ARTICLE Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness A full list of authors and their affiliations appears at the end of the paper. Purpose: Several hundred genetic muscle diseases have been half of the solved cases; while variants in newer disease genes, such described, all of which are rare. Their clinical and genetic as BVES and POGLUT1, were also found. The remaining well- heterogeneity means that a genetic diagnosis is challenging. We characterized unsolved patients (48%) need further investigation. established an international consortium, MYO-SEQ, to aid the Conclusion: Using our unique infrastructure, we developed a work-ups of muscle disease patients and to better understand pathway to expedite muscle disease diagnoses. Our data suggest disease etiology. that exome sequencing should be used for pathogenic variant Methods: Exome sequencing was applied to 1001 undiagnosed detection in patients with suspected genetic muscle diseases, patients recruited from more than 40 neuromuscular disease focusing first on the most common disease genes described here, referral centers; standardized phenotypic information was collected and subsequently in rarer and newly characterized disease genes. for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. Genetics in Medicine (2020) 22:1478–1488; https://doi.org/10.1038/s41436- Results: We identified suspected pathogenic variants in 52% of 020-0840-3 patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, Keywords: next-generation sequencing; targeted exome analysis; RYR1, TTN, COL6A2, and SGCA collectively accounted for over neuromuscular disease; limb-girdle weakness; genetic diagnosis INTRODUCTION genes, respectively, they achieved a diagnostic rate of 43% and Genetic muscle diseases with limb-girdle weakness (LGW) are 27%. However, to date no study has been carried out where highly heterogeneous, rare neuromuscular disorders (NMD). exome sequencing (ES) has been applied to similar limb-girdle In addition to the characteristic progressive pelvic and cohorts; this allowed us not only to determine the impact that shoulder girdle muscle weakness, other manifestations may the panel size has on the detection rate, but also to interrogate include respiratory insufficiency, cardiomyopathy, contrac- the exomes beyond the known NMD genes. tures, and gastrointestinal complications.1 Consequently, these We describe here the application of sequential targeted disorders diminish an individual’s quality of life and can exome sequencing (TES) to a large cohort of undiagnosed reduce life expectancy.2 There is a critical need to fully patients presenting with proximal muscle weakness and/or characterize disease etiology to achieve accurate diagnoses and elevated serum creatine kinase (CK) levels: the MYO-SEQ offer appropriate genetic counseling and disease management. project. We established a partnership between academia, Despite 535 genes known to be associated with 955 distinct industry, and patient organizations, and developed an NMDs,3 many muscle disease patients still remain genetically international network that connected over 40 specialist undiagnosed. This is partly because gene-by-gene and small neuromuscular referral sites. A total of 1001 exomes were panel testing strategies are usually dictated by phenotype, leaving first analyzed for rare pathogenic variants in 169 common little power for the expansion of disease associations and novel NMD genes, and then in a further 260 genes associated with disease characterizations.4 An unbiased methodology has thus rarer muscle conditions. We identify the most common been sought to enhance standard clinical practices. Consistent muscle diseases in this population and suggest that an with an increased affordability over the past decade, next- increased number of patients can be more rapidly tested and generation sequencing (NGS) technologies have been integral in diagnosed through an appropriate TES approach. the diagnosis of disorders such as mitochondrial disease among others.5 Large-scale NGS panels were applied to 504 Italian