Thyroarytenoid Muscle Gene Expression in a Rat Model of Early-Onset Parkinson's Disease.

OBJECTIVES/HYPOTHESIS: Voice disorders in Parkinson's disease (PD) are early-onset, manifest in the preclinical stages of the disease, and negatively impact quality of life. The complete loss of function in the PTEN-induced kinase 1 gene (Pink1) causes a genetic form of early-onset, autosomal recessive PD. Modeled after the human inherited mutation, the Pink1-/- rat demonstrates significant cranial sensorimotor dysfunction including declines in ultrasonic vocalizations. However, the underlying genetics of the vocal fold thyroarytenoid (TA) muscle that may contribute to vocal deficits has not been studied. The aim of this study was to identify differentially expressed genes in the TA muscle of 8-month-old male Pink1-/- rats compared to wildtype controls. STUDY DESIGN: Animal experiment with control. METHODS: High throughput RNA sequencing was used to examine TA muscle gene expression in adult male Pink1-/- rats and wildtype controls. Weighted Gene Co-expression Network Analysis was used to construct co-expression modules to identify biological networks, including where Pink1 was a central node. The ENRICHR tool was used to compare this gene set to existing human gene databases. RESULTS: We identified 134 annotated differentially expressed genes (P < .05 cutoff) and observed enrichment in the following biological pathways: Parkinson's disease (Casp7, Pink1); Parkin-Ubiquitin proteasome degradation (Psmd12, Psmd7); MAPK signaling (Casp7, Ppm1b, Ppp3r1); and inflammatory TNF-α, Nf-κB Signaling (Casp7, Psmd12, Psmd7, Cdc34, Bcl7a, Peg3). CONCLUSIONS: Genes and pathways identified here may be useful for evaluating the specific mechanisms of peripheral dysfunction including within the laryngeal muscle and have potential to be used as experimental biomarkers for treatment development. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2874-E2879, 2021.

DYT-TUBB4A (DYT4 Dystonia): New Clinical and Genetic Observations.

OBJECTIVE: To report 4 novel TUBB4A mutations leading to laryngeal and cervical dystonia with frequent generalization. METHODS: We screened 4 families including a total of 11 definitely affected members with a clinical picture resembling the original description. RESULTS: Four novel variants in the TUBB4A gene have been identified: D295N, R46M, Q424H, and R121W. In silico modeling showed that all variants have characteristics similar to R2G. The variants segregate with the disease in 3 of the families with evidence of incomplete penetrance in 2 of them. All 4 variants would be classified as likely pathogenic. The clinical picture particularly included laryngeal dystonia (often the site of onset), associated with cervical and upper limb dystonia and frequent generalization. Laryngeal dystonia was extremely prevalent (>90%) both in the original cases and in this case series. The hobby horse gait was evident in only 1 patient in this case series. CONCLUSIONS: Our interpretation is that laryngeal involvement is a hallmark feature of DYT-TUBB4A. Nevertheless, TUBB4A mutations remain an exceedingly rare cause of laryngeal or other isolated dystonia.

Unraveling the molecular pathobiology of vocal fold systemic dehydration using an in vivo rabbit model.

Vocal folds are a viscoelastic multilayered structure responsible for voice production. Vocal fold epithelial damage may weaken the protection of deeper layers of lamina propria and thyroarytenoid muscle and impair voice production. Systemic dehydration can adversely affect vocal function by creating suboptimal biomechanical conditions for vocal fold vibration. However, the molecular pathobiology of systemically dehydrated vocal folds is poorly understood. We used an in vivo rabbit model to investigate the complete gene expression profile of systemically dehydrated vocal folds. The RNA-Seq based transcriptome revealed 203 differentially expressed (DE) vocal fold genes due to systemic dehydration. Interestingly, function enrichment analysis showed downregulation of genes involved in cell adhesion, cell junction, inflammation, and upregulation of genes involved in cell proliferation. RT-qPCR validation was performed for a subset of DE genes and confirmed the downregulation of DSG1, CDH3, NECTIN1, SDC1, S100A9, SPINK5, ECM1, IL1A, and IL36A genes. In addition, the upregulation of the transcription factor NR4A3 gene involved in epithelial cell proliferation was validated. Taken together, these results suggest an alteration of the vocal fold epithelial barrier independent of inflammation, which could indicate a disruption and remodeling of the epithelial barrier integrity. This transcriptome provides a first global picture of the molecular changes in vocal fold tissue in response to systemic dehydration. The alterations observed at the transcriptional level help to understand the pathobiology of dehydration in voice function and highlight the benefits of hydration in voice therapy.

