PAX3 mutation suppress otic progenitors proliferation and induce apoptosis by inhibiting WNT1/ß-catenin signaling pathway in WS1 patient iPSC-derived inner ear organoids.

Waardenburg syndrome type 1 (WS1) is a hereditary disease mainly characterized by sensorineural hearing loss, dystopia canthorum, and pigmentary defects. To elucidate molecular mechanisms underlying PAX3-associated hearing loss, we developed inner ear organoids model using induced pluripotent stem cells (iPSCs) derived from WS1 patient and healthy individual. Our results revealed a significant reduction in the size of inner ear organoids, accompanied by an increased level of apoptosis in organoids derived from WS1 patient-iPSCs carrying PAX3 c.214A > G. Transcriptome profiling analysis by RNA-seq indicated that inner ear organoids from WS1 patients were associated with suppression of inner ear development and WNT signaling pathway. Furthermore, the upregulation of the WNT1/ß-catenin pathway which was achieved through the correction of PAX3 isogenic mutant iPSCs using CRISPR/Cas9, contributed to an increased size of inner ear organoids and a reduction in apoptosis. Together, our results provide insight into the underlying mechanisms of hearing loss in WS.

A novel SOX10 mutation causing Waardenburg syndrome type 2 by expressing a truncated and dysfunctional protein in a Chinese child.

OBJECTIVES: This study aimed to identify the causative variants in a patient with Waardenburg syndrome (WS) type 2 using whole exome sequencing (WES). METHODS: The clinical features of the patient were collected. WES was performed on the patient and his parents to screen causative genetic variants and Sanger sequencing was performed to validate the candidate mutation. The AlphaFold2 software was used to predict the changes in the 3D structure of the mutant protein. Western blotting and immunocytochemistry were used to determine the SOX10 mutant in vitro. RESULTS: A de novo variant of SOX10 gene, NM_006941.4: c.707_714del (p. H236Pfs*42), was identified, and it was predicted to disrupt the wild-type DIM/HMG conformation in SOX10. In-vitro analysis showed an increased level of expression of the mutant compared to the wild-type. CONCLUSIONS: Our findings helped to understand the genotype-phenotype association in WS2 cases with SOX10 mutations.

Identification of two variants in PAX3 and FBN1 in a Chinese family with Waardenburg and Marfan syndrome via whole exome sequencing.

Both Warrensburg (WS) and Marfan syndrome (MFS) can impair the vision. Here, we recruited a Chinese family consisting of two WS affected individuals (II:1 and III:3) and five MFS affected individuals( I:1, II:2, III:1, III:2, and III:5) as well as one suspected MFS individual (II:4). Using whole exome sequencing (WES) and subsequent PCR-Sanger sequencing, we identified one novel heterozygous variant NM_000438 (PAX3) c.208 T > C, (p.Cys70Arg) from individuals with WS and one previous reported variant NM_000138 (FBN1) c.2740 T > A, (p.Cys914Ser) from individuals with MFS and co-segregated with the diseases. Real-time PCR and Western blot assay showed that, compared to their wild-type, both mRNAs and proteins of  PAX3 and FBN1 mutants reduced in HKE293T cells. Together, our study identified two disease-causing variants in a same Chinese family with WS and MFS, and confirmed their damaged effects on their genes' expression. Therefore, those findings expand the mutation spectrum of PAX3 and provide a new perspective for the potential therapy.

A 22q13.1 duplication in mosaicism including SOX10.

Waardenburg syndrome (WS) is characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Among the four types, WS Type 2 (WS2) is the only one without a remarkable distinguishing feature. Here, we report a patient initially diagnosed with WS2 who exhibits a 446 kb mosaic duplication in chromosome 22q13.1, encompassing SOX10, and detected using whole genome sequencing in a trio. The patient, a 46,XY boy, presents with profound bilateral sensorineural hearing loss, right heterochromia iridium, left bright blue iris, and skin-depigmented areas in the abdomen and limbs. Vestibular and imaging tests are normal, without inner ear or olfactory bulb malformations. Bilateral cochlear implantation did not prevent language and speech delays. Moderate congenital chronic constipation and neurodevelopmental difficulties were also present. Given the few genes included in this duplicated region (only one OMIM gene with dominant inheritance), this report provides further delineation of the phenotype related to duplications encompassing the entire SOX10 gene.

