Prevalence of genetically confirmed skeletal muscle channelopathies in the era of next generation sequencing.

We provide an up-to-date and accurate minimum point prevalence of genetically defined skeletal muscle channelopathies which is important for understanding the population impact, planning for treatment needs and future clinical trials. Skeletal muscle channelopathies include myotonia congenita (MC), sodium channel myotonia (SCM), paramyotonia congenita (PMC), hyperkalemic periodic paralysis (hyperPP), hypokalemic periodic paralysis (hypoPP) and Andersen- Tawil Syndrome (ATS). Patients referred to the UK national referral centre for skeletal muscle channelopathies and living in UK were included to calculate the minimum point prevalence using the latest data from the Office for National Statistics population estimate. We calculated a minimum point prevalence of all skeletal muscle channelopathies of 1.99/100 000 (95% CI 1.981-1.999). The minimum point prevalence of MC due to CLCN1 variants is 1.13/100 000 (95% CI 1.123-1.137), SCN4A variants which encode for PMC and SCM is 0.35/100 000 (95% CI 0.346 - 0.354) and for periodic paralysis (HyperPP and HypoPP) 0.41/100 000 (95% CI 0.406-0.414). The minimum point prevalence for ATS is 0.1/100 000 (95% CI 0.098-0.102). There has been an overall increase in point prevalence in skeletal muscle channelopathies compared to previous reports, with the biggest increase found to be in MC. This can be attributed to next generation sequencing and advances in clinical, electrophysiological and genetic characterisation of skeletal muscle channelopathies.

Translating genetic and functional data into clinical practice: a series of 223 families with myotonia.

High-throughput DNA sequencing is increasingly employed to diagnose single gene neurological and neuromuscular disorders. Large volumes of data present new challenges in data interpretation and its useful translation into clinical and genetic counselling for families. Even when a plausible gene is identified with confidence, interpretation of the clinical significance and inheritance pattern of variants can be challenging. We report our approach to evaluating variants in the skeletal muscle chloride channel ClC-1 identified in 223 probands with myotonia congenita as an example of these challenges. Sequencing of CLCN1, the gene that encodes CLC-1, is central to the diagnosis of myotonia congenita. However, interpreting the pathogenicity and inheritance pattern of novel variants is notoriously difficult as both dominant and recessive mutations are reported throughout the channel sequence, ClC-1 structure-function is poorly understood and significant intra- and interfamilial variability in phenotype is reported. Heterologous expression systems to study functional consequences of CIC-1 variants are widely reported to aid the assessment of pathogenicity and inheritance pattern. However, heterogeneity of reported analyses does not allow for the systematic correlation of available functional and genetic data. We report the systematic evaluation of 95 CIC-1 variants in 223 probands, the largest reported patient cohort, in which we apply standardized functional analyses and correlate this with clinical assessment and inheritance pattern. Such correlation is important to determine whether functional data improves the accuracy of variant interpretation and likely mode of inheritance. Our data provide an evidence-based approach that functional characterization of ClC-1 variants improves clinical interpretation of their pathogenicity and inheritance pattern, and serve as reference for 34 previously unreported and 28 previously uncharacterized CLCN1 variants. In addition, we identify novel pathogenic mechanisms and find that variants that alter voltage dependence of activation cluster in the first half of the transmembrane domains and variants that yield no currents cluster in the second half of the transmembrane domain. None of the variants in the intracellular domains were associated with dominant functional features or dominant inheritance pattern of myotonia congenita. Our data help provide an initial estimate of the anticipated inheritance pattern based on the location of a novel variant and shows that systematic functional characterization can significantly refine the assessment of risk of an associated inheritance pattern and consequently the clinical and genetic counselling.

Andersen-Tawil syndrome: deep phenotyping reveals significant cardiac and neuromuscular morbidity.

