DHI Increases the Proliferation and Migration of Schwann Cells Through the PI3K/AKT Pathway and the Expression of CXCL12 and GDNF to Promote Facial Nerve Function Repair.

The facial nerve is one of the vulnerable nerves in otolaryngology. Repair and recovery of facial nerve injury have a high priority in clinical practice. The proliferation and migration of Schwann cells are considered of great significance in the process of nerve injury repair. Danhong injection (DHI), as a common drug for cardiovascular and cerebrovascular diseases, has been fully certified in neuroprotection research, but its role in facial nerve injury is still not clear. Our study found that DHI can promote the proliferation and migration of RSC96 cells, a Schwann cell line, and this effect is related to the activation of the PI3K/AKT pathway. LY294002, an inhibitor of PI3K, inhibits the proliferation and migration of RSC96 cells. Further studies have found that DHI can also promote the expression of CXCL12 and GDNF at gene and protein levels, and CXCL12 is, while GDNF is not, PI3K/AKT pathway-dependent. Animal experiments also confirmed that DHI could promote CXCL12 and GDNF expression and promote facial nerve function recovery and myelin regeneration. In conclusion, our in vitro and in vivo experiments demonstrated that DHI could promote the proliferation and migration of Schwann cells through the PI3K/AKT pathway and increase the expression of CXCL12 and GDNF to promote facial nerve function repair.

Distinct Expression Patterns of Apoptosis and Autophagy-Associated Proteins and Genes during Postnatal Development of Spiral Ganglion Neurons in Rat.

Autophagy and apoptosis have a complex interplay in the early embryo development. The development of spiral ganglion neurons (SGNs) in addition to Corti's organ in the mammalian cochlea remains crucial in the first two-week postnatal period. To investigate the roles of apoptosis and autophagy in the development of SGNs, light microscopy was used to observe the morphological changes of SGNs. The number of SGNs was decreased from P1 to P7 and plateaued from P10 to P14. Immunohistochemistry results revealed positive expression of cleaved-caspase3, bcl-2, microtubule-associated protein light chain 3-II (LC3-II), Beclin1, and sequestosome 1 (SQSTM1/P62) in SGNs. The apoptotic bodies and autophagosomes and autolysosomes were also identified by transmission electron microscopy at P1 and P7. Real-time PCR and western blotting results revealed that the apoptotic activity peaked at P7 and the autophagy activity was gradually upregulated along with the development. Taken together, our results for the first time showed that autophagy and apoptosis in SGNs play distinct roles during specific developmental phases in a time-dependent manner.

A dominant variant in DMXL2 is linked to nonsyndromic hearing loss.

PURPOSE: To explore the genetic etiology of deafness in a dominant family with late-onset, progressive, nonsyndromic hearing loss. METHODS: Genome-wide linkage analysis was performed for 21 family members. Candidate pathogenic variants were identified by whole-exome sequencing of selected family members and confirmed by Sanger sequencing of all family members. Cochlear expression of Dmxl2 was investigated by reverse-transcription polymerase chain reaction (RT-PCR) and immunostaining of the organ of Corti from mice. RESULTS: The causative gene was mapped to a 9.68-Mb candidate region on chromosome 15q21.2 (maximum logarithm of the odds score = 4.03) that contained no previously described deafness genes. Whole-exome sequencing identified heterozygous c.7250G>A (p.Arg2417His) in DMXL2 as the only candidate pathogenic variant segregating the hearing loss. In mouse cochlea, expression of DMXL2 was restricted to the hair cells and the spiral ganglion neurons. CONCLUSION: Our data indicated that the p.Arg2417His variant in DMXL2 is associated with dominant, nonsyndromic hearing loss and suggested an important role of DMXL2 in inner ear function.Genet Med advance online publication 22 September 2016.

A nonsense mutation in DHTKD1 causes Charcot-Marie-Tooth disease type 2 in a large Chinese pedigree.

