<scp>QSM</scp> ‐Based Evidence of Brain Iron Accumulation in <scp> <i>THAP1</i> </scp> Dystonia with Biallelic Mutation

The thanatos-associated protein domain-containing apoptosis-associated protein 1 (THAP1) gene was initially identified in Amish–Mennonite families with craniocervical dystonia, referred to as DYT6 dystonia.1, 2 Patients often present with craniocervical or limb-onset segmental dystonia during adolescence. While conventional MRI is often unremarkable, recent studies show iron accumulation in the globus pallidus in some THAP1 cases.3, 4 Advanced MRI techniques have enabled the quantitative assessment of brain iron levels.5 Accordingly, this study aims to assess iron accumulation in a patient with recessive THAP1-related dystonia compared to healthy controls using quantitative susceptibility mapping (QSM). A 13-year-old right-handed girl born to consanguineous parents (first cousins) was referred to assessment for treatment-refractory dystonia. Her older brother and two older sisters were neurologically unaffected. Dystonic contractions began in her left hand at age six and spread gradually over 2 years to her right foot, neck, cranial muscles, right hand, and eventually all extremities. Neurological examination revealed dysphonic speech and impaired saccadic, pursuit, and optokinetic eye movements. She exhibited bradykinesia and generalized dystonia, predominantly affecting the cervical and cranial regions. Axial dystonia presented as rightward truncal deviation and left shoulder elevation, along with distal limb involvement. Occasional blepharospasm, jaw-opening dystonia, and risus sardonicus were noted. Due to severe dystonia, she was unable to walk even with support and had poor sitting balance. Her Burke-Fahn- Marsden Dystonia Rating scale socre (BFMDRS) was 94.5. The difficulty in sitting was therefore attributed to dystonia rather than ataxia, and no clinical signs of cerebellar dysfunction were observed. Nerve conduction and needle EMG studies were also unremarkable. CSF neurofilament level was elevated (497.5 pg/ml; cut-off >380 for age <30 years). The cranial CT scan was normal, without signs of basal ganglia calcification. Brain MRI showed bilateral T2- and SWI-hypointensities in the globus pallidus. Levodopa (62.5 mg/day) induced tongue dyskinesias and was discontinued. Trihexyphenidyl titrated to 15 mg/day was ineffective. Over the two-year follow-up period after implantation of combined GPi and STN DBS, STN stimulation was discontinued due to limited benefit, and only GPi DBS remained active. The patient has been more comfortable under GPi-only stimulation with a BFMDRS score of 87.5, without any stimulation-induced side effects (Table S1). In the proband who underwent WES, a homozygous missense variant in the THAP1 gene (ENST00000254250.7:c.149A>G; p.Tyr50Cys) was identified and classified as likely pathogenic (PM1, PM2, PP3_Moderate). Segregation analysis using Sanger sequencing confirmed heterozygosity for the variant in both parents and the elder brother. Pedigree analysis showed that the proband was homozygous for the variant, while individuals marked with a plus sign (+) were identified as heterozygous carriers (Fig. S1). QSM revealed increased susceptibility in the globus pallidus bilaterally, corresponding to hypointensity on T2*-weighted images in the patient (Fig. 1B,D). Detailed QSM methodology is provided in the Supplementary Methods. The susceptibility values in all the DGM regions of the patient were higher than the average of the control group; however, a statistically significant difference was only detected in the globus pallidus externus. The DGM volumes in the patient were smaller compared to the control group, particularly in the putamen; however, this difference was not statistically significant (Fig. 1E,F). In our study, we provide the first confirmation of iron accumulation in recessive THAP1 mutations using QSM, thereby validating this association through a novel imaging method. However, it remains unclear whether iron accumulation differs between homozygous and heterozygous THAP1 mutations. Within the NBIA spectrum, the patterns and extent of iron accumulation are not yet clearly defined. While this study confirms brain iron accumulation in recessive THAP1 mutation cases, it also highlights the value of QSM as a diagnostic tool in the NBIA group. Additional interpretation and clinical implications of these findings are discussed in detail in the Supplementary Discussion. Approved by the Ethics Committee of Istanbul University, Istanbul Faculty of Medicine (Meeting No: 24, Date: December 1, 2023), for the study titled “Laboratory Evaluation of Findings and Genetic Biomarker Determination in Neurodegenerative Diseases with Brain Iron Accumulation (NBIA)” (File number: 2023/513). Consent for publication was obtained from the patient's family. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. The authors declare no conflict of interest. (1) Research project: A. Conception, B. Organization, C. Execution. (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique. (3) Manuscript Preparation: A. Writing of the first draft, B. Review and Critique. ZY: 1A, 1B, 1C, 3A, 3B, 4. PD: 1A, 1B, 2A, 2C, 3B, 4. GT: 2A, 2B, 3A, 3B. IK: 1C, 3A. NC: 1C. CS: 1C. CT: 1C. SAUI: 3A, 3B. We would also like to extend our sincere thanks to Dr. Pınar Topaloğlu, Dr. Semih Taşdelen, Dr. Zeynep Karakuş, Dr. Pınar Elkoca, Dr. Özgür Öztop Çakmak, Dr. Selçuk Peker, Dr. Yavuz Samancı, and Dr. Erdi Şahin for their valuable contributions. This study was supported in part by the İstanbul University Scientific Research Projects Coordination Unit (Project ID: 40651; Project Code: TSA-2024-40651). The authors declare that there are no conflicts of interest relevant to this work. GT has received financial support in the form of an educational scholarship from the Turkish Neurology Society. ZY, PD, İK, NÇ, CS, ÇT, SAUİ: The authors declare that there are no additional disclosures to report. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1. Supplementary Discussion: Extended discussion of the clinical and imaging findings with reference to relevant literature. Data S2. Supplementary Methods: Detailed description of the QSM methodology used in this study. TABLE S1. Functional and motor scores before and after deep brain stimulation (DBS) are presented. Following the implantation of combined GPi and STN DBS, STN stimulation was discontinued due to limited benefit, and only GPi DBS remained active during the two-year follow-up. The patient was more comfortable under GPi-only stimulation, with a BFMDRS score of 87.5 (improved from 94.5), and without any stimulation-induced side effects. Arrows (↑/↓) indicate worsening or improvement, respectively. Figure S1. Pedigree of the family. The proband, indicated by an arrow, is homozygous for the THAP1 variant (ENST00000254250.7:c.149A>G; p.Tyr50Cys). Individuals marked with a plus sign (+) underwent targeted variant analysis and were all found to be heterozygous carriers of this variant. Individuals IV-5 and IV-6 died at 7 and 8 months of age, respectively, before genetic testing could be performed. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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