Mitochondrial DNA mutations attenuate Bleomycin-induced dermal fibrosis by inhibiting differentiation into myofibroblasts.

Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.

The disease-causing mutation p.F907I reveals a novel pathogenic mechanism for POLγ-related diseases.

Mutations in the catalytic domain of mitochondrial DNA polymerase γ (POLγ) cause a broad spectrum of clinical conditions. POLγ mutations impair mitochondrial DNA replication, thereby causing deletions and/or depletion of mitochondrial DNA, which in turn impair biogenesis of the oxidative phosphorylation system. We here identify a patient with a homozygous p.F907I mutation in POLγ, manifesting a severe clinical phenotype with developmental arrest and rapid loss of skills from 18 months of age. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities, Southern blot of muscle mtDNA demonstrated depletion of mtDNA and the patient deceased at 23 months of age. Interestingly, the p.F907I mutation does not affect POLγ activity on single-stranded DNA or its proofreading activity. Instead, the mutation affects unwinding of parental double-stranded DNA at the replication fork, impairing the ability of the POLγ to support leading-strand DNA synthesis with the TWINKLE helicase. Our results thus reveal a novel pathogenic mechanism for POLγ-related diseases.

Diagnostic value ultrasound signs of stones less than or equal to 10 mm and clinico-radiological variants of ureteric colic.

Objective: To determine the diagnostic value of ultrasound signs of urinary stones less than or equal to 10 mm and to determine clinico-radiological variants of ureteric colic. Methods: A total of 455 ultrasound investigations were performed in patients referring to emergency department with urolithiasis and symptoms suspected of ureteric colic between January 2021 and May 2021. In addition to microscopic evaluation of urine sediment to detect different crystals and non-contrast spiral computed tomography to detect stones, B-mode and color Doppler sonography was performed to assess the presence of acoustic shadow (AS) and twinkle artifacts (TA) as possible signs of stone(s) in ureter. Results: While the sensitivity and specificity of AS and TA were higher than 90% in patients with stones greater than 5 mm; positive prognostic values of these parameters were found to be extremely low for stones with sizes of 1-3 mm with specificity and sensitivity values not exceeding 53%. The sensitivity and specificity of AS and TA in the upper and lower ureters were higher for stones greater than or equal to 5 than for compared to those less than 5 mm. At the same time, the diagnostic values of TA and AS for middle ureter stones were very limited. The most prevalent clinico-radiological variants of ureteric colic were types I, III, and V being observed in 39%, 28% and 21% cases, respectively. Conclusion: Our results demonstrate that TA and AS parameters seem to have a very low sensitivity and specificity in the diagnosis of urinary stones less than 5 mm. The diagnostic value of TA and AS increase significantly in stones greater than or equal to 5 mm. Therefore, clinicians need to be very careful for overestimating the diagnostic values of TA and AS for stones less than 5 mm and non-contrast spiral computed tomography must be the method of choice for patients presenting to emergency department with ureteric colic.

Twinkle artifact in sonographic breast clip visualization.

The 'twinkle' or 'twinkling' artifact represents a phenomenon observed using color Doppler ultrasound that leads to a rapid alternation of color in and immediately behind an echogenic and highly reflective object. It occurs during sonographic examination of kidney stones, and has been also described in clips used for marking breast and axillary lesions.

The N-terminal domain of human mitochondrial helicase Twinkle has DNA-binding activity crucial for supporting processive DNA synthesis by polymerase γ.

Twinkle is the ring-shaped replicative helicase within the human mitochondria with high homology to bacteriophage T7 gp4 helicase-primase. Unlike many orthologs of Twinkle, the N-terminal domain (NTD) of human Twinkle has lost its primase activity through evolutionarily acquired mutations. The NTD has no demonstrated activity thus far; its role has remained unclear. Here, we biochemically characterize the isolated NTD and C-terminal domain (CTD) with linker to decipher their contributions to full-length Twinkle activities. This novel CTD construct hydrolyzes ATP, has weak DNA unwinding activity, and assists DNA polymerase γ (Polγ)-catalyzed strand-displacement synthesis on short replication forks. However, CTD fails to promote multikilobase length product formation by Polγ in rolling-circle DNA synthesis. Thus, CTD retains all the motor functions but struggles to implement them for processive translocation. We show that NTD has DNA-binding activity, and its presence stabilizes Twinkle oligomerization. CTD oligomerizes on its own, but the loss of NTD results in heterogeneously sized oligomeric species. The CTD also exhibits weaker and salt-sensitive DNA binding compared with full-length Twinkle. Based on these results, we propose that NTD directly contributes to DNA binding and holds the DNA in place behind the central channel of the CTD like a "doorstop," preventing helicase slippages and sustaining processive unwinding. Consistent with this model, mitochondrial single-stranded DNA-binding protein (mtSSB) compensate for the NTD loss and partially restore kilobase length DNA synthesis by CTD and Polγ. The implications of our studies are foundational for understanding the mechanisms of disease-causing Twinkle mutants that lie in the NTD.