Charcot-Marie-Tooth (CMT) disease represents a clinically and genetically heterogeneous group of inherited neuropathies. Here, we report a five-generation family of eight affected individuals with CMT disease type 2, CMT2. Genome-wide linkage analysis showed that the disease phenotype is closely linked to chromosomal region 10p13-14, which spans 5.41 Mb between D10S585 and D10S1477. DNA-sequencing analysis revealed a nonsense mutation, c.1455T>G (p.Tyr485(∗)), in exon 8 of dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1) in all eight affected individuals, but not in other unaffected individuals in this family or in 250 unrelated normal persons. DHTKD1 mRNA expression levels in peripheral blood of affected persons were observed to be half of those in unaffected individuals. In vitro studies have shown that, compared to wild-type mRNA and DHTKD1, mutant mRNA and truncated DHTKD1 are significantly decreased by rapid mRNA decay in transfected cells. Inhibition of nonsense-mediated mRNA decay by UPF1 silencing effectively rescued the decreased levels of mutant mRNA and protein. More importantly, DHTKD1 silencing was found to lead to impaired energy production, evidenced by decreased ATP, total NAD(+) and NADH, and NADH levels. In conclusion, our data demonstrate that the heterozygous nonsense mutation in DHTKD1 is one of CMT2-causative genetic alterations, implicating an important role for DHTKD1 in mitochondrial energy production and neurological development.

A Novel Missense Mutation of NOG Interferes With the Dimerization of NOG and Causes Proximal Symphalangism Syndrome in a Chinese Family.

OBJECTIVES: NOG is an antagonist to bone morphogenetic proteins and plays an important role in proper bone and joint development. Dominant mutations in NOG may lead to a series of symphalangism spectrum disorders. In this study, we aimed to identify the genetic cause and the pathogenic mechanism of an autosomal dominant disorder with cosegregating proximal symphalangism and conductive hearing impairment in a Chinese family. METHODS: Mutation screening of NOG was performed in the affected family members by polymerase chain reaction (PCR) amplification and direct sequencing. Western blotting analysis of NOG was performed in the leukocyte samples of the family members. RESULTS: A novel p.W150C heterozygous mutation in NOG was identified cosegregating with the proximal symphalangism disorder in the family. Western blotting analysis showed that the p.W150C mutation interferes with the dimerization of the mutant NOG. CONCLUSIONS: Our results agreed with previously published results of in vitro studies and suggested that impaired dimerization of mutant NOG is an important pathogenic mechanism for the NOG-related symphalangism spectrum disorder.

Mutation spectrum and differential gene expression in cystic and solid vestibular schwannoma.

PURPOSE: We sought to characterize the mutation spectrum of NF2 and the differential gene expression in cystic and solid vestibular schwannomas. METHODS: We collected tumor tissue and blood samples of 31 cystic vestibular schwannomas and 114 solid vestibular schwannomas. Mutation screening of NF2 was performed in both tumor and blood DNA samples of all patients. cDNA microarray was used to analyze the differential gene expression between 11 cystic vestibular schwannomas and 6 solid vestibular schwannomas. Expression levels of top candidate genes were verified by quantitative reverse transcription PCR. RESULTS: NF2 mutations were identified in 34.5% of sporadic vestibular schwannomas, with all mutations being exclusively somatic. No significant difference was found between the mutation detection rates of cystic vestibular schwannoma (35.5%) and solid vestibular schwannoma (34.2%). cDNA microarray analysis detected a total of 46 differentially expressed genes between the cystic vestibular schwannoma and solid vestibular schwannoma samples. The significantly decreased expression of four top candidate genes, C1orf130, CNTF, COL4A3, and COL4A4, was verified by quantitative reverse transcription PCR. CONCLUSION: NF2 mutations are not directly involved in the cystic formation of vestibular schwannoma. In addition, the differential gene expression of cystic vestibular schwannoma reported in our study may provide useful insights into the molecular mechanism underlying this process.

Molecular etiology of non-dominant, non-syndromic, mild-to-moderate childhood hearing impairment in Chinese Hans.

Childhood hearing impairment (HI) is genetically heterogeneous. Compared with the severe-to-profound HI, the molecular etiology of mild-to-moderate HI in children has been less well characterized, especially for those not inherited in the dominant mode. In this study, we recruited 114 probands with non-dominant, non-syndromic, mild-to-moderate childhood HI. Sequencing of GJB2, SLC26A4, and MTRNR1 identified causative mutations in 30.7% (35/114), 4.4% (5/114), and 4.4% (5/114) of subjects, respectively. A majority (62.9%) of bi-allelic GJB2 mutations have non-truncating mutations in at least one allele. In 10 multiplex probands with no GJB2, SLC26A4, and MTRNR1 mutations identified, targeted next-generation sequencing (NGS) of 79 known deafness genes did not identify any additional causes. Our data showed that the molecular etiology of mild-to-moderate childhood HI is considerably different from what reported for severe-to-profound HI and far from complete for those inherited in non-dominant modes.