MATERIALS AND METHODS undiagnosed muscular dystrophy and myopathy patients,6 and Patient recruitment more recently to 4655 limb-girdle muscular dystrophy (LGMD) We utilized the infrastructure established by TREAT-NMD patients in the United States.7 By investigating 93 and 35 NMD (https://treat-nmd.org/) to recruit 43 NMD referral centers Correspondence: Volker Straub (volker.straub@ncl.ac.uk) These authors contributed equally: Ana Töpf, Katherine Johnson Submitted 25 November 2019; revised 5 May 2020; accepted: 8 May 2020 Published online: 11 June 2020 1478 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE ARTICLE from throughout Europe and the Middle East (Figure S1). muscle biopsies further investigated, and clinical histories Singleton patients matching the inclusion criteria of unex- reassessed by the referring clinicians (Figure S2). plained limb-girdle weakness and/or elevated CK were The remaining unsolved cases were subsequently analyzed included in the project. Informed consent was obtained for for likely pathogenic variants in a further 260 genes (Table S2) all patients. Anonymized clinical information was entered on including those associated with neuropathies, cardiomyopa- the PhenoTips platform (https://phenotips.org/) using NMD- thies, and other phenotypes presenting with muscle weakness specific forms. Clinical features, histological findings, electro- to identify rarer neuromuscular conditions. This extended myography results, CK levels, mode of inheritance, and gene list also included genes newly associated with muscle disease onset and progression were recorded using Human disease. ES data were also analyzed for copy-number variants Phenotype Ontology terms. DNA samples were submitted to (CNVs)11 and SMN1 exon 7 deletions12 (Supplementary the Newcastle Medical Research Council (MRC) Centre Materials and Methods). For those cases carrying a single Biobank for Neuromuscular Diseases for which ethical heterozygous pathogenic variant in the most common approval was granted by the National Research Ethics Service (exclusively recessive) genes found in our cohort (i.e., CAPN3, (NRES) Committee North East–Newcastle & North Tyneside DYSF, ANO5, and SGCA) we further interrogated the 1 (reference 08/H0906/28). noncoding regions of the gene captured in the ES data. In addition, for the DYSF carriers we Sanger sequenced two Exome sequencing, data analysis, and interpretation previously reported pathogenic intronic variants.13 Finally, ES was performed at the Broad Institute of MIT and one patient was also subjected to genome sequence (GS) and Harvard’s Genomics Platform (Cambridge, MA, USA) using RNA sequencing (RNA-Seq), as described elsewhere.14 1234567890():,; a 38-Mb targeted Illumina exome capture, as described previously.8 Data were first filtered for variants in 169 NMD- RESULTS causing genes (Table S1), with a minor allele frequency in the Demographics of 1001 patients with suspected genetic ExAC control population9 of ≤1% and a moderate to high muscle disease Ensembl Variant Effect Predictor. The resulting shortlists of One thousand and one patients were recruited into the MYO- single-nucleotide variants (SNVs) and small insertions/ SEQ project, with the majority (93%) inferred to be of deletions (indels) were examined for pathogenicity by two European or Middle Eastern ancestry. The study cohort independent data analysts (M.B./J.D./K.J./M.M./A.T. at the comprised 545 (54%) males and 456 (46%) females and John Walton Muscular Dystrophy Research Centre originated from 972 families. Disease onset for 42% of the [JWMDRC] and L.X./E.M.E./E.V./T.M. at the Broad Insti- patients was in adulthood (Figure S3A) and the mean age at tute). We deemed SNVs and indels pathogenic if they had the time of recruitment was 39 years (Figure S3D). Proximal been previously and consistently reported as pathogenic in muscle weakness, either in isolation or with distal muscle ClinVar (two gold stars review status; https://www.ncbi.nlm. weakness, affected most participants (77%; Figure S3B), while nih.gov/clinvar/) and/or published