Andersen-Tawil syndrome is a neurological channelopathy caused by mutations in the KCNJ2 gene that encodes the ubiquitously expressed Kir2.1 potassium channel. The syndrome is characterized by episodic weakness, cardiac arrythmias and dysmorphic features. However, the full extent of the multisystem phenotype is not well described. In-depth, multisystem phenotyping is required to inform diagnosis and guide management. We report our findings following deep multimodal phenotyping across all systems in a large case series of 69 total patients, with comprehensive data for 52. As a national referral centre, we assessed point prevalence and showed it is higher than previously reported, at 0.105 per 100 000 population in England. While the classical phenotype of episodic weakness is recognized, we found that a quarter of our cohort have fixed myopathy and 13.5% required a wheelchair or gait aid. We identified frequent fat accumulation on MRI and tubular aggregates on muscle biopsy, emphasizing the active myopathic process underpinning the potential for severe neuromuscular disability. Long exercise testing was not reliable in predicting neuromuscular symptoms. A normal long exercise test was seen in five patients, of whom four had episodic weakness. Sixty-seven per cent of patients treated with acetazolamide reported a good neuromuscular response. Thirteen per cent of the cohort required cardiac defibrillator or pacemaker insertion. An additional 23% reported syncope. Baseline electrocardiograms were not helpful in stratifying cardiac risk, but Holter monitoring was. A subset of patients had no cardiac symptoms, but had abnormal Holter monitor recordings which prompted medication treatment. We describe the utility of loop recorders to guide management in two such asymptomatic patients. Micrognathia was the most commonly reported skeletal feature; however, 8% of patients did not have dysmorphic features and one-third of patients had only mild dysmorphic features. We describe novel phenotypic features including abnormal echocardiogram in nine patients, prominent pain, fatigue and fasciculations. Five patients exhibited executive dysfunction and slowed processing which may be linked to central expression of KCNJ2. We report eight new KCNJ2 variants with in vitro functional data. Our series illustrates that Andersen-Tawil syndrome is not benign. We report marked neuromuscular morbidity and cardiac risk with multisystem involvement. Our key recommendations include proactive genetic screening of all family members of a proband. This is required, given the risk of cardiac arrhythmias among asymptomatic individuals, and a significant subset of Andersen-Tawil syndrome patients have no (or few) dysmorphic features or negative long exercise test. We discuss recommendations for increased cardiac surveillance and neuropsychometry testing.

Andersen-Tawil Syndrome Presenting with Complete Heart Block.

Andersen-Tawil syndrome (ATS) is a rare autosomal dominant neuromuscular disorder due to mutations in the KCNJ2 gene. The classical phenotype of ATS consists of a triad of periodic paralysis, cardiac conduction abnormalities and dysmorphic features. Episodes of either muscle weakness or cardiac arrhythmia may predominate however, and dysmorphic features may be subtle, masking the true breadth of the clinical presentation, and posing a diagnostic challenge. The severity of cardiac involvement varies but includes reports of life-threatening events or sudden cardiac death, usually attributed to ventricular tachyarrhythmias. We report the first case of advanced atrioventricular (AV) block in ATS and highlight clinical factors that may delay diagnosis.

Sodium channel myotonia may be associated with high-risk brief resolved unexplained events.

Brief resolved unexplained events (BRUEs) have numerous and varied causes posing a challenge to investigation and management. A subset of infants with the neuromuscular disorder sodium channel myotonia, due to mutations in the SCN4A gene, experience apnoeic events due to laryngospasm (myotonia) of the upper airway muscles that may present as a BRUE. We sought to ascertain the frequency, severity and outcome of infants carrying the G1306E SCN4A mutation commonly associated with this presentation. We report 12 new cases of individuals with the G1306E mutation from three unrelated families and perform a literature review of all published cases. Infants with the G1306E mutation almost universally experience laryngospasm and apnoeic events. The severity varies significantly, spans both low and high-risk BRUE categories or can be more severe than criteria for a BRUE would allow. At least a third of cases require intensive care unit (ICU) care. Seizure disorder is a common erroneous diagnosis. Apnoeas are effectively reduced or abolished by appropriate treatment with anti-myotonic agents. Probands with the G1306E mutation who are family planning need to be counselled for the likelihood of post-natal complications. There is readily available and extremely effective treatment for the episodic laryngospasm and apnoea caused by this mutation. Proactively seeking clinical evidence of myotonia or muscle hypertrophy with consideration of CK and EMG in high risk BRUEs or more complex apnoeic events may reduce avoidable and prolonged ICU admissions, patient morbidity and potentially mortality.

Myotonia in a patient with a mutation in an S4 arginine residue associated with hypokalaemic periodic paralysis and a concomitant synonymous CLCN1 mutation.