G protein coupled oestrogen receptor 1, aromatase, 17ß-HSD and cAMP level in mutational falsetto.

OBJECTIVES: Puberphonia or mutational falsetto (MF) is seen more in males, and hormonal changes are considered to be among the aetiological causes. Therefore, the aim of this study was to investigate the molecules [G protein-coupled oestrogen receptor 1 (GPER-1), aromatase, 17-beta-hydroxysteroid dehydrogenase (17ß-HSD), cyclic adenosine monophosphate (cAMP) levels] related to receptors and pathways in patients with MF. METHODS: The study included 30 MF patients and a control group of 30 healthy individuals. Voice recordings were made of the MF patients and acoustic analyses were applied. The serum GPER-1, aromatase, 17ß-HSD, cAMP levels and TSH, estradiol, prolactin, progesterone, and testosterone levels were evaluated in venous blood samples. RESULTS: In the MF patients, the GPER-1 level determined of mean 3.68 (1.95-4.26) pg/ml, 17 beta dehydrogenase of 5.25 (2.73-6.77) ng/ml, and cAMP of 24.62 (11.62-30.35) ng/ml were statistically signficantly higher than those of the control group (p = 0.008, p = 0.002, p = 0.003, respectively). The aromatase level in the MF patients was found to be 3.48 (2.01-4.91) and the difference between the two groups was not statistically significant (p = 0.067). CONCLUSION: The GPER-1, 17ß-HSD, and cAMP levels were found to be higher in the MF patients than in the control group, suggesting that they could be of importance in the diagnosis and treatment of MF.

Matrix Metalloproteinases (MMP-2,-3,-9) Gene Polymorphisms in Cases of Benign Vocal Fold Lesions and Laryngeal Carcinoma.

BACKGROUND/AIM: The matrix metalloproteinases (MMP) play an important role in the physiological and pathological remodeling of tissues including carcinogenesis. The study's aim was to assess the relations between MMP-2(-735C/T), MMP-2(-1306C/T), MMP-9(-1562C/T), and MMP-3(-11715A/6A) polymorphisms, and clinical/morphological manifestation of laryngeal squamous cell carcinoma (LSCC) and benign vocal fold lesions (BVFL). PATIENTS AND METHODS: Two hundred and seventeen patients with LSCC and BVFL and 458 controls were included in this study. The genotyping was performed using the real-time polymerase chain reaction method. RESULTS: The MMP-2(-1306C/T) C/T genotype was significantly rarer among the patients with moderate-poorly differentiated LSCC compared to the control group, however the MMP-3(-11715A/6A) 6A/6A genotype was significantly more frequent compared to controls. Smoking and 6A/6A genotype of MMP-3(-11715A/6A) polymorphism were associated with increased odds of LSCC risk. No associations between MMP genotypes and BVFL were found. CONCLUSION: Smoking and MMP-3 (-11715A/6A) 6A/6A genotype may cause a higher risk for developing LSCC.

Associations Between Vocal Symptoms and Genetic Variants in the Oxytocin Receptor and Arginine Vasopressin 1A Receptor Gene.

Purpose: Oxytocin and arginine vasopressin are associated with different aspects of the stress response. As stress is regarded as a risk factor for vocal symptoms, we wanted to explore the association between the oxytocin receptor gene (OXTR) and arginine vasopressin 1A receptor gene (AVPR1A) single-nucleotide polymorphisms (SNPs) and vocal symptoms. We also wanted to explore whether such effects might be mediated by cortisol because oxytocin and vasopressin are associated with cortisol levels. Method: A population-based sample (N = 657) of Finnish twins (born 1961-1989) completed a web questionnaire on the occurrence of vocal symptoms. A total of 170 participants submitted saliva samples for hormone analysis. A total of 20 OXTR and AVPR1A SNPs were analyzed. Results: Three OXTR polymorphisms (rs2270465, rs2268493, rs7632287) and 2 AVPR1A polymorphisms (rs1587097, rs1042615) showed nominal effects (p < .05) on vocal symptoms, of which 1 (rs1587097) remained significant after correcting for multiple testing (p = .003). We found potential mediation of the effect of the OXTR rs2268493 polymorphism on vocal symptoms through levels of cortisol. Conclusions: The associations between variants of OXTR and AVPR1A and vocal symptoms indicate that oxytocin and vasopressin might influence vocal symptoms. The effect of oxytocin seems to be partly mediated through cortisol actions.