literature. In line with 68% of the cohort had increased serum CK levels (Figure S3C). American College of Medical Genetics and Genomics Based on family history, 46% of patients were sporadic cases, (ACMG) guidelines,10 pathogenicity of novel variants was while 14%, 7%, and 1% showed recessive, dominant, and X- evaluated based on (1) computational data, i.e., predicted to linked inheritance, respectively; for the remaining 32%, no be deleterious by all, or the majority, of the in silico tools used; indication of inheritance pattern was stated by the referring (2) population data, i.e., have a frequency in the control clinician. population compatible with the variant being rare disease- causing, according to its inheritance mode; and (3) allelic Initial screening detected suspected pathogenic variants in data, i.e., be in trans with a pathogenic variant; however, as 47% of participants phase could rarely be established for putative compound Using the World Muscle Society gene table (http://www. heterozygous cases we refer to these as double heterozygous. musclegenetable.fr/) we generated a list of 169 genes known to Variants of uncertain significance are not discussed here. To be associated with limb-girdle muscle disease (Table S1) and be considered solved, a patient was required to carry two initially restricted our search for likely causal variants to only known or suspected pathogenic variants in a recessive gene, or these genes. As a result, 468 patients (47%) had rare known or (at least) one known or suspected pathogenic variant in a suspected pathogenic variants across 72 genes that we dominant gene. By suspected pathogenic variants we refer to considered were causal; these cases were deemed solved rare and damaging variants identified in a gene associated (Table 1, Fig. 1). with a disease that matches the patients’ clinical presentation, disease onset and progression, magnetic resonance image Frequency of NMD genes varied greatly in our cohort (MRI) findings, and/or muscle histopathology. The findings The most common disease in our cohort was LGMD R1 were disclosed to the submitting centers in tailored reports, calpain3-related, caused by autosomal recessive pathogenic signposting the patients to relevant disease registries. Where variants in CAPN3. Variants in CAPN3 and seven other genes necessary and possible, the suspected pathogenic variants —DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA— were confirmed and segregated through Sanger sequencing, accounted for over half of the solved cases (n = 260). The GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1479 ARTICLE Table 1 Breakdown of genes in which suspected causal variants were identified in the MYO-SEQ cohort. Gene Number of patients Gene Number of patients Gene Number of patients Gene Number of patients CAPN3 78 POGLUT1a 5 PYROXD1a 2 HSPB1a 1 RYR1 26 SCN4A 5 SGCB 2 HSPB8a 1 DYSF 45 CLCN1 3 ACADVL 1 INPP5Ka 1 ANO5 33 DNM2 4 BAG3 1 LPIN1 1 DMD 31 MYOT 4 BVESa 1 MATR3 1 TTN 25 RAPSN 4 CACNA1Sa 1 MGME1a 1 COL6A2 17 DNAJB6 3 CHRND 1 MMEa 1 SGCA 16 GMPPB 3 COL12A1 1 MYF6 1 COL6A3 12 MTM1 3 CPT2 1 MYH14 1 FLNC 12 SEPN1 3 CRYAB 1 NEB 1 DES 10 TCAP 3 DOK7 1 PFKM 1 GAA 10 TRAPPC11 3 DPM3 1 PGK1 1 LAMA2 10 ACTA1 2 DYNC1H1a 1 POLG 1 POMT2 10 AGL 2 EGR2a 1 PTPLA 1 COL6A1 10 ATP2A1 2 ETFDH 1 SYNE2 1 LMNA 9 COLQ 2 FKTN 1 TMEM8Ca 1 MYH7 9 FHL1 2 GBE1 1 TNNT3 1 FKRP 8 PHKA1 2 GFPT1 1 TPM2 1 SGCG 8 POMK 2 GNE 1 TTRa 1 SMCHD1 8 POMT1 2 GYG1 1 VCP 1 TRIM32 7 PRPS1a 2 HNRNPA1a 1 VMA21 1 CAV3 5 PYGM 2 HNRNPDL 1 Suspected causative variants were identified in 87 genes in 489 patients: 72 genes during the first round of analysis using a gene list of 169 neuromuscular disorder (NMD) genes, and 15 additional genes when using a gene list extended by a further 260 NMD genes. a Fifteen additional genes identified. other 208 solved cases had suspected pathogenic variants end of the spectrum with an earlier disease onset. Both sets of across 64 additional genes. Importantly, we identified a stop- parents were heterozygous for the respective variants. The gain TTN founder pathogenic variant (p.