The sarcolemmal voltage gated sodium channel NaV1.4 conducts the key depolarizing current that drives the upstroke of the skeletal muscle action potential. It contains four voltage-sensing domains (VSDs) that regulate the opening of the pore domain and ensuing permeation of sodium ions. Mutations that lead to increased NaV1.4 currents are found in patients with myotonia or hyperkalaemic periodic paralysis (HyperPP). Myotonia is also caused by mutations in the CLCN1gene that result in loss-of-function of the skeletal muscle chloride channel ClC-1. Mutations affecting arginine residues in the fourth transmembrane helix (S4) of the NaV1.4 VSDs can result in a leak current through the VSD and hypokalemic periodic paralysis (HypoPP), but these have hitherto not been associated with myotonia. We report a patient with an Nav1.4 S4 arginine mutation, R222Q, presenting with severe myotonia without fulminant paralytic episodes. Other mutations affecting the same residue, R222W and R222G, have been found in patients with HypoPP. We show that R222Q channels have enhanced activation, consistent with myotonia, but also conduct a leak current. The patient carries a concomitant synonymous CLCN1 variant that likely worsens the myotonia and potentially contributes to the amelioration of muscle paralysis. Our data show phenotypic variability for different mutations affecting the same S4 arginine that have implications for clinical therapy.

A novel ATP1A2 mutation in a patient with hypokalaemic periodic paralysis and CNS symptoms.

Hypokalaemic periodic paralysis is a rare genetic neuromuscular disease characterized by episodes of skeletal muscle paralysis associated with low serum potassium. Muscle fibre inexcitability during attacks of paralysis is due to an aberrant depolarizing leak current through mutant voltage sensing domains of either the sarcolemmal voltage-gated calcium or sodium channel. We report a child with hypokalaemic periodic paralysis and CNS involvement, including seizures, but without mutations in the known periodic paralysis genes. We identified a novel heterozygous de novo missense mutation in the ATP1A2 gene encoding the α2 subunit of the Na+/K+-ATPase that is abundantly expressed in skeletal muscle and in brain astrocytes. Pump activity is crucial for Na+ and K+ homeostasis following sustained muscle or neuronal activity and its dysfunction is linked to the CNS disorders hemiplegic migraine and alternating hemiplegia of childhood, but muscle dysfunction has not been reported. Electrophysiological measurements of mutant pump activity in Xenopus oocytes revealed lower turnover rates in physiological extracellular K+ and an anomalous inward leak current in hypokalaemic conditions, predicted to lead to muscle depolarization. Our data provide important evidence supporting a leak current as the major pathomechanism underlying hypokalaemic periodic paralysis and indicate ATP1A2 as a new hypokalaemic periodic paralysis gene.

Hypokalaemic periodic paralysis and myotonia in a patient with homozygous mutation p.R1451L in NaV1.4.

Dominantly inherited channelopathies of the skeletal muscle voltage-gated sodium channel NaV1.4 include hypokalaemic and hyperkalaemic periodic paralysis (hypoPP and hyperPP) and myotonia. HyperPP and myotonia are caused by NaV1.4 channel overactivity and overlap clinically. Instead, hypoPP is caused by gating pore currents through the voltage sensing domains (VSDs) of NaV1.4 and seldom co-exists clinically with myotonia. Recessive loss-of-function NaV1.4 mutations have been described in congenital myopathy and myasthenic syndromes. We report two families with the NaV1.4 mutation p.R1451L, located in VSD-IV. Heterozygous carriers in both families manifest with myotonia and/or hyperPP. In contrast, a homozygous case presents with both hypoPP and myotonia, but unlike carriers of recessive NaV1.4 mutations does not manifest symptoms of myopathy or myasthenia. Functional analysis revealed reduced current density and enhanced closed state inactivation of the mutant channel, but no evidence for gating pore currents. The rate of recovery from inactivation was hastened, explaining the myotonia in p.R1451L carriers and the absence of myasthenic presentations in the homozygous proband. Our data suggest that recessive loss-of-function NaV1.4 variants can present with hypoPP without congenital myopathy or myasthenia and that myotonia can present even in carriers of homozygous NaV1.4 loss-of-function mutations.

Dysfunction of NaV1.4, a skeletal muscle voltage-gated sodium channel, in sudden infant death syndrome: a case-control study.