White coat color of a Black Angus calf attributed to an occurrence of the delR217 variant of MITF.

A white calf, with minimal pigmented markings, was born to two registered Black Angus parents. Given the possibility of an unknown recessive or de novo dominant mutation, whole-genome sequencing was conducted on the trio of individuals. A 3-bp in-frame deletion in MITF was identified; this mutation was unique to the calf but identical to the delR217 variant reported in both humans and murine models of Waardenburg syndrome type 2A and Tietz syndrome. Given the coat color phenotype and identity of the mutation, our data support that this calf represents the first instance of this recurring MITF mutation in cattle.

Biallelic variants in PAX3 cause Klein syndrome.

Waardenburg syndrome is a group of genetic conditions that can cause hearing loss and pigmentation deficiency of the hair, skin, and eyes. Klein-Waardenburg syndrome (Waardenburg syndrome type 3) represents a distinct presentation of Waardenburg syndrome type 1 and includes musculoskeletal abnormalities in addition to dystopia canthorum hearing loss and pigmentary changes. Heterozygous or homozygous variants in the PAX3 gene cause Klein-Waardenburg syndrome. Here we report on a new severely affected child, with a homozygous PAX3 variant (c.251C>T; p.Ser84Phe), review the features of the syndrome, and propose a new classification. The designation of Waardenburg syndrome should be given only to patients with monoallelic pathogenic variants in PAX3 whether or not musculoskeletal abnormalities are present. Patients with biallelic PAX3 variants should be outlined as a distinct group and designated Klein syndrome.

Diagnosis and genetic analysis of a case of Waardenburg syndrome type 2 with hypogonadotropic hypogonadism caused by SOX10 gene deletion.

Hypogonadotropic hypogonadism (HH) is a disease defined by dysfunction of the hypothalamic- pituitary-gonadal hormone axis, leading to low sex hormone levels and impaired fertility. HH with anosmia or hyposmia is known as Kallmann syndrome (KS). Waardenburg syndrome (WS) is a rare autosomal dominant genetic disorder characterized by sensorineural hearing loss and abnormal pigmentation. In this report, we collected the clinical data of a patient with hypogonadotropic hypogonadism and congenital hearing loss of unknown cause. The patient had no obvious secondary sexual characteristics development after puberty, and had a heterozygous deletion (at least 419 kb) in 22q13.1 region (Chr.22:38106433-38525560), which covered the SOX10 gene. The abnormalities were not found in gene sequencing analysis of both the parents and sister of the proband. By summarizing and analyzing the characteristics of this case, we further discussed the molecular biological etiological association between HH and WS type 2. This case also enriches the clinical data of subsequent genetic studies, and provides a reference for the diagnosis and treatment of such diseases.

A novel case of concurrent occurrence of demyelinating-polyneuropathy-causing PMP22 duplication and SOX10 gene mutation producing severe hypertrophic neuropathy.

BACKGROUND: Hereditary motor and sensory neuropathy, also referred to as Charcot-Marie-Tooth disease (CMT), is most often caused by a duplication of the peripheral myelin protein 22 (PMP22) gene. This duplication causes CMT type 1A (CMT1A). CMT1A rarely occurs in combination with other hereditary neuromuscular disorders. However, such rare genetic coincidences produce a severe phenotype and have been reported in terms of "double trouble" overlapping syndrome. Waardenburg syndrome (WS) is the most common form of a hereditary syndromic deafness. It is primarily characterized by pigmentation anomalies and classified into four major phenotypes. A mutation in the SRY sex determining region Y-box 10 (SOX10) gene causes WS type 2 or 4 and peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, WS, and Hirschsprung disease. We describe a 11-year-old boy with extreme hypertrophic neuropathy because of a combination of CMT1A and WS type 2. This is the first published case on the co-occurrence of CMT1A and WS type 2. CASE PRESENTATION: The 11-year-old boy presented with motor developmental delay and a deterioration in unstable walking at 6 years of age. In addition, he had congenital hearing loss and heterochromia iridis. The neurological examination revealed weakness in the distal limbs with pes cavus. He was diagnosed with CMT1A by the fluorescence in situ hybridization method. His paternal pedigree had a history of CMT1A. However, no family member had congenital hearing loss. His clinical manifestation was apparently severe than those of his relatives with CMT1A. In addition, a whole-body magnetic resonance neurography revealed an extreme enlargement of his systemic cranial and spinal nerves. Subsequently, a genetic analysis revealed a heterozygous frameshift mutation c.876delT (p.F292Lfs*19) in the SOX10 gene. He was eventually diagnosed with WS type 2. CONCLUSIONS: We described a patient with a genetically confirmed overlapping diagnoses of CMT1A and WS type 2. The double trouble with the genes created a significant impact on the peripheral nerves system. Severe phenotype in the proband can be attributed to the cumulative effect of mutations in both PMP22 and SOX10 genes, responsible for demyelinating neuropathy.