Insights Into the Role of Collagen in Vocal Fold Health and Disease.

As one of the key fibrous proteins in the extracellular matrix, collagen plays a significant role in the structural and biomechanical characteristics of the vocal fold. Anchored fibrils of collagen create secure structural regions within the vocal folds and are strong enough to sustain vibratory impact and stretch during phonation. This contributes tensile strength, density, and organization to the vocal folds and influences health and pathogenesis. This review offers a comprehensive summary for a current understanding of collagen within normal vocal fold tissues throughout the life span as well as vocal pathology and wound repair. Further, collagen's molecular structure and biosynthesis are discussed. Finally, collagen alterations in tissue injury and repair and the incorporation of collagen-based biomaterials as a method of treating voice disorders are reviewed.

Acute exposure to vibration is an apoptosis-inducing stimulus in the vocal fold epithelium.

Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120min) or a control group (120min of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120min of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120min of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases.

H-ABC syndrome and DYT4: Variable expressivity or pleiotropy of TUBB4 mutations?

Recently, mutations in the TUBB4A gene have been found to underlie hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) syndrome, a rare neurodegenerative disorder of infancy and childhood. TUBB4A mutations also have been described as causative of DYT4 ("hereditary whispering dysphonia"). However, in DYT4, brain imaging has been reported to be normal and, therefore, H-ABC syndrome and DYT4 have been construed to be different disorders, despite some phenotypic overlap. Hence, the question of whether these disorders reflect variable expressivity or pleiotropy of TUBB4A mutations has been raised. We report four unrelated patients with imaging findings either partially or totally consistent with H-ABC syndrome, who were found to have TUBB4A mutations. All four subjects had a relatively homogenous phenotype characterized by severe generalized dystonia with superimposed pyramidal and cerebellar signs, and also bulbar involvement leading to complete aphonia and swallowing difficulties, even though one of the cases had an intermediate phenotype between H-ABC syndrome and DYT4. Genetic analysis of the TUBB4A gene showed one previously described and two novel mutations (c.941C>T; p.Ala314Val and c.900G>T; p.Met300Ile) in the exon 4 of the gene. While expanding the genetic spectrum of H-ABC syndrome, we confirm its radiological heterogeneity and demonstrate that phenotypic overlap with DYT4. Moreover, reappraisal of previously reported cases would also argue against pleiotropy of TUBB4A mutations. We therefore suggest that H-ABC and DYT4 belong to a continuous phenotypic spectrum associated with TUBB4A mutations.

Phenotype of matrin-3-related distal myopathy in 16 German patients.

OBJECTIVE: To characterize the phenotype of patients with distal myopathy with vocal cord and pharyngeal weakness due to the p.S85C mutation in the matrin-3 gene (MATR3, Mendelian Inheritance in Man 164015). Recently, it has been suggested that patients with this mutation may suffer from familial amyotrophic lateral sclerosis. METHODS: Sixteen patients from 6 families with late onset distal myopathy associated with the p.S85C MATR3 mutation were characterized. RESULTS: Patients had a predominantly distal muscle weakness, most severely affecting ankle and wrist dorsiflexion. Relevant proximal and axial weakness was found in 6 and respiratory impairment in 5 patients. Dysphagia was diagnosed in 6 and mild voice abnormalities were found in 7 patients. However, laryngoscopy revealed normal vocal cord function. Creatine kinase was normal or mildly elevated. Electromyographically, spontaneous activity was found in 10 of 14 patients and complex repetitive discharges in 9 of 14 patients. Magnetic resonance imaging revealed severe fatty degeneration of distal and upper posterior leg and of paraspinal muscles. Histopathology ranged from mild myopathic to severe dystrophic changes including vacuoles. Absence of sarcomeres in the perinuclear region and abnormal invaginations of nuclei were found ultrastructurally. Haplotype analysis showed a common disease-specific haplotype of the 6 families and suggested that these families form a separate cluster. INTERPRETATION: In contrast to the 2 previously reported families, MATR3-related distal myopathy might be associated with relevant axial, proximal, and respiratory muscle weakness but without vocal cord palsy. There were no clinical, electrophysiological, or histopathological signs of lower motor neuron involvement.

Whispering dysphonia (DYT4 dystonia) is caused by a mutation in the TUBB4 gene.

