Similarities were noted between images presented in this article 1 and two additional articles published by the same research group 2, 3: * The schematics of postanal day-3 and adult cochlear sensory ...epithelia presented in Fig 1A 1 are identical to the schematics of postanal day-3 and adult cochlear sensory epithelia presented in Fig 1 2 and the schematics presented in Gene Expression Patterns 3. * The immunohistochemistry images of undifferentiated CGR8 mouse embryonic stem cells in Fig 1B 1 are identical to the immunohistochemistry images of undifferentiated CGR8 mouse embryonic stem cells in Fig 4D–4F 2. The following information has been added to the Materials and Methods section: “Culture of mouse embryonic stem cells (mESCs) The undifferentiated mESCs (CGR8 line kindly provided by Bernard Binetruy, Aix-Marseille University, France) were expanded in the absence of feeder cells in DMEM culture medium (Gibco by Life Technologies) supplemented with LIF (leukemia inhibitory factor) on gelatin-coated plates. The undifferentiated and untreated cells used for immunohistochemistry were harvested from passage 2 cell cultures and fixed in paraformaldehyde 4% in Phosphate Buffer Solution for 20 min at room temperature.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Similarities were noted between images presented in this article 1 and two additional articles published by the same research group 2, 3: * The schematics of postanal day-3 and adult cochlear sensory ...epithelia presented in Fig 1 1 are identical to the schematics of postanal day-3 and adult cochlear sensory epithelia presented in Fig 1A 2 and the schematics presented in Gene Expression Patterns 3. * The immunohistochemistry images of undifferentiated CGR8 mouse embryonic stem cells in Fig 4D–4F 1 are identical to the immunohistochemistry images of undifferentiated CGR8 mouse embryonic stem cells in Fig 1B 2. “Culture of mouse embryonic stem cells (mESCs) The undifferentiated mESCs (CGR8 line kindly provided by Bernard Binetruy, Aix-Marseille University, France) were expanded in the absence of feeder cells in DMEM culture medium (Gibco by Life Technologies) supplemented with LIF (leukemia inhibitory factor) on gelatin-coated plates. The correct sentence now reads: “We thank Drs T. Van de Water (University of Miami) for his critical reading, B. Binetruy (University Aix Marseille) for the ES CGR8 cells, M. Narita (Cambridge Institute) for the HMGA2 antibody and A. Dos Santos (UMR 7260, University Aix Marseille) for cell culture.” * The authors have indicated that in addition to data within the paper and its Supporting Information files additional data are accessible at the gene expression Omnibus (GEO) repository https://www.ncbi.nlm.nih.gov/geo through the provisional accession series number GSE32963.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
: Presbycusis, an age-related hearing impairment (ARHI) disease, is the most common cause for HI in adults worldwide. One of the best candidate genes for ARHI susceptibility is Cadherin 23 (
) which ...encodes stereocilia tip-links of the inner ear sensory hair cell. Although alterations in the methylation status of CpG dinucleotides across various genes were reported to be associated with HI, methylation changes in
gene have not been reported previously.
: This study aimed at investigating whether DNA methylation level of
gene at intragenic CpG island overlapping an exonic-intronic region at position chr10:73565570-73565827 (GRCh37/hg19) could be risk factor associated with ARHI.
: We screened for methylation changes in this particular position for
gene in 50 blood samples of elderly women affected with presbycusis and healthy control cohort. Methylation of CpG sites were assessed using Quantitative methylation-specific PCR (qMSP) following sodium bisulfite DNA conversion chemistry. Methylation levels were normalized against
reference gene.
: DNA methylation analysis for the common CpG islands in
gene revealed 3.27-folds significant increase (
< 0.0001) in methylation profile for ARHI women as compared to healthy controls with an elevated risk odds ratio (OR) of 2.219 95% CI 1.071-4.597.
: Our study is the first of its kind to prove that higher CpG site methylation levels in
gene are likely to be associated with ARHI.