Four mutations in MITF, SOX10 and PAX3 genes were identified as genetic causes of waardenburg syndrome in four unrelated Iranian patients: case report.

BACKGROUND: Waardenburg syndrome (WS) is a rare genetic disorder. The purpose of this study was to investigate clinical and molecular characteristics of WS in four probands from four different Iranian families. CASE PRESENTATION: The first patient was a 1-year-old symptomatic boy with congenital hearing loss and heterochromia iridis with a blue segment in his left iris. The second case was a 1.5-year-old symptomatic girl who manifested congenital profound hearing loss, brilliant blue eyes, and skin hypopigmentation on the abdominal region at birth time. The third patient was an 8-month-old symptomatic boy with developmental delay, mild atrophy, hypotonia, brilliant blue eyes, skin hypopigmentation on her hand and foot, Hirschsprung disease, and congenital profound hearing loss; the fourth patient was a 4-year-old symptomatic boy who showed dystopia canthorum, broad nasal root, synophrys, skin hypopigmentation on her hand and abdomen, brilliant blue eyes, and congenital profound hearing loss. Whole exome sequencing (WES) was used for each proband to identify the underlying genetic factor. Sanger sequencing was performed for validation of the identified mutations in probands and the available family members. A novel heterozygous frameshift mutation, c.996delT (p.K334Sfs*15), on exon 8 of the MITF gene was identified in the patient of the first family diagnosed with WS2A. Two novel de novo heterozygous mutations including a missense mutation, c.950G > A (p.R317K), on exon 8 of the MITF gene, and a frameshift mutation, c.684delC (p.E229Sfs*57), on the exon 3 of the SOX10 gene were detected in patients of the second and third families with WS2A and PCWH (Peripheral demyelinating neuropathy, Central dysmyelinating leukodystrophy, Waardenburg syndrome, Hirschsprung disease), respectively. A previously reported heterozygous frameshift mutation, c.1024_1040del AGCACGATTCCTTCCAA, (p.S342Pfs*62), on exon 7 of the PAX3 gene was identified in the patient of the fourth family with WS1. CONCLUSIONS: An exact description of the mutations responsible for WS provides useful information to explain the molecular cause of clinical features of WS and contributes to better genetic counseling of WS patients and their families.

Identification of a novel heterozygous mutation in the MITF gene in an Iranian family with Waardenburg syndrome type II using next-generation sequencing.

BACKGROUND: Waardenburg syndrome (WS) is a genetically heterogeneous syndrome with both autosomal recessive and dominant inheritance. WS causes skin and iris pigmentation accumulation and sensorineural hearing loss, in varying degrees. There are four WS types with different characteristics. WS1 and WS2 are the most common and have a dominant inheritance. WS2 is caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. METHODS: An Iranian couple with hearing loss was recruited in the present study. First, they were screened for GJB2 and GJB6 gene mutations, and then whole-exome sequencing 100X was performed along with bioinformatics analysis. RESULTS: A novel pathogenic heterozygous mutation, c.425T>A; p.L142Ter, was detected in the MITF gene's exon 4. Bioinformatics analysis predicted c.425T>A; p.L142Ter as a possible pathogenic variation. It appears that the mutated transcript level declines through nonsense-mediated decay. It probably created a significantly truncated protein and lost conserved and functional domains like basic helix-loop-helix-zipper proteins. Besides, the variant was utterly co-segregated with the disease within the family. CONCLUSIONS: We investigated an Iranian family with congenital hearing loss and identified a novel pathogenic variant c.425T>A; p. L142Ter in the MITF gene related to WS2. This variant is a nonsense mutation, probably leading to a premature stop codon. Our data may be beneficial in upgrading gene mutation databases and identifying WS2 causes.