BACKGROUND: Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant death in high-income countries. Central respiratory system dysfunction seems to contribute to these deaths. Excitation that drives contraction of skeletal respiratory muscles is controlled by the sodium channel NaV1.4, which is encoded by the gene SCN4A. Variants in NaV1.4 that directly alter skeletal muscle excitability can cause myotonia, periodic paralysis, congenital myopathy, and myasthenic syndrome. SCN4A variants have also been found in infants with life-threatening apnoea and laryngospasm. We therefore hypothesised that rare, functionally disruptive SCN4A variants might be over-represented in infants who died from SIDS. METHODS: We did a case-control study, including two consecutive cohorts that included 278 SIDS cases of European ancestry and 729 ethnically matched controls without a history of cardiovascular, respiratory, or neurological disease. We compared the frequency of rare variants in SCN4A between groups (minor allele frequency <0·00005 in the Exome Aggregation Consortium). We assessed biophysical characterisation of the variant channels using a heterologous expression system. FINDINGS: Four (1·4%) of the 278 infants in the SIDS cohort had a rare functionally disruptive SCN4A variant compared with none (0%) of 729 ethnically matched controls (p=0·0057). INTERPRETATION: Rare SCN4A variants that directly alter NaV1.4 function occur in infants who had died from SIDS. These variants are predicted to significantly alter muscle membrane excitability and compromise respiratory and laryngeal function. These findings indicate that dysfunction of muscle sodium channels is a potentially modifiable risk factor in a subset of infant sudden deaths. FUNDING: UK Medical Research Council, the Wellcome Trust, National Institute for Health Research, the British Heart Foundation, Biotronik, Cardiac Risk in the Young, Higher Education Funding Council for England, Dravet Syndrome UK, the Epilepsy Society, the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, and the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program.

Atypical periodic paralysis and myalgia: A novel RYR1 phenotype.

OBJECTIVE: To characterize the phenotype of patients with symptoms of periodic paralysis (PP) and ryanodine receptor (RYR1) gene mutations. METHODS: Cases with a possible diagnosis of PP but additional clinicopathologic findings previously associated with RYR1-related disorders were referred for a tertiary neuromuscular clinical assessment in which they underwent detailed clinical evaluation, including neurophysiologic assessment, muscle biopsy, and muscle MRI. Genetic analysis with next-generation sequencing and/or targeted Sanger sequencing was performed. RESULTS: Three cases with episodic muscle paralysis or weakness and additional findings compatible with a RYR1-related myopathy were identified. The McManis test, used in the diagnosis of PP, was positive in 2 of 3 cases. Genetic analysis of known PP genes was negative. RYR1 analysis confirmed likely pathogenic variants in all 3 cases. CONCLUSIONS: RYR1 mutations can cause late-onset atypical PP both with and without associated myopathy. Myalgia and cramps are prominent features. The McManis test may be a useful diagnostic tool to indicate RYR1-associated PP. We propose that clinicopathologic features suggestive of RYR1-related disorders should be sought in genetically undefined PP cases and that RYR1 gene testing be considered in those in whom mutations in SCN4A, CACNA1S, and KCNJ2 have already been excluded.

Skeletal Muscle Channelopathies: Rare Disorders with Common Pediatric Symptoms.

OBJECTIVE: To ascertain the presenting symptoms of children with skeletal muscle channelopathies to promote early diagnosis and treatment. STUDY DESIGN: Retrospective case review of 38 children with a skeletal muscle channelopathy attending the specialist pediatric neuromuscular service at Great Ormond Street Hospital over a 15-year period. RESULTS: Gait disorder and leg cramps are a frequent presentation of myotonic disorders (19 of 29). Strabismus or extraocular myotonia (9 of 19) and respiratory and/or bulbar symptoms (11 of 19) are common among those with sodium channelopathy. Neonatal hypotonia was observed in periodic paralysis. Scoliosis and/or contractures were demonstrated in 6 of 38 children. School attendance or ability to engage fully in all activities was often limited (25 of 38). CONCLUSIONS: Children with skeletal muscle channelopathies frequently display symptoms that are uncommon in adult disease. Any child presenting with abnormal gait, leg cramps, or strabismus, especially if intermittent, should prompt examination for myotonia. Those with sodium channel disease should be monitored for respiratory or bulbar complications. Neonatal hypotonia can herald periodic paralysis. Early diagnosis is essential for children to reach their full educational potential.