Potential biomarkers in peripheral blood mononuclear cells of patients with sporadic Ménière's disease based on proteomics.

BACKGROUND: Ménière's disease (MD) mainly refers to the endolymphatic hydrops in membranous labyrinth of the inner ear. Application of the mass spectrometry-based proteomics techniques has not been applied in the field of MD. OBJECTIVES: To search for potential differential proteins to identify the disease biomarkers and reveal disease bioinformatics-related mechanisms through applying protein technology to analyze the expression changes of peripheral blood mononuclear cells (PBMCs) in sporadic MD patients. MATERIAL AND METHODS: 15 MD patients and 15 healthy individuals were enrolled. PBMCs from them were extracted, and their protein expression was identified and compared by LC-MS/MS and spectra analysis. RESULTS: There was significant difference in protein expression between MD patients and the control group. GO and KEGG analysis showed that endocytosis was involved in MD patients. Western blot results of CHMP1A and MMP9 protein showed that the expression of CHMP1A was higher in the MD group than that in the control group, while MMP9 was down-regulated. Immunohistochemistry confirmed that CHMP1A and MMP9 were expressed in the endolymphatic sacs of MD patients and in the inner ear of adult mice. CONCLUSIONS AND SIGNIFICANCE: Endocytosis may be involved in the pathogenesis of sporadic MD, furthermore CHMP1A, VPS4A, FCN3 and MMP9 could be considered as potential biomarkers.

背景：梅尼埃病（MD）主要是指内耳耳膜迷路中的内淋巴积液。 基于质谱的蛋白质组学技术尚未在MD治疗上得到应用。目的：通过应用蛋白质技术分析散发性MD患者外周血单核细胞（PBMcs）的表达变化, 寻找潜在的分异性蛋白来识别该病生物标志物, 揭示该病生物信息学相关机制。材料和方法：纳入 15 名 MD 患者和 15 名健康人。 提取PBMcs, 通过lc-Ms/Ms和光谱分析鉴定并比较其蛋白表达。结果：MD患者与对照组的蛋白表达存在显著差异。 GO和KeGG分析表明, MD患者具有内吞作用。 chMP1a和MMP9蛋白的蛋白质印迹结果显示, MD组chMP1a的表达高于对照组, 而MMP9表达下降。 免疫组织化学证实, chMP1a和MMP9在MD患者的内淋巴囊和成年小鼠的内耳中有表达。结论及意义：内吞作用可能参与散发性MD的发病机制, chMP1a、VPs4a、FcN3和MMP9可作为潜在的生物标志物。.

Kölliker's organ-supporting cells and cochlear auditory development.

The Kölliker's organ is a transient cellular cluster structure in the development of the mammalian cochlea. It gradually degenerates from embryonic columnar cells to cuboidal cells in the internal sulcus at postnatal day 12 (P12)-P14, with the cochlea maturing when the degeneration of supporting cells in the Kölliker's organ is complete, which is distinct from humans because it disappears at birth already. The supporting cells in the Kölliker's organ play a key role during this critical period of auditory development. Spontaneous release of ATP induces an increase in intracellular Ca2+ levels in inner hair cells in a paracrine form via intercellular gap junction protein hemichannels. The Ca2+ further induces the release of the neurotransmitter glutamate from the synaptic vesicles of the inner hair cells, which subsequently excite afferent nerve fibers. In this way, the supporting cells in the Kölliker's organ transmit temporal and spatial information relevant to cochlear development to the hair cells, promoting fine-tuned connections at the synapses in the auditory pathway, thus facilitating cochlear maturation and auditory acquisition. The Kölliker's organ plays a crucial role in such a scenario. In this article, we review the morphological changes, biological functions, degeneration, possible trans-differentiation of cochlear hair cells, and potential molecular mechanisms of supporting cells in the Kölliker's organ during the auditory development in mammals, as well as future research perspectives.

A liquid chromatography-mass spectroscopy-based untargeted metabolomic study of the rat cochlear nucleus at various stages of maturity.