[Gln35879Ter]) in 14 mother of the homozygous VCP patient remained asympto- patients from a Serbian subpopulation.8 Without this variant, matic at the time of examination, while the father presented TTN would be outnumbered by COL6A2 and COL6A3 with proximal weakness and atrophy of the upper and lower (collagen VI–related myopathies), SGCA (LGMD R3 α- limbs, and rimmed vacuoles on histopathology,16 as would be sarcoglycan–related), and FLNC (myofibrillar myopathy 5 expected for this previously reported dominant variant.17 On and distal myopathy 4). Some diseases were notably absent the other hand, the parents of the homozygous FLNC patient from our cohort; for example, no POMGnT1 (LGMD R15 were both clinically unaffected on examination. This would POMGnT1–related) or ISPD (LGMD R20 ISPD-related) suggest a different pathological mechanism for this novel patients were identified, highlighting the extreme rarity of FLNC variant and functional work is underway (unpublished these diseases. When the data were stratified by sex, there data by University Duisburg–Essen). appeared to be a skewed ratio of males (n = 22; 69%) to females (n = 10; 31%) harboring suspected pathogenic Solved rate differed with geographical location of the variants in ANO5 (p < 0.0691), confirming previously participating center reported findings.15 When the data were stratified by geographic location, there was a clear distinction between regions with and without the Homozygous variants associated with established infrastructure to sufficiently prescreen patients prior to autosomal dominant inheritance submission. The detection rate for referring centers in Eastern A proband from a consanguineous pedigree harbored a Europe and the Middle East was as high as 95%, including, for previously reported dominant pathogenic variant (c.476G>A example, 57 LGMD R1 calpain3-related and 32 LGMD R2 p.[Arg159His]) in VCP in homozygosity, while another dysferlin-related cases. Conversely, the detection rate for proband carried a novel homozygous variant (c.1325C>G p. Western Europe was as low as 35%, with only 23 LGMD R1 [Pro442Arg]) in FLNC; both genes have established autosomal calpain3-related (p < 0.00001) and 15 LGMD R2 dysferlin- dominant inheritance. In both cases, the clinical presentation related cases (p < 0.00001), and a larger proportion of rarer was in keeping with what was expected, but at the more severe genes and phenotypes (Fig. 2). 1480 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE ARTICLE CAPN3 8% DYSF 4% ANO5 3% DMD 3% RYR1 2% TTN 2% Unsolved COL6A2 2% 48% SGCA 2% One gene - other 19% Transcriptomics and GS Two genes 0.1% 2% CNV SMN1 deletion 3% 0.5% Extended gene list 2% Fig. 1 Summary of the solved rate and sequential analysis in the MYO-SEQ cohort. Suspected pathogenic variants were detected in a total of 520 patients (52%). Initially, 468 (47%) patients were solved through a screening of the MYO-SEQ gene list of 169 neuromuscular disorder (NMD) genes; of these, 450 (45%) had pathogenic variants in one of 72 genes and 18 (2%) had pathogenic variants in two genes. Pathogenic variants in eight of these genes alone accounted for half of the solved patients (26%); the most common disease detected in our cohort was limb-girdle muscular dystrophy (LGMD) R1 calpain3-related (CAPN3). Variants in 64 genes accounted for the remaining patients with one causal gene. The analysis strategy was extended by a further 260 genes, solving an additional 2% of the cohort. Copy-number variations (CNVs) were detected in 26 (3%) patients, SMN1 deletions in five (0·5%) patients and transcriptional perturbations in one patient. n = 1001. GS genome sequencing. Analysis of an extended gene list increased the diagnostic POMT2, COL6A1, LMNA, MYH7, and FKRP) account for an yield to 49% additional 10%, followed by a very small but nonetheless For the remaining unsolved cases, we extended the analysis to meaningful increase in solved rate as very rare and/or a total of 429 disease-causing genes (Table S2). This resulted unexpected disease genes, as well as