Identification and functional analysis of a novel missense mutation of PAX3 associated with Waardenburg syndrome type I.

PURPOSE: Waardenburg syndrome type 1 (WS1) is a rare genetic disorder characterized by dystopia canthorum, abnormal iris pigmentation, and congenital hearing loss with variable penetrance.WS1 is caused by mutations in paired box gene 3 (PAX3). The current study aimed to investigate the genetic cause of hearing loss in a four-generation Chinese WS1 family. METHODS: The phenotype of the study family was characterized using clinical evaluation and pedigree analysis. Target region high-throughput sequencing system was designed to screen the all coding exons and flanking intronic sequences of the six WS-associated genes. Sanger sequencing was used to identify the causative nucleotide changes and perform the co-segregating analysis. The expression, subcellular distribution, and transcriptional activity of the mutant PAX3 protein were analysis to reveal the functional consequences of the mutation. RESULTS: Based on diagnostic criteria, the proband of this pedigree classified as WS1. We identified a novel missense mutation (c.117 C > A, p. Asn39Lys) in exon 2 of the PAX3 gene. In vitro, the Asn39Lys PAX3 retained nuclear distribution ability. However, it failed to activate the melanocyte inducing transcription factor (MITF) promoter and impaired the function of WT PAX3. CONCLUSIONS: Our study reports a novel missense PAX3 mutation in a Chinese family and shows haploinsufficiency may be the underlying mechanism for the WS1 phenotype.

A novel frameshift mutation in SOX10 causes Waardenburg syndrome with peripheral demyelinating neuropathy, visual impairment and the absence of Hirschsprung disease.

Waardenburg syndrome (WS) is a group of genetic disorders associated with varying components of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and eyes. There exist four different WS subtypes, each defined by the absence or presence of additional features. One of the genes associated with WS is SOX10, a key transcription factor for the development of neural crest-derived lineages. Here we report a 12-year-old boy with a novel de novo SOX10 frameshift mutation and unique combination of clinical features including primary peripheral demyelinating neuropathy, hearing loss and visual impairment but absence of Hirschsprung disease and the typical pigmentary changes of hair or skin. This expands the spectrum of currently recognized phenotypes associated with WS and illustrates the phenotypic heterogeneity of SOX10-associated WS.

Phenotypic diversity and genetic complexity of PAX3-related Waardenburg syndrome.

Waardenburg syndrome subtypes 1 and 3 are caused by pathogenic variants in PAX3. We investigated 12 individuals from four unrelated families clinically diagnosed with Waardenburg syndrome type 1/3. Novel pathogenic variants identified in PAX3 included single nucleotide variants (c.166C>T, c.829C>T), a 2-base pair deletion (c.366_367delAA) and a multi-exonic deletion. Two novel variants, c.166C>T and c.829C>T and a previously reported variant, c.256A>T in PAX3 were evaluated for their nuclear localization and ability to activate MITF promoter. The coexistence of two subtypes of Waardenburg syndrome with pathogenic variants in PAX3 and EDNRB was seen in one of the affected individuals. Multiple genetic diagnoses of Waardenburg syndrome type 3 and autosomal recessive deafness 1A was identified in an individual. We also review the phenotypic and genomic spectrum of individuals with PAX3-related Waardenburg syndrome reported in the literature.

Expression of unfolded protein response markers in the pheochromocytoma with Waardenburg syndrome: a case report.