OBJECTIVE: A study was undertaken to identify the gene underlying DYT4 dystonia, a dominantly inherited form of spasmodic dysphonia combined with other focal or generalized dystonia and a characteristic facies and body habitus, in an Australian family. METHODS: Genome-wide linkage analysis was carried out in 14 family members followed by genome sequencing in 2 individuals. The index patient underwent a detailed neurological follow-up examination, including electrophysiological studies and magnetic resonance imaging scanning. Biopsies of the skin and olfactory mucosa were obtained, and expression levels of TUBB4 mRNA were determined by quantitative real-time polymerase chain reaction in 3 different cell types. All exons of TUBB4 were screened for mutations in 394 unrelated dystonia patients. RESULTS: The disease-causing gene was mapped to a 23cM region on chromosome 19p13.3-p13.2 with a maximum multipoint LOD score of 5.338 at markers D9S427 and D9S1034. Genome sequencing revealed a missense variant in the TUBB4 (tubulin beta-4; Arg2Gly) gene as the likely cause of disease. Sequencing of TUBB4 in 394 unrelated dystonia patients revealed another missense variant (Ala271Thr) in a familial case of segmental dystonia with spasmodic dysphonia. mRNA expression studies demonstrated significantly reduced levels of mutant TUBB4 mRNA in different cell types from a heterozygous Arg2Gly mutation carrier compared to controls. INTERPRETATION: A mutation in TUBB4 causes DYT4 dystonia in this Australian family with so-called whispering dysphonia, and other mutations in TUBB4 may contribute to spasmodic dysphonia. Given that TUBB4 is a neuronally expressed tubulin, our results imply abnormal microtubule function as a novel mechanism in the pathophysiology of dystonia.

Insights into the role of elastin in vocal fold health and disease.

Elastic fibers are large, complex, and surprisingly poorly understood extracellular matrix macromolecules. The elastin fiber, generated from a single human gene--elastin, is a self assembling integral protein that endows critical mechanical proprieties to elastic tissues and organs such as the skin, lungs, and arteries. The biology of elastic fibers is complex because they have multiple components, a tightly regulated developmental deposition, a multistep hierarchical assembly, and unique biomechanical functions. Elastin is present in vocal folds, where it plays a pivotal role in the quality of phonation. This review article provides an overview of the genesis of elastin and its wide-ranging structure and function. Specific distribution within the vocal fold lamina propria across the lifespan in normal and pathological states and its contribution to vocal fold biomechanics will be examined. Elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. The properties of various elastin-based materials will be discussed and their current and future applications evaluated. A new level of understanding of the biomechanical properties of vocal fold elastin composites and their molecular basis should lead to new strategies for elastic fiber repair and regeneration in aging and disease.

Objective assessment of nasality in Flemish adults with neurofibromatosis type 1.

When characterizing the speech of a patient with neurofibromatosis type 1 (NF1), hypernasality is often mentioned. As few studies applied technically assisted evaluations of nasality in NF1 patients, the aim of the present study was to document the nasal resonance of adults with NF1 using nasometry. The nasometric data obtained from the NF1 patients were compared with the nasalance scores of a healthy control group and with normative data. The final study group consisted of 24 adult NF1 patients and 16 controls, all living in the East Flemish part of Belgium. Nasalance scores were obtained while the participants sustained three vowels (/a:/, /i./, and /u./) and one consonant (/m/) and read three standard nasalance passages. Despite the inter- and intra-subject variability, we observed that NF1 patients as a group exhibited higher mean nasalance scores than controls. This finding was especially clear in males. Potential genotype-phenotype correlations between NF1 mutation type and hypernasality were examined but could not be demonstrated. Conversely, comparison of the nasometric data obtained from the NF1 patients with magnetic resonance imaging findings showed some degree of interesting correlation. We conclude that, notwithstanding the small sample size for some analyses, nasality is an area of interest in the NF1 population. As altered nasality influences speech intelligibility, nasality requires attention during follow-up visits, particularly when it concerns a male NF1 patient.

Molecular characterization of microduplication 22q11.2 in a girl with hypernasal speech.