Structural insight and characterization of human Twinkle helicase in mitochondrial disease.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

The mitochondrial replisome replicates the 16.6 kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

TWNK in Parkinson's Disease: A Movement Disorder and Mitochondrial Disease Center Perspective Study.

BACKGROUND: Parkinsonian features have been described in patients harboring variants in nuclear genes encoding for proteins involved in mitochondrial DNA maintenance, such as TWNK. OBJECTIVES: The aim was to screen for TWNK variants in an Italian cohort of Parkinson's disease (PD) patients and to assess the occurrence of parkinsonism in patients presenting with TWNK-related autosomal dominant progressive external ophthalmoplegia (TWNK-adPEO). METHODS: Genomic DNA of 263 consecutively collected PD patients who underwent diagnostic genetic testing was analyzed with a targeted custom gene panel including TWNK, as well as genes causative of monogenic PD. Genetic and clinical data of 18 TWNK-adPEO patients with parkinsonism were retrospectively analyzed. RESULTS: Six of 263 PD patients (2%), presenting either with isolated PD (n = 4) or in combination with bilateral ptosis (n = 2), carried TWNK likely pathogenic variants. Among 18 TWNK-adPEO patients, 5 (28%) had parkinsonism. CONCLUSIONS: We show candidate TWNK variants occurring in PD without PEO. This finding will require further confirmatory studies. © 2022 Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Mitochondrial DNA homeostasis impairment and dopaminergic dysfunction: A trembling balance.

Maintenance of mitochondrial DNA (mtDNA) homeostasis includes a variety of processes, such as mtDNA replication, repair, and nucleotides synthesis, aimed at preserving the structural and functional integrity of mtDNA molecules. Mutations in several nuclear genes (i.e., POLG, POLG2, TWNK, OPA1, DGUOK, MPV17, TYMP) impair mtDNA maintenance, leading to clinical syndromes characterized by mtDNA depletion and/or deletions in affected tissues. In the past decades, studies have demonstrated a progressive accumulation of multiple mtDNA deletions in dopaminergic neurons of the substantia nigra in elderly population and, to a greater extent, in Parkinson's disease patients. Moreover, parkinsonism has been frequently described as a prominent clinical feature in mtDNA instability syndromes. Among Parkinson's disease-related genes with a significant role in mitochondrial biology, PARK2 and LRRK2 specifically take part in mtDNA maintenance. Moreover, a variety of murine models (i.e., "Mutator", "MitoPark", "PD-mitoPstI", "Deletor", "Twinkle-dup" and "TwinkPark") provided in vivo evidence that mtDNA stability is required to preserve nigrostriatal integrity. Here, we review and discuss the clinical, genetic, and pathological background underlining the link between impaired mtDNA homeostasis and dopaminergic degeneration.

CLPP Depletion Causes Diplotene Arrest; Underlying Testis Mitochondrial Dysfunction Occurs with Accumulation of Perrault Proteins ERAL1, PEO1, and HARS2.

Human Perrault syndrome (PRLTS) is autosomal, recessively inherited, and characterized by ovarian insufficiency with hearing loss. Among the genetic causes are mutations of matrix peptidase CLPP, which trigger additional azoospermia. Here, we analyzed the impact of CLPP deficiency on male mouse meiosis stages. Histology, immunocytology, different OMICS and biochemical approaches, and RT-qPCR were employed in CLPP-null mouse testis. Meiotic chromosome pairing and synapsis proceeded normally. However, the foci number of the crossover marker MLH1 was slightly reduced, and foci persisted in diplotene, most likely due to premature desynapsis, associated with an accumulation of the DNA damage marker γH2AX. No meiotic M-phase cells were detected. Proteome profiles identified strong deficits of proteins involved in male meiotic prophase (HSPA2, SHCBP1L, DMRT7, and HSF5), versus an accumulation of AURKAIP1. Histone H3 cleavage, mtDNA extrusion, and cGAMP increase suggested innate immunity activation. However, the deletion of downstream STING/IFNAR failed to alleviate pathology. As markers of underlying mitochondrial pathology, we observed an accumulation of PRLTS proteins ERAL1, PEO1, and HARS2. We propose that the loss of CLPP leads to the extrusion of mitochondrial nucleotide-binding proteins to cytosol and nucleus, affecting late meiotic prophase progression, and causing cell death prior to M-phase entry. This phenotype is more severe than in mito-mice or mutator-mice.