BACKGROUND: It is clinically emergent to further understand the pathological mechanism to advance therapeutic strategy for endocrine tumors. A high amount of secretory protein with tumorigenic triggers are thought to induce unfolded protein response in endoplasmic reticulum in endocrine tumors, but its evidence is limited. CASE PRESENTATION: A 40-year-old woman had an approximately 10-year history of intermittent headaches. After the incidental detection of a mass in her right adrenal gland by CT scan, she was admitted to our hospital. She had been diagnosed as type 1 Waardenburg syndrome with the symptoms of dystopia canthorum, blue iris, and left sensorineural hearing loss. Urinary catecholamine levels were markedly elevated. 123I-MIBG scintigraphy showed uptake in the mass in her adrenal gland. After the adrenalectomy, her headaches disappeared and urinary catecholamine levels decreased to normal range within 2 weeks. Genome sequencing revealed germline mutation of c.A175T (p.Ile59Phe) in transcription factor PAX3 gene and somatic novel mutation of c.1893_1898del (p. Asp631_Leu633delinsGlu) in proto-oncogene RET in her pheochromocytoma. RNA expression levels of RET were increased 139 times in her pheochromocytoma compared with her normal adrenal gland. Those of unfolded protein response markers, Bip/GRP78, CHOP, ATF4, and ATF6, were also increased in the pheochromocytoma. CONCLUSION: We report a rare case of pheochromocytoma with type 1 Waardenburg syndrome. This is the first case to show the activation of unfolded protein response in the pheochromocytoma with the novel somatic mutation in RET gene. Our findings may support that unfolded protein response is activated in endocrine tumors, which potentially could be a candidate of therapeutic target.

Genome-wide association studies for iris pigmentation and heterochromia patterns in Large White pigs.

Eye colour genetics have been extensively studied in humans since the rediscovery of Mendel's laws. This trait was first interpreted using simplistic genetic models but soon it was realised that it is more complex. In this study, we analysed eye colour variability in a Large White pig population (n = 897) and report the results of GWASs based on several comparisons including pigs having four main eye colour categories (three with both pigmented eyes of different brown grades: pale, 17.9%; medium, 14.8%; and dark, 54.3%; another one with both eyes completely depigmented, 3.8%) and heterochromia patterns (heterochromia iridis - depigmented iris sectors in pigmented irises, 3.2%; heterochromia iridum - one whole eye iris of depigmented phenotype and the other eye with the iris completely pigmented, 5.9%). Pigs were genotyped with the Illumina PorcineSNP60 BeadChip and GEMMA was used for the association analyses. The results indicated that SLC45A2 (on chromosome 16, SSC16), EDNRB (SSC11) and KITLG (SSC5) affect the different grades of brown pigmentation of the eyes, the bilateral eye depigmentation defect and the heterochromia iridis defect recorded in this white pig population respectively. These genes are involved in several mechanisms affecting pigmentation. Significant associations for the eye depigmented patterns were also identified for SNPs on two SSC4 regions (including two candidate genes: NOTCH2 and PREX2) and on SSC6, SSC8 and SSC14 (including COL17A1 as candidate gene). This study provided useful information to understand eye pigmentation mechanisms, further valuing the pig as animal model to study complex phenotypes in humans.

[Analysis of genetic variation in patients with Waardenburg syndrome type â ¡ by next generation sequencing].

Objective: To detect gene mutation sassociated with deafness in four Waardenburg syndrome (WS) type Ⅱ patients, and to explore the possible mechanism of molecular genetics. Methods: All patients with WS were identified at the genetic and prenatal diagnosis center of the First Affiliated Hospital of Zhengzhou University from August 2015 to December 2018.Clinical materials and peripheral blood were collected from patients and family members. The genes associated with deafness of the patients were tested by next generation sequencing(NGS). And suspected mutations were verified by Sanger sequencing. Results: All patients carried heterozygous mutations in SOX10, they were c.355_356insTCAGGCAGCGC, c.1106_1107insTGGGGCCCCCCACACTA, c.511T>C (p.Y171H), c.91_100del. According to the guidelines for genetic variation of the Amercian College of Medical Genetics and Genomics (ACMG), three frameshift mutations were pathogenic mutations, one missense mutation was likely pathogenic mutation. Conclusion: Application of next generation sequencing technologies make gene diagnosis of Waardenburg syndrome efficiently and accurately.

Locus and allelic heterogeneity and phenotypic variability in Waardenburg syndrome.

