<i>PRKRAP1</i> Pseudogene Complicating the Diagnosis of Young‐Onset Dystonia Due to <i>PRKRA</i> Gene Disease‐Causing Variants (<scp>DYT‐</scp><i>PRKRA</i>)

A genetic etiology of isolated dystonia is suspected if there is an early-onset dystonia or a positive family history. However, the current overall yield of a genetic etiological diagnosis remains below 30%.1 We present a case of a 13-year-old Caucasian male, the only child from a non-consanguineous couple, without family history of neurological diseases, with no previous medical history, and normal psychomotor development. At the age of 8, he started developing involuntary twisting movements that resulted in abnormal postures, first affecting his right lower-limb, then progressing to cervical region and right upper-limb, mildly impairing his daily activities and manifesting mostly while exercising. At 9 years old, the dystonic movements progressed to left hemi-body and axial region, and he developed a slight slurred speech. There were no cognitive or behavioral symptoms, and he maintained school performance with adjustments. At the age of 11, his examination revealed a generalized dystonia (Fig. 1, Video 1) resulting in gait impairment, frequent falls, and important disruption of daily activities. There were no ophthalmological abnormalities, other cranial nerve signs, pyramidal signs, parkinsonism, cerebellar features, or myoclonus. Initial therapeutic management included levodopa/carbidopa (up to 300 mg per day), clonazepam, baclofen, and tetrabenazine, all without sustained response. Complementary examination included a normal brain and cervical magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis. Laboratory work-up showed normal hepatic function, negative systemic autoimmunity, normal urinary, and serum copper studies and serum ceruloplasmin. Abdominal ultrasound was unremarkable. A next-generation-sequencing (NGS) exome-based panel for dystonia was performed with the results summarized in Table 1. Our focus was initially on the ATP7B gene variants because the therapeutic approach is significantly different. However, his clinical presentation, copper kinetics, and brain MRI ruled out Wilson's disease. Considering age of onset and clinical presentation with prominent cervical dystonia, associated with truncal and limb involvement, in the absence of development delay, cognitive regression or other neurological signs, genetic causes of dystonia were the most likely diagnosis—in particular, mutations on DYT-TOR1A, DYT-THAP1, DYT-KMT2B, DYT-PRKRA, DYT-COL6A3, and DYT-GNAL. Despite no definite molecular diagnosis at that time, the absence of levodopa (l-dopa) response, an insufficient response to other agents and the increased disability prompted the quest for other dystonia treatments. He was submitted to deep-brain-stimulation (DBS) surgery at 11, targeting bilateral globus pallidus internus (GPi), with a good motor response (Video 2). Nonetheless, the results of his genetic tests remained inconclusive. Both DYT-PRKRA and DYT-COL6A3 were possibilities, however, the COL6A3 gene variant was of uncertain significance, whereas the PRKRA gene variant had been previously described as pathogenic, but was present in heterozygous state. The investigation for possible etiologies that may have escape standard NGS or Sanger sequencing lead the genetic lab to clarify the contribution of the PRKRAP1 pseudogene for the heterozygous NGS result in this particular patient.2 Polymerase chain reaction (PCR) amplification with intronic flanking primers followed by Sanger sequencing allowed the detection of the variant in the other allele of the functional PRKRA gene, confirming the homozygous state for the c.665C>T;p.(Pro222Leu), and establishing a final diagnosis of DYT-PRKRA. NGS revolutionized the molecular diagnosis of dystonia, because it allows the generation of enormous amounts of sequence data in a short time at an affordable cost, providing a high quality and efficient replacement for conventional sequencing.3 The first step in NGS consists in enrichment of the target gene sequences by multiplex PCR amplification or probe hybridization. If a PCR-based strategy is selected, specific PCR primers are designed, whereas if a capture approach is used, the DNA should be fragmented first and then specific probes are designed to capture each fragment of the target. During this step is crucial to amplify and/or capture selectively only the active gene. The NGS data analysis pipelines are not able to determine if a given sequence derives from the functional gene or the highly homologous pseudogene. Furthermore, reads generated by the pseudogene sequencing might align with the active gene, resulting in false-positive or false-negative results. Pseudogenes are not a new topic in movement disorders—the most recognized example is GBAP1 pseudogene, which shares a high homology with GBA gene and makes genetic characterization by NGS analysis challenging. This motivated implementation of adequate strategies to avoid misdetection of GBA recombinant disease-causing variants.3 To our knowledge, this is the first case of DYT-PRKRA reported in the literature, in which the presence of a pseudogene delayed molecular diagnosis. This finding is of utmost clinical importance because it may not routinely be taken into account when the PRKRA gene is analyzed. PRKRA gene mutations were first described in 2008 in Brazilian patients,4, 5 although European descent families were recognized.6, 7 The only case identified in Portugal is of a Brazilian descendent child.8 The clinical presentation of our case is in keeping with one of the phenotypes previously described.9 Despite frequently referred as a dystonia-parkinsonism syndrome, parkinsonism is only reported in approximately half of the cases and is usually a late feature.4, 9 GPi-DBS was reported in four DYT-PRKRA patients,7, 10 but our patient is the youngest described in literature, therefore expanding the number of patients benefiting from surgery. We thank the following members of the technical team, who greatly contributed to the characterization and multidisciplinary care of the patient: Joana Almeida (Psychologist), Alexandra Pedruco (Psychologist), Henriqueta Araújo (Occupational Therapist), Vera Ribeiro (Occupational Therapist), Isabel Lucas (Speech Therapist), and Dulcínia Lages (Special Education Teacher). (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. J.A.R.: 1B, 1C, 3A M.S.: 1A, 1B, 1C, 3A I.A.: 1C, 3B F.M.: 1C, 3B R.P.: 1C, 3B F.P.: 1B, 1C, 3B Ethical Compliance Statement: 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 parents gave written informed consent for publication of their child clinical report, and also for publication of images and videos. Even so, and because of the fact he is a minor, we made our best efforts to anonymize the patient. The authors confirm that the approval of an institutional review board was not required for this work. Funding Sources and Conflicts of Interest: The authors report no sources of funding and no conflicts of interest. Financial Disclosures for the Previous 12 Months: JAR has been participating on advisory boards for Biogen, Roche and Novartis, and received speaking fees from Biogen. FM received speaking honoraria for BIAL. FP received research grants from Biogen, Merck, Roche and Novartis. He has been participating on advisory boards for Merck and Novartis. Fig. S1 Integrative genomics viewer (IGV) alignment, pedigree, and electropherogram of the PRKRA variant described. (A) IGV view of c.665C>T substitution in the proband; (B) Sanger sequencing electropherograms of the homozygous c.665C>T variant in the proband and the presence of the variant in the heterozygous state in both parents. A black circle indicates the affected proband; a circle and square with a black dot correspond to carriers of the c.665C>T variant. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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