Hmga2 protein belongs to the non-histone chromosomal high-mobility group (HMG) protein family. HMG proteins have been shown to function as architectural transcription regulators, facilitating ...enhanceosome formation on a variety of mammalian promoters. Hmga2 are expressed at high levels in embryonic and transformed cells. Terminally differentiated cells, however, have been reported to express only minimal, if any, Hmga2. Our previous affymetrix array data showed that Hmga2 is expressed in the developing and adult mammalian cochleas. However, the spatio-temporal expression pattern of Hmga2 in the murine cochlea remained unknown. In this study, we report the expression of Hmga2 in developing and adult cochleas using immunohistochemistry and quantitative real time PCR analysis. Immunolabeling of Hmga2 in the embryonic, postnatal, and mature cochleas showed broad Hmga2 expression in embryonic cochlea (E14.5) at the level of the developing organ of Corti in differentiating hair cells, supporting cells, in addition to immature cells in the GER and LER areas. By postnatal stage (P0-P3), Hmga2 is predominantly expressed in the hair and supporting cells, in addition to cells in the LER area. By P12, Hmga2 immunolabeling is confined to the hair cells and supporting cells. In the adult ear, Hmga2 expression is maintained in the hair and supporting cell subtypes (i.e. Deiters' cells, Hensen cells, pillar cells, inner phalangeal and border cells) in the cochlear epithelium. Using quantitative real time PCR, we found a decrease in transcript level for Hmga2 comparable to other known inner ear developmental genes (Sox2, Atoh1, Jagged1 and Hes5) in the cochlear epithelium of the adult relative to postnatal ears. These data provide for the first time the tissue-specific expression and transcription level of Hmga2 during inner ear development and suggest its potential dual role in early differentiation and maintenance of both hair and supporting cell phenotypes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal ...cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Loss of hair cells in the mammalian cochlea leads to permanent sensori-neural hearing loss. Hair cells degenerate and their places are taken by phalangeal scars formed by non-sensory supporting ...cells. Current data indicate that early postnatal post-mitotic supporting cells can proliferate and differentiate into hair cell-like cells in culture.
In this study, we used GFAP and nestin promoter-GFP transgenic mice in combination with other stem cell markers to characterize supporting cell subtypes in the postnatal day-3 (P3) and adult organs of Corti with potential stem/progenitor cell phenotype.
In P3 organ of Corti, we show GFAP-GFP signal in all the supporting cell subtypes while the nestin-GFP was restricted to the supporting cells in the inner hair cell area. At this stage, GFAP and selected stem/progenitor markers displayed overlapping expression pattern in the supporting cell population.
In the adult, GFAP expression is down-regulated from the supporting cells in the outer hair cell area and nestin expression is down-regulated in the supporting cells of the inner hair cell area. Sox2 and Jagged1 expression is maintained in the mature supporting cells, while Abcg2 was down-regulated in these cells. In contrast, GFAP and Abcg2 expression was up-regulated in the inner sulcus limbal cells outside the mature organ of Corti’s area. Using quantitative reverse transcription-PCR, we found a decrease in transcripts for Jagged1 and Sox2 in adult cochleae. Our findings suggest that the loss of regenerative capacity of the adult organ of Corti is related to down-regulation of stem/progenitor key-markers from the mature supporting cells.
Context: Presbycusis, an age-related hearing impairment (ARHI), represents the most common sensory disability in adults. Today, the molecular mechanisms underlying presbycusis remain unclear. This is ...in particular due to the fact that ARHI is a multifactorial complex disorder resulting from several genomic factors interacting with lifelong cumulative effects of: disease, diet, and environment.
Objective: Identification of novel biomarkers for presbycusis.
Materials and methods: We selectively ascertained 18 elderly unrelated women lacking environmental and metabolic risk factors. Subsequently, we screened for methylation map changes in blood samples of women with presbycusis as compared to controls, using reduced representation bisulfite sequencing. We focused on hypermethylated cytosine bases located in gene promoters and the first two exons. To elucidate the related gene expression changes, we performed transcriptomic study using gene expression microarray.
Results: Twenty-seven genes, known to be expressed in adult human cochlea, were found in the blood cells to be differentially hypermethylated with significant (p < 0.01) methylation differences (>30%) and down-expressed with fold change >1.2 (FDR <0.05). Functional annotation and qRT-PCR further identified P2RX2, KCNQ5, ERBB3 and SOCS3 to be associated with the progression of ARHI.
Discussion and conclusion: Down-expressed genes associated with DNA hypermethylation could be used as biomarkers for understanding complex pathogenic mechanisms underlying presbycusis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries ...where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. Addressing the extreme genetic heterogeneity of HI using classic molecular methods would be expensive and time-consuming. We designed a cost-effective North African Deafness chip for rapid and simultaneous analysis of 58 mutations using multiplex PCR coupled with dual-color arrayed primer extension. These mutations are found in North African HI patients and are distributed over 31 exons and five introns in 21 distinct genes. Assay specificity was initially optimized using 103 archived DNA samples of known genotypes. Blind validation of HI-unrelated patients revealed mutant alleles in 13 samples, and these mutations were confirmed by Sanger sequencing. The North African Deafness chip allows for simultaneous genotyping of eight different samples, at a minimal cost and in a single day, and is therefore amenable to large-scale molecular screening of HI in North Africa.
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