Waardenburg syndrome (WS) is a disorder of neural crest cell migration characterized by auditory and pigmentary abnormalities. We investigated a cohort of 14 families (16 subjects) either by targeted sequencing or whole-exome sequencing. Thirteen of these families were clinically diagnosed with WS and one family with isolated non-syndromic hearing loss (NSHL). Intra-familial phenotypic variability and non-penetrance were observed in families diagnosed with WS1, WS2 and WS4 with pathogenic variants in PAX3, MITF and EDNRB, respectively. We observed gonosomal mosaicism for a variant in PAX3 in an asymptomatic father of two affected siblings. For the first time, we report a biallelic pathogenic variant in MITF in a subject with WS2 and a biallelic variant in EDNRB was noted in a subject with WS2. An individual with isolated NSHL carried a pathogenic variant in MITF. Blended phenotype of NSHL and albinism was observed in a subject clinically diagnosed to have WS2. A phenocopy of WS1 was observed in a subject with a reported pathogenic variant in GJB2, known to cause isolated NSHL. These novel and infrequently reported observations exemplify the allelic and genetic heterogeneity and show phenotypic diversity of WS.

Biology of human melanocyte development, Piebaldism, and Waardenburg syndrome.

Melanocyte development is orchestrated by a complex interconnecting regulatory network of genes and synergistic interactions. Piebaldism and Waardenburg syndrome are neurocristopathies that arise from mutations in genes involved in this complex network. Our understanding of melanocyte development, Piebaldism, and Waardenburg syndrome has improved dramatically over the past decade. The diagnosis and classification of Waardenburg syndrome, first proposed in 1992 and based on phenotype, have expanded over the past three decades to include genotype. This review focuses on the current understanding of human melanocyte development and the evaluation and management of Piebaldism and Waardenburg syndrome. Management is often challenging and requires a multidisciplinary approach.

[Ophthalmologic manifestations of Waardenburg syndrome]. / Oftal'mologicheskie proiavleniia sindroma Vaardenburga.

The Waardenburg syndrome is a group of rare genetic diseases, which clinical manifestations include neurosensory hearing loss, diminished pigmentation of forelock in the frontal region, iris heterochromia, medial canthus dystopia, and the presence of such changes in first-line relatives. The article presents a clinical case of type I Waardenburg syndrome, which developed de novo in a family. This case is unique in its combination of complete bilateral iris heterochromia and impaired choroidal pigmentation. The choroid did not only have hypopigmentation zones, but also large areas of hyper- and depigmentation. Such choroidal changes in Waardenburg patients has not been described in literature before. The diagnosis was confirmed by OCT of the anterior and posterior segments, angio-OCT, fluorescein angiography, indocyanine green angiography, fundus autofluorescence, and electrophysiological studies. The main ophthalmologic diagnostic criterion of Waardenburg syndrome in the present case, beside iris heterochromia, was the detection of iris thickness changes in hyper- and hypopigmentation areas with completely preserved structural and functional properties of the retina and choroid.

Cochlear morphology in the developing inner ear of the porcine model of spontaneous deafness.

BACKGROUND: Auditory function and cochlear morphology have previously been described in a porcine model with spontaneous WS2-like phenotype. In the present study, cochlear histopathology was further investigated in the inner ear of the developing spontaneous deafness pig. RESULTS: We found that the stria vascularis transformed into a complex tri-laminar tissue at embryonic 85 days (E85) in normal pigs, but not in the MITF-/- pigs. As the neural crest (NC) of cochlea was derived by melanocytes. MITF mutation caused failure of development of melanocytes which caused a subsequent collapse of cochlear duct and deficits of the epithelium after E100. Furthermore, the spiral ganglion neurons of cochlea in the MITF-/- pigs began to degenerate at postnatal 30 days (P30). Thus, our histopathological results indicated that the malformation of the stria vascularis was a primary defect in MITF-/- induced WT pigs which was resulted from the loss of NC-derived melanocytes. Subsequently, the cochleae underwent secondary degeneration of the vestibular organs. As the degeneration of spiral ganglion neurons happened after P30, it suggests that WS patients should be considered as candidates for cochlear implant. CONCLUSIONS: Our porcine model of MITF-M mutation may provide a crucial animal model for cochlear implant, cell therapy in patients with congenital hereditary hearing loss.