The cochlear nucleus receives numerous inputs from auditory and nonauditory systems. This extensive innervation of the cochlear nucleus is involved in sound source localization and the integration of auditory signals with other sensory modalities. The dorsal cochlear nucleus may also have an important role in tinnitus. Although its gross anatomy and function have been extensively studied, the metabolome of the cochlear nucleus remains poorly understood, particularly at different stages of auditory maturity. Here, we present a protocol for untargeted metabolomics analysis of the rat cochlear nucleus, then discuss differences in the metabolome of the rat cochlear nucleus between postnatal day (PD) 14 (hearing onset) and PD60 (hearing maturation). Cochlear nucleus samples collected from rats at PD14 or PD60 were analyzed by liquid chromatography-tandem mass spectrometry (LCMS). In total, 344 metabolites were identified. Principal component analysis and orthogonal partial least-square discriminant analysis showed that the metabolic profiles at these two stages had distinct distribution patterns. Moreover, 91 significantly differential metabolites (62 upregulated and 29 downregulated) were identified at PD60 vs. PD14. N-acetylaspartylglutamic acid (NAAG), γ-aminobutyric acid (GABA), taurine, adenosine monophosphate (AMP), and choline were significantly upregulated at PD60. Pathway enrichment analysis suggested that alanine, aspartate, and glutamate metabolism; glycine, serine, and threonine metabolism; the mammalian target of rapamycin (mTOR) signaling pathway; and the AMP-activated protein kinase (AMPK) signaling pathway may be involved in key developmental events during maturation of the cochlear nucleus. Taken together, the metabolic profiles identified in this study could lead to the identification and understanding of specific key biomarkers and metabolic pathways involved in the maturation of hearing. Moreover, LC-MS-based metabolomics provides an alternative approach for the characterization of auditory maturation and auditory diseases.

Molecular etiology study of hearing loss in 13 Chinese Han families.

Hearing loss affecting about 2/1000 newborns is the most common congenital disease. Genetic defects caused approximately 70% of patients who have non-syndromic hearing loss. We recruited 13 Chinese Han deafness families who tested negative for GJB2, SLC26A4, and mitochondrial 12S rRNA. The probands of each family were performed whole-exome sequencing (WES) or targeted next-generation sequencing (NGS) for known deafness genes to study for pathogenic causes. We found four novel mutations of CDH23, one novel mutation of MYO15A, one novel mutation of TMC1, one novel mutation of PAX3, and one novel mutation of ADGRV1, one novel CNV of ADGRV1, and one novel CNV of STRC. Hearing loss is a highly hereditary and heterogeneous disease. The results in the limited samples of this study show that Usher and Waardenburg syndrome-related genes account for a major proportion are strongly associated with Chinese Han hearing loss patients negative for GJB2, SLC26A4, and mitochondrial 12S rRNA, followed by STRC resulting in mild to moderate deafness.

Genetic etiological analysis of auditory neuropathy spectrum disorder by next-generation sequencing.

Objective: Auditory neuropathy spectrum disease (ANSD) is caused by both environmental and genetic causes and is defined by a failure in peripheral auditory neural transmission but normal outer hair cells function. To date, 13 genes identified as potentially causing ANSD have been documented. To study the etiology of ANSD, we collected 9 probands with ANSD diagnosed in the clinic and performed targeted next-generation sequencing. Methods: Nine probands have been identified as ANSD based on the results of the ABR tests and DPOAE/CMs. Genomic DNA extracted from their peripheral blood was examined by next-generation sequencing (NGS) for a gene panel to identify any potential causal variations. For candidate pathogenic genes, we performed co-segregation among all family members of the pedigrees. Subsequently, using a mini-gene assay, we examined the function of a novel splice site mutant of OTOF. Results: We analyzed nine cases of patients with ANSD with normal CMs/DPOAE and abnormal ABR, discovered three novel mutants of the OTOF gene that are known to cause ANSD, and six cases of other gene mutations including TBC1D24, LARS2, TIMM8A, MITF, and WFS1. Conclusion: Our results extend the mutation spectrum of the OTOF gene and indicate that the genetic etiology of ANSD may be related to gene mutations of TBC1D24, LARS2, TIMM8A, MITF, and WFS1.

Pathological mechanisms of connexin26-related hearing loss: Potassium recycling, ATP-calcium signaling, or energy supply?