Twinkle artifact in renal ultrasound, is it a solid point for the diagnosis of renal stone in children?

Background: Twinkle artifact, also known as color Doppler comet-tail artifact, occurs behind very strong, granular, and irregular reflecting interfaces such as crystals, stones, or calcification. This is visualized as a random mixture of red and blue pixels in the high-frequency shift spectrum located deep to the interface. Study results have suggested that the sonographic twinkling artifact may aid in the detection of renal stones with a variety of reference standard imaging modalities, including abdominal radiography, excretory urography, gray-scale sonography, and CT. Material and methods: Our retrospective observational study included children who had undergone abdomen/renal ultrasound for kidneys stones in our radiology department between 2013 and 2019. Presence of the twinkle artifact, and stone numbers and sizes were documented. CT examinations done <3 months prior to or after US were retrospectively assessed to confirm the presence of kidney stones as a reference standard. Results: Thirty-three abdominal renal US scans of 33 patients (21 males, 12 females) fulfilled the entry criteria. The interval between the US and CT was <3 months for all patients. The median overall age of the patients was 4 years (IQR: 3.125, range: 1- 165 months), The median number of days between the US and CT was 13 (IQR: 26, range: 0-81 days). US detected 33 hyperechoic foci suspected to be stones; 26 were confirmed as true positive (i.e. showed the twinkle artifact and were seen in CT), 4 were false positive (showed the twinkle artifact but were not seen in CT), and 3 were false negative (did not show the twinkle artifact but were seen in CT). The overall median stone size was 2 mm in the right kidney, and 5 mm in the left kidney (IQR: 6,11 mm), respectively. Twinkle artifact sensitivity was found to be 89.7% (95% CI 39.574%-90%). The twinkle artifact was assessed in all true-positive stones, determining a relatively high PPV of 26/29 (86.7%) for the twinkle artifact. The twinkle artifact was not dependent on stone size. Specificity for the twinkle artifact could not be calculated due to a lack of true negatives. Conclusion: The twinkle artifact is a sensitive US tool for detecting pediatric kidney and ureter stones, but with a small risk of false positive findings.

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.

Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse.

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Binding Protein.

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.

Does twinkle artifact truly represent a kidney stone on renal ultrasound?

INTRODUCTION: Nephrolithiasis is detected on ultrasound by the presence of an echogenic focus, posterior acoustic shadowing and/or twinkle artifact (TA). TA has been shown to be highly predictive of nephrolithiasis in adults with renal colic and ureteral stones. We sought to evaluate if TA is reliable for diagnosing nephrolithiasis in the pediatric population. METHODS: We reviewed renal ultrasound reports indicating presence or absence of TA associated with a single echogenic focus in the kidney or ureter. Exclusion criteria were age >18, multiple echogenic foci or medullary calcinosis, no follow-up, or TA located outside the kidney or ureter. Stone was confirmed either by CT within 3 months of color Doppler ultrasound, visualization on ureteroscopy, or patient report of passing the stone. RESULTS: Five hundred and ninety-nine ultrasound reports were reviewed and 293 met inclusion criteria. Sixty-nine had diffuse twinkle without echogenic focus and 224 showed TA with single echogenic focus. 135 patients had confirmatory information available (Summary Table). Nephrolithiasis was diagnosed using TA and confirmed on confirmatory studies for 49 ultrasounds. The majority of confirmed stones were in the kidney (n = 40; 82%) and mean size of confirmed stones on ultrasound was 5 mm (range 1.5-10). Sensitivity, specificity, positive predictive value and negative predictive value of TA for detecting nephrolithiasis were 83%, 78%, 74% and 86% respectively. CONCLUSIONS: Compared to the adult literature, TA in children has lower sensitivity, specificity and positive predictive value, but similar negative predictive value for diagnosing nephrolithiasis. This may be related to renal location and smaller stone size. The presence of TA should be weighed in the setting of other clinical and radiographic evidence of nephrolithiasis.

Use of the color Doppler twinkle artifact for teaching ultrasound guided peripheral vascular access.

