19]. It should be noted that the stapes footplate is an essential part of the otic capsule by the embryonic point of view [1]. At the later stages of embryonic development, RELN mRNA expression may not be required, since synaptic formation and neural integration process are laid oV within the otic capsule [1, 19]. Furtheromore, post-embryonic bone remodeling is totally absent in the otic capsule in order to prevent the biomechanical aspects of the organ of Corti [1]. Finally, reelin is a large stucture potein (»3,500 AA) having a long half-life time expression without active transcription of RELN-derived mRNA and it could persist throughout life [21, 22]. Nevertheless, in view of persisting reelin detectability in human stapes footplates a complicated question arises: what could be the role of a protein


coordinating synaptic formation and axonal development in the pathogenesis of otosclerotic bone remodeling disorder? Certainly, our hypothesis requires further examinations in the future (Fig. 5). In conclusion, expression of RELN-speciWc mRNA should be excluded in ankylotic stapes footplates and in bone-speciWc controls. In spite of passive reelin expression, current observations query the genetic association between RELN-speciWc SNPs and the pathogenesis of otosclerosis. In the light of our results, we do think that further studies are necessary to determine the role of RELN in the pathophysiology of otosclerosis. Acknowledgments Thanks are due to Bálint Liktor MD (BajcsyZsilinszky Hospital, Department of Otolaryngology Head and Neck Surgery, Budapest, Hungary) and Professor Péter Molnár MD PhD (University of Debrecen, Medical and Health Science Center, Department of Pathology) for collecting of a series of ankylotic stapes and cadaver brain specimens. This work was supported by grants from the Hungarian ScientiWc Research Fund (OTKA PD75371, K81480) and Mecenatúra Fund of Debrecen University (DE OEC Mec 17/2008). ConXict of interest

None.

References 1. Karosi T, Szekanecz Z, Sziklai I (2009) Otosclerosis: an autoimmune disease? Autoimmun Rev 9(2):95–101 2. Karosi T, Csomor P, Petkó M, Liktor B, Szabó LZ, Pytel J, Jóri J, Sziklai I (2009) Histopathology of nonotosclerotic stapes Wxations. Otol Neurotol 30(8):1058–1066 3. Lehnerdt G, Metz KA, Trellakis S, Jahnke K, Neumann A (2007) Signaling by way of type IB and II bone morphogenetic protein receptors regulates bone formation in otospongiosis. Laryngoscope 117(5):812–816 4. Lehnerdt G, Unkel C, Metz KA, Jahnke K, Neumann A (2008) Immunohistochemical evidence of BMP-2, -4 and -7 activity in otospongiosis. Acta Otolaryngol 128(1):13–17 5. Karosi T, Szalmás A, Csomor P, Kónya J, Petkó M, Sziklai I (2008) Disease-associated novel CD46 splicing variants and pathologic bone remodeling in otosclerosis. Laryngoscope 118(9):1669–1676 6. Csomor P, Szalmás A, Kónya J, Sziklai I, Karosi T (2010) Restriction analysis of otosclerosis-associated CD46 splicing variants. Eur Arch Otorhinolaryngol 267(2):219–226 7. McKenna MJ, Kristiansen AG, Haines J (1996) Polymerase chain reaction ampliWcation of a measles virus sequence from human temporal bone sections with active otosclerosis. Am J Otol 17(6):827–830 8. Arnold W, Niedermeyer HP, Lehn N, Neubert W, HöXer H (1996) Measles virus in otosclerosis and the speciWc immune response of the inner ear. Acta Otolaryngol 116(5):705–709 9. Imauchi Y, Jeunemaître X, Boussion M, Ferrary E, Sterkers O, Grayeli AB (2008) Relation between renin-angiotensin-aldosterone system and otosclerosis: a genetic association and in vitro study. Otol Neurotol 29(3):295–301 10. Schrauwen I, Thys M, Vanderstraeten K, Fransen E, Ealy M, Cremers CW, Dhooge I, Van de Heyning P, OVeciers E, Smith RJ, Van Camp G (2009) No evidence for association between the renin-angiotensin-aldosterone system and otosclerosis in a large Belgian-Dutch population. Otol Neurotol 30(8):1079–1083