Hereditary deafness is one of the most common human birth defects. GJB2 gene mutation is the most genetic etiology. Gap junction protein 26 (connexin26, Cx26) encoded by the GJB2 gene, which is responsible for intercellular substance transfer and signal communication, plays a critical role in hearing acquisition and maintenance. The auditory character of different Connexin26 transgenic mice models can be classified into two types: profound congenital deafness and late-onset progressive hearing loss. Recent studies demonstrated that there are pathological changes including endocochlear potential reduction, active cochlear amplification impairment, cochlear developmental disorders, and so on, in connexin26 deficiency mice. Here, this review summarizes three main hypotheses to explain pathological mechanisms of connexin26-related hearing loss: potassium recycling disruption, adenosine-triphosphate-calcium signaling propagation disruption, and energy supply dysfunction. Elucidating pathological mechanisms underlying connexin26-related hearing loss can help develop new protective and therapeutic strategies for this common deafness. It is worthy of further study on the detailed cellular and molecular upstream mechanisms to modify connexin (channel) function.

Failure Of Hearing Acquisition in Mice With Reduced Expression of Connexin 26 Correlates With the Abnormal Phasing of Apoptosis Relative to Autophagy and Defective ATP-Dependent Ca2+ Signaling in Kölliker's Organ.

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2 loxP/loxP; ROSA26 CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker's organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker's organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function.

YTHDF1 Protects Auditory Hair Cells from Cisplatin-Induced Damage by Activating Autophagy via the Promotion of ATG14 Translation.

N6-methyladenosine (m6A) has been recognized as a common type of post-transcriptional epigenetic modification. m6A modification and YTHDF1, one of its reader proteins, have been documented to play a pivotal role in numerous human diseases via regulating mRNA splicing, translation, stability, and subcellular localization. The chemotherapeutic drug cisplatin (CDP) can damage sensory hair cells (HCs) and result in permanent sensorineural hearing loss. However, whether YTHDF1-mediated modification of mRNA is potentially involved in CDP-induced injury in sensory hair cells was not fully clarified. This study investigated the potential mechanisms for the modification of YTHDF1 in CDP-induced damage in HCs. Here, we discovered that YTHDF1's expression level statistically increased significantly after treating with CDP. Apoptosis and cell death of HCs induced by CDP were exacerbated after the knockdown of YTHDF1, while overexpression of YTHDF1 in HCs alleviated their injury induced by CDP. Moreover, YTHDF1 expression correlated with cisplatin-induced autophagy with statistical significance in HCs; namely, YTHDF1's overexpression enhanced the activation of autophagy, while its deficiency suppressed autophagy and, at the same time, increased the loss of HCs after CDP damage. WB analysis and qRT-PCR results of autophagy-related genes indicated that YTHDF1 promoted the translation of autophagy-related genes ATG14, thus boosting autophagy. Therefore, CDP-induced YTHDF1 expression protected HCs against CDP-induced apoptosis by upregulating the translation of autophagy-related genes ATG14, along with enhancing autophagy. Based on these findings, it can be inferred that YTHDF1 is potentially a target for ameliorating drug-induced HCs damage through m6A modification.

Pseudo-Temporal Analysis of Single-Cell RNA Sequencing Reveals Trans-Differentiation Potential of Greater Epithelial Ridge Cells Into Hair Cells During Postnatal Development of Cochlea in Rats.

The hair cells of the cochlea play a decisive role in the process of hearing damage and recovery, yet knowledge of their regeneration process is still limited. Greater epithelial ridge (GER) cells, a type of cell present during cochlear development that has the characteristics of a precursor sensory cell, disappear at the time of maturation of hearing development. Its development and evolution remain mysterious for many years. Here, we performed single-cell RNA sequencing to profile the gene expression landscapes of rats' cochlear basal membrane from P1, P7, and P14 and identified eight major subtypes of GER cells. Furthermore, single-cell trajectory analysis for GER cells and hair cells indicated that among the different subtypes of GER, four subtypes had transient cell proliferation after birth and could transdifferentiate into inner and outer hair cells, and two of them mainly transdifferentiated into inner hair cells. The other two subtypes eventually transdifferentiate into outer hair cells. Our study lays the groundwork for elucidating the mechanisms of the key regulatory genes and signaling pathways in the trans-differentiation of GER cell subtypes into hair cells and provides potential clues to understand hair cell regeneration.

