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Next-Generation Sequencing Approach to Hyperckemia a 2-Year Cohort Study

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Next-Generation Sequencing Approach to Hyperckemia a 2-Year Cohort Study ARTICLE OPEN ACCESS Next-generation sequencing approach to hyperCKemia A 2-year cohort study Anna Rubegni, MD, Alessandro Malandrini, MD, Claudia Dosi, MD, Guja Astrea, MD, Jacopo Baldacci, BS, Correspondence Carla Battisti, MD, Giulia Bertocci, BSc, M. Alice Donati, MD, M. Teresa Dotti, MD, Antonio Federico, MD, Dr. Santorelli [email protected] Fabio Giannini, MD, Salvatore Grosso, MD, Renzo Guerrini, MD, Sara Lenzi, MD, Maria A. Maioli, MD, Federico Melani, MD, Eugenio Mercuri, MD, Michele Sacchini, MD, Simona Salvatore, MD, Gabriele Siciliano, MD, Deborah Tolomeo, MD, Paola Tonin, MD, Nila Volpi, MD, Filippo M. Santorelli, MD, and Denise Cassandrini, PhD Neurol Genet 2019;5:e352. doi:10.1212/NXG.0000000000000352 Abstract Objective Next-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic or paucisymptomatic hyperCKemia to investigate whether this technique might allow detection of the genetic basis of the condition. Methods Sixty-six patients with undiagnosed asymptomatic or paucisymptomatic hyperCKemia, referred to tertiary neuromuscular centers over an approximately 2-year period, were analyzed using a customized, targeted sequencing panel able to investigate the coding exons and flanking intronic regions of 78 genes associated with limb-girdle muscular dystrophies, rhabdomyolysis, and metabolic and distal myopathies. Results A molecular diagnosis was reached in 33 cases, corresponding to a positive diagnostic yield of 50%. Variants of unknown significance were found in 17 patients (26%), whereas 16 cases (24%) remained molecularly undefined. The major features of the diagnosed cases were mild proximal muscle weakness (found in 27%) and myalgia (in 24%). Fourteen patients with a molecular diagnosis and mild myopathic features on muscle biopsy remained asymptomatic at a 24-month follow-up. Conclusions This study of patients with undiagnosed hyperCKemia, highlighting the advantages of NGS used as a first-tier diagnostic approach in genetically heterogeneous conditions, illustrates the ongoing evolution of molecular diagnosis in the field of clinical neurology. Isolated hyper- CKemia can be the sole feature alerting to a progressive muscular disorder requiring careful surveillance. From the IRCCS Fondazione Stella Maris (A.R., G.A., J.B., G.B., S.L., F.M.S., D.C.), Pisa, Italy; Department of Medicine (A.M., C.B., M.T.D., A.F., F.G., S.S., N.V.), Surgery and Neurosciences, University of Siena; Department of Clinical and Experimental Medicine (C.D., G.S., D.T.), University of Pisa; Metabolic Disease Unit (M.A.D., M.S.), AOU Meyer Children Hospital, Florence; Department of Molecular and Developmental Medicine (S.G.), University of Siena, Siena; Pediatric Neurology (R.G., F.M.), AOU Meyer Children Hospital, Florence; Neurophysiopathology Multiple Sclerosis Center Hospital Binaghi (M.A.M.), Cagliari; Pediatric Neurology and Nemo Clinical Centre (E.M.), Fondazione Policlinico Universitario “A. Gemelli IRCSS”, Universit`a Cattolica del Sacro Cuore, Rome; and Department of Neurosciences (P.T.), Biomedicine and Movement Sciences, University of Verona, Italy. Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary CK = creatine kinase; EFNS = European Federation of Neurologic Societies; HPO = Human Phenotype Ontology; LGMD = limb-girdle muscular dystrophy; NGS = next-generation sequencing; ULN = upper limit of normal; VUS = variants of unknown significance. Creatine kinase (CK) levels can be mildly and transiently the EFNS criteria) were consecutively referred to the neu- increased as a result of muscle injury or exercise, even in rology, pediatric, or neuropediatric units of 7 Italian tertiary healthy individuals. Persistent elevation of serum CK (termed neuromuscular centers for clinical and diagnostic purposes. hyperCKemia) is defined, according to European Federation All 66 met the inclusion criteria for our study: (1) persistent of Neurological Societies (EFNS) guidelines (ean.org/ serum CK elevation at rest and (2) values higher than twice Guideline-Reference-Center.2699.0.html), as the presence of the ULN on at least 2 occasions after refraining from muscular serum CK values beyond 1.5 times the upper limit of normal exercise for at least 72 hours before CK measurement. For (ULN) in least 2 measurements,1 and it is a common reason each patient, we collected clinical and laboratory data and the for referrals to specialized neuromuscular