439 11. Van Den Bogaert K, Govaerts PJ, De Leenheer EM, Schatteman I, Verstreken M, Chen W, Declau F, Cremers CW, Van De Heyning PH, OVeciers FE, Somers T, Smith RJ, Van Camp G (2002) 2002. Otosclerosis: a genetically heterogeneous disease involving at least three diVerent genes. Bone 30(4):624–630 12. Tomek MS, Brown MR, Mani SR, Ramesh A, Srisailapathy CR, Coucke P, Zbar RI, Bell AM, McGuirt WT, Fukushima K, Willems PJ, Van Camp G, Smith RJ (1998) Localization of a gene for otosclerosis to chromosome 15q25–q26. Hum Mol Genet 7(2):285–290 13. Thys M, Van Den Bogaert K, Iliadou V, Vanderstraeten K, Dieltjens N, Schrauwen I, Chen W, Eleftheriades N, Grigoriadou M, Pauw RJ, Cremers CR, Smith RJ, Petersen MB, Van Camp G (2007) A seventh locus for otosclerosis, OTSC7, maps to chromosome 6q13–16.1. Eur J Hum Genet 15(3):362–368 14. Van Den Bogaert K, Govaerts PJ, Schatteman I, Brown MR, Caethoven G, OVeciers E, Somers T, Declau F, Coucke P, Van de Heyning P, Smith RJ, Van Camp G (2001) A second gene for otosclerosis, OTSC2, maps to chromosome 7q34–36. Am J Hum Genet 68(2):495–500 15. Van Den Bogaert K, De Leenheer EM, Chen W, Lee Y, Nurnberg P, Pennings RJ, Vanderstraeten K, Thys M, Cremers CW, Smith RJ, Van Camp G (2004) A Wfth locus for otosclerosis, OTSC5, maps to chromosome 3q22–24. J Med Genet 41(6):450–453 16. Chen W, Meyer NC, McKenna MJ, PWster M, McBride DJ Jr, Fukushima K, Thys M, Camp GV, Smith RJ (2007) Single-nucleotide polymorphisms in the COL1A1 regulatory regions are associated with otosclerosis. Clin Genet 71(5):406–414 17. Thys M, Schrauwen I, Vanderstraeten K, Janssens K, Dieltjens N, Van Den Bogaert K, Fransen E, Chen W, Ealy M, Claustres M, Cremers CR, Dhooge I, Declau F, Claes J, Van de Heyning P, Vincent R, Somers T, OVeciers E, Smith RJ, Van Camp G (2007) The coding polymorphism T263I in TGF-beta1 is associated with otosclerosis in two independent populations. Hum Mol Genet 16(17):2021–2030 18. Schrauwen I, Ealy M, Huentelman MJ, Thys M, Homer N, Vanderstraeten K, Fransen E, Corneveaux JJ, Craig DW, Claustres M, Cremers CW, Dhooge I, Van de Heyning P, Vincent R, OVeciers E, Smith RJ, Van Camp G (2009) A genome-wide analysis identiWes genetic variants in the RELN gene associated with otosclerosis. Am J Hum Genet 84(3):328–338 19. Nielsen KB, Søndergaard A, Johansen MG, Schauser K, Vejlsted M, Nielsen AL, Jørgensen AL, Holm IE (2010) Reelin expression during embryonic development of the pig brain. BMC Neurosci 15(11):75 20. Roberts RC, Xu L, Roche JK, Kirkpatrick B (2005) Ultrastructural localization of reelin in the cortex in post-mortem human brain. J Comp Neurol 482(3):294–308 21. De Silva U, D’Arcangelo G, Braden VV, Chen J, Miao GG, Curran T, Green ED (1997) The human reelin gene: isolation, sequencing, and mapping on chromosome 7. Genome Res 7(2):157–164 22. Förster E, Bock HH, Herz J, Chai X, Frotscher M, Zhao S (2010) Emerging topics in reelin function. Eur J Neurosci 31(9):1511–1518 23. Persico AM, D’Agruma L, Maiorano N, Totaro A, Militerni R, Bravaccio C, Wassink TH, Schneider C, Melmed R, Trillo S, Montecchi F, Palermo M, Pascucci T, Puglisi-Allegra S, Reichelt KL, Conciatori M, Marino R, Quattrocchi CC, Baldi A, Zelante L, Gasparini P, Keller F (2001) Collaborative linkage study of autism. Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder. Mol Psychiatry 6(2):150–159 24. Hong SE, Shugart YY, Huang DT, Shahwan SA, Grant PE, Hourihane JO, Martin ND, Walsh CA (2000) Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations. Nat Genet 26(1):93–96 25. Wedenoja J, Loukola A, Tuulio-Henriksson A, Paunio T, Ekelund J, Silander K, Varilo T, Heikkilä K, Suvisaari J,

123

440 Partonen T, Lönnqvist J, Peltonen L (2008) Replication of linkage on chromosome 7q22 and association of the regional Reelin gene with working memory in schizophrenia families. Mol Psychiatry 13(7):673–684 26. Fatemi SH, Snow AV, Stary JM, Araghi-Niknam M, Reutiman TJ, Lee S, Brooks AI, Pearce DA (2005) Reelin signaling is impaired in autism. Biol Psychiatry 57(7):777–787 27. Maurin JC, Couble ML, Didier-Bazes M, Brisson C, Magloire H, Bleicher F (2004) Expression and localization of reelin in human odontoblasts. Matrix Biol 23(5):277–285 28. Schrauwen I, Ealy M, Fransen E, Vanderstraeten K, Thys M, Meyer NC, Cosgarea M, Huber A, Mazzoli M, PWster M, Smith RJ, Van

123

Eur Arch Otorhinolaryngol (2012) 269:431–440 Camp G (2010) Genetic variants in the RELN gene are associated with otosclerosis in multiple European populations. Hum Genet 127(2):155–162 29. Khalfallah A, Schrauwen I, Mnaja M, Fransen E, Lahmar I, Ealy M, Dhouib L, Ayadi H, Charfedine I, Driss N, Ghorbel A, Smith RJ, Masmoudi S, Van Camp G (2010) Genetic variants in RELN are associated with otosclerosis in a non-European population from Tunisia. Ann Hum Genet 74(5):399–405 30. Priyadarshi S, Panda KC, Panda AK, Ramchander PV (2010) Lack of association between SNP rs3914132 of the RELN gene and otosclerosis in India. Genet Mol Res 9(3):1914–1920