Prevalence study of genetically defined skeletal muscle channelopathies in England.

OBJECTIVES: To obtain minimum point prevalence rates for the skeletal muscle channelopathies and to evaluate the frequency distribution of mutations associated with these disorders. METHODS: Analysis of demographic, clinical, electrophysiologic, and genetic data of all patients assessed at our national specialist channelopathy service. Only patients living in the United Kingdom with a genetically defined diagnosis of nondystrophic myotonia or periodic paralysis were eligible for the study. Prevalence rates were estimated for England, December 2011. RESULTS: A total of 665 patients fulfilled the inclusion criteria, of which 593 were living in England, giving a minimum point prevalence of 1.12/100,000 (95% confidence interval [CI] 1.03-1.21). Disease-specific prevalence figures were as follows: myotonia congenita 0.52/100,000 (95% CI 0.46-0.59), paramyotonia congenita 0.17/100,000 (95% CI 0.13-0.20), sodium channel myotonias 0.06/100,000 (95% CI 0.04-0.08), hyperkalemic periodic paralysis 0.17/100,000 (95% CI 0.13-0.20), hypokalemic periodic paralysis 0.13/100,000 (95% CI 0.10-0.17), and Andersen-Tawil syndrome (ATS) 0.08/100,000 (95% CI 0.05-0.10). In the whole sample (665 patients), 15 out of 104 different CLCN1 mutations accounted for 60% of all patients with myotonia congenita, 11 out of 22 SCN4A mutations for 86% of paramyotonia congenita/sodium channel myotonia pedigrees, and 3 out of 17 KCNJ2 mutations for 42% of ATS pedigrees. CONCLUSION: We describe for the first time the overall prevalence of genetically defined skeletal muscle channelopathies in England. Despite the large variety of mutations observed in patients with nondystrophic myotonia and ATS, a limited number accounted for a large proportion of cases.

New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics.

OBJECTIVE: The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so. METHODS: An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies. RESULTS: This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing. CONCLUSIONS: We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.

Sodium and chloride channelopathies with myositis: coincidence or connection?

INTRODUCTION: A proximal myopathy develops in some patients with muscle channelopathies, but the causative molecular mechanisms are unknown. METHODS: We reviewed retrospectively all clinical and muscle biopsy findings of 3 patients with channelopathy and additional myositis. Direct DNA sequencing was performed. RESULTS: Pathogenic mutations were identified in each case. Biopsies demonstrated inflammatory infiltrates. CONCLUSIONS: Clinicians should consider muscle biopsy in channelopathy patients with severe myalgia and/or subacute weakness and accompanying elevated creatine kinase. Chance association of myositis and channelopathy is statistically unlikely. An alternative hypothesis suggests that inflammatory insults could contribute to myopathy in some patients.

Caprin-1 is a target of the deafness gene Pou4f3 and is recruited to stress granules in cochlear hair cells in response to ototoxic damage.

The POU4 family of transcription factors are required for survival of specific cell types in different sensory systems. Pou4f3 is essential for the survival of auditory sensory hair cells and several mutations in human POU4F3 cause hearing loss. Thus, genes regulated by Pou4f3 are likely to be essential for hair cell survival. We performed a subtractive hybridisation screen in an inner-ear-derived cell line to find genes with differential expression in response to changes in Pou4f3 levels. The screen identified the stress-granule-associated protein Caprin-1 as being downregulated by Pou4f3. We demonstrated that this regulation occurs through the direct interaction of Pou4f3 with binding sites in the Caprin-1 5' flanking sequence, and describe the expression pattern of Caprin-1 mRNA and protein in the cochlea. Moreover, we found Caprin-1-containing stress granules are induced in cochlear hair cells following aminoglycoside-induced damage. This is the first report of stress granule formation in mammalian hair cells and suggests that the formation of Caprin-1-containing stress granules is a key damage response to a clinically relevant ototoxic agent. Our results have implications for the understanding of aminoglycoside-induced hearing loss and provide further evidence that stress granule formation is a fundamental cellular stress response.

Refined exercise testing can aid DNA-based diagnosis in muscle channelopathies.