We present a 12-year-old girl with karyotype 46,XX. A comparative genomic hybridization array revealed a 3.172-Mb microduplication on 22q11.2. This chromosome 22q11.2 region microduplication has been described in patients with variable phenotypes; a large majority of them have identical 3-Mb duplications. The girl presented mild mental motor retardation, facial dysmorphism consisting of a long narrow face, widely spaced eyes, downslanting palpebral fissures, broad nasal base, short philtrum, thin upper lip, micro/retrognathia, low set and retroverted ears, microcephaly, high-arched palate, hypoplastic teeth, and hypernasal speech. She had delayed psychomotor development and behavioral problems. Molecular characterization of patients differs greatly among reports and detailed molecular characterization and documentation are needed to better understand the effects of these duplications. This description of the phenotype of a patient with microduplication on 22q11.2 will contribute to the growing knowledge regarding deletions and duplications of the 22q11.2 region; this is important to conclude whether 22q11.2 duplication is a microduplication syndrome or not.

Whispering dysphonia in an Australian family (DYT4): a clinical and genetic reappraisal.

The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role of known causes of dystonia has not been comprehensively investigated in this family, nor has the possible relationship with Wilson disease (WND) in 2 siblings. Eighteen family members were neurologically examined, and DNA samples were obtained. Linkage analysis was performed to DYT1, DYT6, DYT7, DYT11, DYT13, DYT15, and ATP7B with microsatellite markers and the THAP1 (DYT6), PRKRA (DYT16), and ATP7B (WND) genes were sequenced. Reevaluation of the family identified 9 living affected family members, 6 of whom are newly affected. Phenotypic expression was variable, ranging from isolated spasmodic dysphonia (often with mild craniocervical dystonia) to severe generalized dystonia. Two newly described features included an extrusional tongue dystonia and a unique "hobby horse gait." Genetic analyses excluded all tested loci. Haplotype analysis of the ATP7B region resulted in three different combinations of the two parental alleles in the 8 investigated siblings of the 2 deceased WND patients, indicating that the fourth combination (of two mutated alleles) had occurred only in the deceased WND patients. On these two alleles, we identified a missense (c.2297C>G; p.T766R) and a splice-site mutation (IVS5+1G>T). The c.2297C>G mutation was detected in 3 affected and 4 unaffected family members, whereas the IVS5+1G>T mutation was detected in 1 affected and unaffected family member. Five DYT4 patients carried neither mutation. DYT4 is a familial form of dystonia unrelated to known dystonia genes and loci. ATP7B mutations do not segregate with the dystonia phenotype, indicating two independent genetic diseases in this family.

Sensory ataxic neuropathy with dysarthria and ophthalmoparesis (SANDO) in late life due to compound heterozygous POLG mutations.

Missense mutations in the gene for polymerase gamma 1 (POLG1) cause a number of phenotypically heterogeneous mitochondrial diseases, most commonly progressive external ophthalmoplegia, and are characterized by the accumulation of multiple, large-scale deletions of mitochondrial DNA. The triad of sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO) has been demonstrated in a small subset of patients with POLG1 mutations. We report a sporadic case of an 80-year-old compound heterozygote man who presented with SANDO and was found to have three known pathogenic mutations in the POLG1 gene (p.T251I/p.P587L/p.G848S). To our knowledge, none of these mutations have been demonstrated previously in SANDO. This patient's late presentation illustrates that a mitochondrial disorder should be considered regardless of age if the clinical symptoms warrant.

Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts.

The composition and organization of the vocal fold extracellular matrix (ECM) provide the viscoelastic mechanical properties that are required to sustain high-frequency vibration during voice production. Although vocal injury and pathology are known to produce alterations in matrix physiology, the mechanisms responsible for the development and maintenance of vocal fold ECM are poorly understood. The objective of this study was to investigate the effect of physiologically relevant vibratory stimulation on ECM gene expression and synthesis by fibroblasts encapsulated within hyaluronic acid hydrogels that approximate the viscoelastic properties of vocal mucosa. Relative to static controls, samples exposed to vibration exhibited significant increases in mRNA expression levels of HA synthase 2, decorin, fibromodulin and MMP-1, while collagen and elastin expression were relatively unchanged. Expression levels exhibited a temporal response, with maximum increases observed after 3 and 5 days of vibratory stimulation and significant downregulation observed at 10 days. Quantitative assays of matrix accumulation confirmed significant increases in sulphated glycosaminoglycans and significant decreases in collagen after 5 and 10 days of vibratory culture, relative to static controls. Cellular remodelling and hydrogel viscosity were affected by vibratory stimulation and were influenced by varying the encapsulated cell density. These results indicate that vibration is a critical epigenetic factor regulating vocal fold ECM and suggest that rapid restoration of the phonatory microenvironment may provide a basis for reducing vocal scarring, restoring native matrix composition and improving vocal quality.