Single-Cell RNA Sequencing Analysis Reveals Greater Epithelial Ridge Cells Degeneration During Postnatal Development of Cochlea in Rats.

Greater epithelial ridge cells, a transient neonatal cell group in the cochlear duct, which plays a crucial role in the functional maturation of hair cell, structural development of tectorial membrane, and refinement of audio localization before hearing. Greater epithelial ridge cells are methodologically homogeneous, while whether different cell subtypes are existence in this intriguing region and the degeneration mechanism during postnatal cochlear development are poorly understood. In the present study, single-cell RNA sequencing was performed on the cochlear duct of postnatal rats at day 1 (P1) and day 7 (P7) to identify subsets of greater epithelial ridge cell and progression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to examine genes enriched biological processes in these clusters. We identified a total of 26 clusters at P1 and P7 rats and found that the cell number of five cell clusters decreased significantly, while four clusters had similar gene expression patterns and biological properties. The genes of these four cell populations were mainly enriched in Ribosome and P13K-Akt signal pathway. Among them, Rps16, Rpsa, Col4a2, Col6a2, Ctsk, and Jun are particularly interesting as their expression might contribute to the greater epithelial ridge cells degeneration. In conclusion, our study provides an important reference resource of greater epithelial ridge cells landscape and mechanism insights for further understanding greater epithelial ridge cells degeneration during postnatal rat cochlear development.

Identification of MYO6 copy number variation associated with cochlear aplasia by targeted sequencing.

Copy number variation is an extensively studied cause of hereditary diseases. However, its role in hereditary sensorineural deafness has been rarely reported. Using targeted sequencing, SNP array and qPCR, we found a novel 622.2 kb duplication of 6q14.1 in a patient with congenital sensorineural hearing loss and cochlear aplasia. The duplication included MYO6 and IMPG1 genes. FISH study confirmed that this duplication was inherited from the patient's mosaic mother.

Self-Assembled ZnO Nanoparticle Capsules for Carrying and Delivering Isotretinoin to Cancer Cells.

ZnO@polymer core-shell nanoparticles are assembled into novel capsule shells with diameters of about 100 nm to load isotretinoin (ISO) with a capacity as high as 34.6 wt %. Although ISO, a widely used drug for acne treatment, by itself is not suitable for treating cancer because of its hydrophobicity, our ZnO-ISO composite showed much stronger anticancer activity. The improved cytotoxicity is ascribed to the synergistic effects of the ZnO@polymer and ISO, where the ZnO@polymer helps in the accumulation of ISO in cancer cells on the one hand, and on the other hand, ISO is released completely through ZnO decomposition under acidic conditions of cancer cells. Such a pH-triggered drug-delivery system exhibits a much improved killing of cancer cells in vitro in comparison with the benchmarks, Nintedanib and Crizotinib, two commercial drugs clinically applied against lung cancer.

Molecular etiology and genotype-phenotype correlation of Chinese Han deaf patients with type I and type II Waardenburg Syndrome.

Waardenburg syndrome (WS) characterized by sensorineural hearing loss and pigmentary abnormalities is genetically heterogeneous and phenotypically variable. This study investigated the molecular etiology and genotype-phenotype correlation of WS in 36 Chinese Han deaf probands and 16 additional family members that were clinically diagnosed with WS type I (WS1, n = 8) and type II (WS2, n = 42). Mutation screening of six WS-associated genes detected PAX3 mutations in 6 (86%) of the 7 WS1 probands. Among the 29 WS2 probands, 13 (45%) and 10 (34%) were identified with SOX10 and MITF mutations, respectively. Nineteen of the 26 detected mutations were novel. In WS2 probands whose parental DNA samples were available, de novo mutations were frequently seen for SOX10 mutations (7/8) but not for MITF mutations (0/5, P = 0.005). Excessive freckle, a common feature of WS2 in Chinese Hans, was frequent in WS2 probands with MITF mutations (7/10) but not in those with SOX10 mutations (0/13, P = 4.9 × 10-4). Our results showed that mutations in SOX10 and MITF are two major causes for deafness associated with WS2. These two subtypes of WS2 can be distinguished by the high de novo rate of the SOX10 mutations and the excessive freckle phenotype exclusively associated with the MITF mutations.