centers. results of familial segregation analyses and previous genetic tests. MRI scans of thigh and calf muscles were performed in Increased serum CK can be present in the absence of obvious 26 patients, EMG in 26, and muscle biopsy in 57, in all cases clinical signs.2 In individuals with a normal neurologic ex- using routine clinical methods.5 Before this study, we had amination, the condition is termed asymptomatic or isolated performed multiplex ligation-dependent probe amplifica- hyperCKemia, and it can signal the presence of several un- tion analysis in all the patients to exclude multiexon rear- suspected metabolic, cardiac, rheumatic, or endocrine con- rangements in the DMD gene and tested the levels of acid ditions. When no underlying cause is found, it is referred to as alpha-glucosidase from dried blood spots6 to detect possible idiopathic hyperCKemia. The clinical management of idio- undiagnosed late-onset Pompe disease. Patients with bor- pathic hyperCKemia is unclear; although clinically asymp- derline enzyme values or biallelic mutations in the GAA tomatic, affected patients are potentially susceptible to gene were not included in this study. DNA samples were malignant hyperthermia.3 analyzed at a single center. In asymptomatic hyperCKemia, definition of the correct di- None of the 66 individuals had a medical history of anesthesia- agnosis may be time consuming, and success is not guaran- related complications or a family history of cardiovascular teed. Next-generation sequencing (NGS) has recently been events. Any family history of neuromuscular disorders or mild proposed as a cost-effective strategy for the molecular di- muscle complaints was carefully recorded. Occasional causes of agnosis of inherited neuromuscular disorders.4 The efforts to hyperCKemia, such as malignancies, drug and alcohol abuse, define the molecular etiology in hyperCKemia come from the rheumatic, thyroid, and parathyroid disorders, infections, and need to refine follow-up avoiding unnecessary examinations hematologic diseases, were all excluded. All patients underwent and to improve counseling in the family. We set out to explore routine serum chemistry, including serum myoglobin mea- whether NGS might allow detection of the molecular basis of surement. No participant was under statin treatment or taking hyperCKemia, addressing this question in a consecutive series other drugs potentially capable of inducing hyperCKemia. of adults and children recruited at 7 Italian tertiary neuro- muscular centers over an approximately 2-year period. NGS workflow and sequencing analyses We used the SureSelect technology (Agilent, Santa Clara, CA) and SureDesign software (earray.chem.agilent.com/ Methods suredesign/) to design a multiexon amplicon panel containing Standard protocol approvals, registrations, a total of 78 genes known to be associated with limb-girdle and patient consents muscular dystrophies (LGMDs), rhabdomyolysis, and meta- 7 This study was approved by the Tuscany Regional Pediatric bolic and distal myopathies ; the panel spanned more than Ethics committee. All the procedures complied with the 259 Mbp, with gene coverage >99%. To analyze the data Helsinki Declaration of 1975. Genetic studies and muscle obtained from our study, we used a routine bioinformatic 8 biopsies were performed with written informed consent. All pipeline that adopts the Ingenuity Variant analysis suite participants (including parents or legal guardians in case of (Qiagen, apps.ingenuity.com). To assign pathogenicity, we minor patients) were provided pre- and post-test genetic set up a precise Alissa (Agilent) pipeline using the following counseling as routine in our neurogenetic clinics. criteria: a sequence quality score greater than 30, a read depth greater than 30, and rare occurrence in publicly available Patients and study design polymorphic data sets (with a minor allele frequency <0.01% Over an approximately 2-year period (May 2016–August for autosomal dominant and <0.1% for autosomal recessive 2018), 66 patients presenting with hyperCKemia (meeting genes) with less than 1 occurrence in homozygosity in 2 Neurology: Genetics | Volume 5, Number 5 | October 2019 Neurology.org/NG gnomADv2.1 (gnomad.broadinstitute.org/; macarthurlab. Most of the patients were Italian, adults (≥16 years), and ap- org/2018/10/17/gnomad-v2-1). As reported previously, we parently sporadic cases. Although none of them had obvious determined predictably or probably deleterious scores using neuromuscular disorders, 16 had a positive family history of an in silico pipeline using
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