Pipis, Menelaos

Poh, Roy

Polke, James M

Record, Christopher J

Reilly, Mary M

English

UCL Discovery

Beware next generation sequencing gene panels as the first line genetic test in Charcot-Marie-Tooth disease Christopher J Record1, Menelaos Pipis1, Roy Poh2, James M Polke2, Mary M Reilly1 1 Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK  2 Neurogenetics Laboratory, UCL Queen Square Institute of Neurology, London, UK  Corresponding author: Professor Mary M Reilly. Centre for Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, WC1N 3BG, UK. m.reilly@ucl.ac.uk  ORCID Christopher Record 0000-0002-9802-2683 Menelaos Pipis 0000-0003-0511-6515 Mary Reilly 0000-0003-0686-905X  Author contributions CJR study design, acquisition, analysis, and interpretation of data and drafting of manuscript. MP, RP, JMP: acquisition, analysis and interpretation of data and revision of manuscript. MMR: study design and revision of manuscript.  Word count: 670 Reference count: 2   Dear Editor, The testing strategy for genetic conditions has evolved in recent years. Initially, sequential single gene tests were the mainstay. This was followed by gene panels performed through targeted gene panel sequencing. Now in many countries ‘virtual panels’ are applied to whole exome (WES) or whole genome sequencing (WGS) as first line tests, where multiple genes can be tested in parallel. Improved reliability and cost efficiency of WES or WGS, combined with advancing bioinformatic technology, mean that next generation sequencing (NGS), which includes WES and WGS, is preferable. One exception is for diseases where there is a common genetic diagnosis, and a single gene test is still more cost-efficient e.g. Charcot-Marie-Tooth disease (CMT) type 1A. Another is where the pathogenic genetic defects are not easily detectable with NGS, including some complex copy number variants (CNVs; large deletions, duplications, rearrangements or translocations e.g. deletion of exon 7 and 8 of SMN1 in spinal muscular atrophy) or repeat expansions (e.g. amyotrophic lateral sclerosis caused by repeat expansion in C9orf72). Many CNVs are now reliably detected by bioinformatic pipelines, but historical pipelines were less robust and CNVs were missed. For these reasons, multiplex ligation-dependent probe amplification (MLPA) is still commonly used as the first line single gene test in these settings. CMT1A is by far the most common form of CMT, accounting for up to 62% of genetic diagnoses.[1] It is caused by a 1.5Mb duplication in the short arm of chromosome 17, incorporating PMP22. For this reason, in patients with demyelinating CMT (CMT1), MLPA of chromosome 17 remains the first line genetic test in most laboratories. In the past, other methods have been employed including microsatellite analysis, though this method comes with around a 2% false negative rate.[2] In the last three years our specialist service has diagnosed CMT1A in three patients referred for a diagnostic opinion because the common genetic causes of CMT had been excluded, in whom we have unexpectedly detected PMP22 duplication through NGS. The first was a patient in their 60s with typical CMT1, in whom no sequencing variants were detected in our CMT panel, though abnormal PMP22 dosage was flagged through our diagnostic laboratory’s CNV analysis of exome sequencing. The assumption was made that the patient had previously undergone PMP22 dosage analysis, since a CMT ‘genetic screen’ had been performed prior to referral; in the UK this usually includes MLPA for CMT1A and a NGS CMT gene panel. The second, another patient in their 60s, was referred with a diagnosis of severe axonal CMT (CMT2) and had been enrolled in the 100,000 Genomes Project (100K GP) with no primary findings detected. Our neurophysiology surprisingly demonstrated a demyelinating neuropathy, and analysis of the 100K GP data in the research environment showed 1.5x the read depth of PMP22 compared with other parts of the genome (Figure 1). Lastly, a patient in their 40s was referred with a ‘normal PMP22 dosage’. On assessment the patient’s phenotype was typical for CMT1A. Reviewing the original PMP22 dosage test, done in an external laboratory, we noted this was negative but had been done by microsatellite analysis. They had been enrolled in the 100K GP, again with no primary findings reported, and retrospective review of this data suggested PMP22 duplication, as seen in the second case. MLPA subsequently confirmed PMP22 duplication in all three cases. In conclusion, our cases act as a reminder to neurologists and geneticists, in an era of gene therapies where a molecular diagnosis is more important than ever, that NGS is not always the right test. Firstly, careful review of prior testing (‘Has PMP22 dosage been done?’) can avoid unnecessary further expensive tests, and the possibility that the diagnosis will still be missed (Case 1). Secondly, patient phenotype is critical to guide testing (‘Is the clinical diagnosis correct’?); MLPA is still the first line test for CMT1A (Case 2). Lastly, the method of prior testing must be scrutinised (‘Exactly what test was done?’), considering that both microsatellite analysis and older NGS pipelines can miss the PMP22 duplication (Case 3).   Acknowledgements MMR is grateful to the Medical Research Council (MRC MR/S005021/1), the National Institutes of Neurological Diseases and Stroke and office of Rare Diseases (U54NS065712 and 1UOINS109403-01 and R21TROO3034), Muscular Dystrophy Association (MDA510281) and the Charcot Marie Tooth Association (CMTA) for their support. The INC (U54NS065712) is a part of the NCATS Rare Diseases Clinical Research Network (RDCRN). This research was also supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre (MMR). Part of this research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support.   Ethics statements Ethics approval: Participant data was collected in line with the ethically approved study ‘Charcot-Marie-Tooth Disease and related disorders: A Natural History Study’, reviewed by the London Queen Square Research Ethics Committee (REC No.: 09/H0716/61). Patient consent for publication: not applicable   Funding CJR was supported by an MRC strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) MR/S005021/1 and the National Institutes of Neurological Diseases and Stroke and office of Rare Diseases (U54NS065712).   Competing interests None declared.   References 1  Fridman V, Bundy B, Reilly MM, et al. CMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: A cross-sectional analysis. J Neurol Neurosurg Psychiatry 2015;86:873–8. doi:10.1136/jnnp-2014-308826 2  Rowland JS, Barton DE, Taylor GR, et al. A comparison of methods for gene dosage analysis in HMSN type 1. J Med Genet 2001;38:90–5. doi:10.1136/jmg.38.2.90 Figure 1 Integrated Genomics Viewer (IGV) showing our patient’s bam file with 1.5x the read depth of PMP22 (right pane) compared with other parts of the genome (chromosome 1, left pane). This indicates a duplication of chromosome 17 in the region of PMP22, confirmed by multiplex ligation-dependent probe amplification. 

Beware next-generation sequencing gene panels as the first-line genetic test in Charcot-Marie-Tooth disease

https://discovery.ucl.ac.uk/10160888/2/Record_Beware%20next-generation%20sequencing%20gene%20panels%20as%20the%20first-line%20genetic%20test%20in%20Charcot-Marie-Tooth%20disease_AAM.pdf

Beware next-generation sequencing gene panels as the first-line genetic test in Charcot-Marie-Tooth disease

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