BACKGROUND: The optimal method for teaching ultrasound guided peripheral IV (USGPIV) insertion is unknown. Poor needle tip visualization has been cited for USGPIV failure. Twinkle artifact (TA), visualized with color Doppler, is used in other clinical settings. Our objective was to investigate whether teaching students USGPIV placement utilizing TA would enhance needle tip visualization and improve first pass success. METHODS: This was a prospective, randomized study of premedical and preclinical medical students without prior USGPIV experience. Students were given a standardized didactic session on USGPIV placement before being randomized and separated to learn and practice USGPIV with or without TA (control). The students were given 5 min to perform USGPIV on phantom models. The primary outcome was the rate of first pass success. Secondary outcomes included total time to cannulation, rate of posterior venous wall puncture, and total number of attempts. RESULTS: Rates of first pass success were similar in both the TA (82%) and control groups (57%), p = 0.095. There was a difference in the mean time to cannulation. The TA group achieved success at 50.76 s (SD 26.93) while the control group achieved success at 85.30 s (SD 65.47), p = 0.048. CONCLUSION: In this study of utilizing TA to aid in USGPIV placement, students were able to achieve successful cannulation in a shorter amount of time. There was no significant difference in first pass success. Future studies should utilize a larger sample size and evaluate the utility of TA when placing USGPIV on patients.

Structural Variation of the Turtle Mitochondrial Control Region.

The present study describes the most comprehensive comparison of turtle mtD-loop regions to date. The primary structure was compared from DNA sequences accessed from GenBank from 48 species in 13 families of extant turtles, and secondary structures of the mtD-loop region were inferred from thermal stabilities, using the program Mfold, for each superfamiliy of turtles. Both primary and secondary structures were found to be highly variable across the order. The Cryptodira showed conservation in the primary structure at conserved sequence blocks (CSBs), but the Pleurodira displayed limited conservation of primary structural characters, other than the coreTAS, a binding site for the helicase TWINKLE, which was highly conserved in the Central and Right Domains across the order. No secondary structure was associated with a TAS, but an AT-rich fold (secondary structure) near the 3' terminus of the mtD-loop region was detected in all turtle superfamilies. Mapping of character states of structural features of the mtD-loop region revealed that most character states were autapomorphies and inferred a number of homoplasies. The Left Domain of turtles, containing no highly conserved structural elements, likely does not serve a functional role; therefore, the Central Domain in turtles is likely equivalent to the Left Domain of mammals. The AT-rich secondary structural element near the 3' terminus of the mtD-loop region may be conserved across turtles because of a functional role, perhaps containing the Light Strand Promotor, or perhaps interacting with the TWINKLE-coreTAS complex in the Central and Right Domains to regulate mtDNA replication and transcription.

The 'twinkle' artifact - A novel method of clip identification to facilitate targeted axillary surgery following neoadjuvant chemotherapy in breast cancer patients.

OBJECTIVES: A targeted surgical approach which accurately determines axillary status after neoadjuvant chemotherapy (NAC) may allow appropriate de-escalation of surgical treatment. It is postulated that the combined use of a particular marker clip, ULTRACOR®TWIRL™ (Bard, Inc.), and specific ultrasound settings for its identification, may overcome the challenges of pre-operative localisation with size diminution of nodes following NAC. Hence, this feasibility study was performed. METHODS: Three patients with locally advanced breast cancer (LABC), who had the Twirl marker clip placed in biopsy-proven metastatic axillary lymph nodes and completed treatment were included in this preliminary study. Preoperative sonographic identification of the clip and associated node was done on the day of surgery, confirmed by eliciting the 'twinkle' artifact, followed by hookwire localisation and resection. The 'twinkle' artifact was demonstrated within each specimen after resection. RESULTS: All three patients who completed treatment for LABC had successful identification and resection of clipped node aided by the 'twinkle artifact'. CONCLUSION: The 'twinkle' artifact is a novel and useful aid in identifying the Twirl™ clip marking a lymph node in the post-NAC setting, with the potential to be used intraoperatively.

A novel mutation of Twinkle in Perrault syndrome: A not rare diagnosis?

Perrault syndrome is a rare disorder characterized by ovarian dysgenesis, bilateral sensorineural hearing loss and associated with mutations in six mitochondrial proteins. Additional neurological features were also described. Herein, we report on a 27-year-old woman with Perrault syndrome (PS), moderate ataxia and axonal sensory-motor peripheral neuropathy in whom we identified compound heterozygous mutations in the TWNK gene (p.Val507Ile and the novel p.Phe248Ser variant). Fewer than 30 patients with PS have been reported worldwide. Neurological involvement is more frequently associated with mutations in TWNK and indicates possible genotype-phenotype correlations. TWNK mutations should be searched in patients with sensory ataxia, early onset bilateral sensorineural hearing loss, and ovarian dysfunction in women.