OBJECTIVE: To improve the accuracy of genotype prediction and guide genetic testing in patients with muscle channelopathies we applied and refined specialized electrophysiological exercise test parameters. METHODS: We studied 56 genetically confirmed patients and 65 controls using needle electromyography, the long exercise test, and short exercise tests at room temperature, after cooling, and rewarming. RESULTS: Concordant amplitude-and-area decrements were more reliable than amplitude-only measurements when interpreting patterns of change during the short exercise tests. Concordant amplitude-and-area pattern I and pattern II decrements of >20% were 100% specific for paramyotonia congenita and myotonia congenita, respectively. When decrements at room temperature and after cooling were <20%, a repeat short exercise test after rewarming was useful in patients with myotonia congenita. Area measurements and rewarming distinguished true temperature sensitivity from amplitude reduction due to cold-induced slowing of muscle fiber conduction. In patients with negative short exercise tests, symptomatic eye closure myotonia predicted sodium channel myotonia over myotonia congenita. Distinctive "tornado-shaped" neuromyotonia-like discharges may be seen in patients with paramyotonia congenita. In the long exercise test, area decrements from pre-exercise baseline were more sensitive than amplitude decrements-from-maximum-compound muscle action potential (CMAP) in patients with Andersen-Tawil syndrome. Possible ethnic differences in the normative data of the long exercise test argue for the use of appropriate ethnically-matched controls. INTERPRETATION: Concordant CMAP amplitude-and-area decrements of >20% allow more reliable interpretation of the short exercise tests and aid accurate DNA-based diagnosis. In patients with negative exercise tests, specific clinical features are helpful in differentiating sodium from chloride channel myotonia. A modified algorithm is suggested.

Stridor as a neonatal presentation of skeletal muscle sodium channelopathy.

OBJECTIVE: To describe stridor as the presenting feature in a neonate with the skeletal muscle sodium channelopathy paramyotonia congenita. DESIGN: Case report. SETTING: Outpatient neuromuscular clinics at Great Ormond Street Hospital for Children and the Medical Research Council Centre for Neuromuscular Disease at the National Hospital for Neurology and Neurosurgery, London, England. PATIENT: A child carrying the Thr1313Met SCN4A mutation associated with paramyotonia congenita. INTERVENTION: Supportive care in the neonatal period and administration of mexiletine hydrochloride at age 4 years. MAIN OUTCOME MEASURE: The association of stridor and paramyotonia congenita was made retrospectively following the diagnosis in the infant's mother; the child is now regularly reviewed at the pediatric outpatient clinic. RESULTS: Persistent stridor was present for the first 6 months of life, and episodic stridor can still be exacerbated by intercurrent respiratory tract infection, cold, laughter, or crying. Common symptoms of paramyotonia congenita have been apparent from age 1 year and are beginning to respond to a recent trial of mexiletine. CONCLUSIONS: To our knowledge, neonatal stridor has not previously been reported in skeletal muscle sodium channelopathies. The recognition that infants inheriting mutations known to cause paramyotonia congenita are inherently at risk for developing neonatal complications following an uneventful labor is important for all training neurologists so they can advise expectant mothers and pediatric and obstetric colleagues appropriately.

Transcriptional regulation by Barhl1 and Brn-3c in organ-of-Corti-derived cell lines.

Barhl1 and Brn-3c have been identified as transcription factors that are essential for survival and maintenance of hair cells of the inner ear. Little is known about the mechanism of how Brn-3c or Barhl1 may regulate transcription in the inner ear. In this study, the transcriptional function of both Brn-3c and Barhl1 was investigated in the organ-of-Corti-derived cell lines, OC-1 and OC-2. We examined regulatory domains in these transcription factors by linking regions of Barhl1 and Brn-3c to the DNA binding domain of the heterologous transcription factor GAL4 and assayed their effect on a heterologous promoter containing GAL4 DNA binding sites by co-transfection into OC-1 and OC-2 cell lines. Brn-3c was found to contain an independent N-terminal activation domain that is sufficient to activate gene transcription in the organ of corti derived cell lines. Barhl1 on the other hand was found to act as a transcriptional repressor with repressive activity not restricted to a particular domain of Barhl1. In addition, we analyzed the effect of Barhl1 on the promoters of the neurotrophin genes NT-3 and BDNF in OC-1 and OC-2 cell lines. However, Barhl1 was not found to directly regulate neurotrophin promoter constructs in these cells.