those recently described, in a 2% increase in detection rate to an overall 49% (n = 488), are detected (Table 1). across 87 muscle disease–associated genes; 15 genes more than the initial analysis. These genes were associated with Expanding exome sequencing beyond single base pair neuropathies (EGR2, DYNC1H1, HSPB1, HSPB8, PRPS1, and changes and small indels increased the detection yield by a MME), multisystemic disorders presenting with muscle further 3% weakness (HNRNPA1, INPP5K, TMEM8C, and TTR), chan- Further to point variants and small indels, we detected nelopathies (CACNA1S), and mitochondrial disease suspected pathogenic CNVs in 26 (3%) additional patients. Of (MGME1), all detected in patients who had been initially these, 17 carried copy-number zero deletions (at exon or clinically diagnosed as LGW (n = 12). In addition, three genes multiexon level) in autosomal recessive genes (DYSF, SGCA, only recently associated with muscular dystrophy (BVES and SGCB, SGCD, SGCG, and LPIN1; n = 10) and in X-linked POGLUT1)18,19 and myofibrillar myopathy (PYROXD1)20 genes (DMD, EMD, and FHL1; n = 7, all males). Eight cases were found in eight patients. Thus, after the eight most carried a heterozygous CNV in combination with a suspected common disease genes, accounting for 25% of the full cohort, pathogenic SNV in the same gene and one case, presenting the next ten genes (COL6A3, FLNC, DES, GAA, LAMA2, with a classical dominant myofibrillar myopathy phenotype, GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1481 ARTICLE Fig. 2 Solved rate by country of origin. The MYO-SEQ solved rate (in dark gray) was higher in countries such as Egypt and Turkey where the infrastructure for genetic testing for prescreening is not as widely available, and lower in Western European countries where genetic prescreening of common limb-girdle muscular dystrophy (LGMD) genes is routinely performed. Calculated for referring centers submitting more than 20 samples. carried a heterozygous deletion in DES. Some of these CNVs remaining 401 were novel and had not been yet reported at were recurring; we identified a 10.4-kb deletion in CAPN3 in the time of the analysis (Fig. 3c). Of the novel variants, 285 five unrelated patients, and a 0.7-kb and 0.12-kb deletion in were seen only once and, notably, 116 were seen multiple SGCD and SGCG, respectively, in two unrelated patients each times in our patient population (Fig. 3d). These novel variants (Table S3). In addition, we looked for homozygous deletions will be submitted to ClinVar to aid future genetic diagnoses. of exon 7 in SMN1, causative of spinal muscular atrophy. Such deletions were detected in five patients (0.5%), all of Single heterozygous pathogenic variants in the most which were confirmed by multiplex ligation-dependent probe common recessive genes account for 2.5% of the unsolved amplification (MLPA). Finally, through additional RNA-Seq cases and GS, we were able to identify an intronic change resulting To unravel the remaining unsolved patients, we looked at in a 73-bp intron inclusion in DMD leading to a premature those who carried single reported pathogenic variants in the stop codon in one patient with a clear Becker-like phenotype four most common exclusively recessive genes found in our and supporting family history, but with no evident causal cohort. We found 25 (2.5%) heterozygous carriers: 13 CAPN3, exonic DMD variant,14 highlighting that strong phenotypic 9 ANO5, 2 SGCA, and 1 DYSF. These cases were accounted clues should always be followed up. for by 16 pathogenic variants: 10 CAPN3, 4 ANO5, 1 DYSF, and 1 SGCA. These variants were all more frequent in our Over 400 novel suspected pathogenic variants were cohort of patients than in the control population. For identified example, a rare SGCA variant (c.229C>T; p.[Arg77Cys]) In total, we identified 865 SNV and indel changes in 429 occurred four times more frequently in the disease population genes that were most likely to contribute to the 496 (p = 0.02), the common Eastern European CAPN3 variant participants’ diseases. A breakdown of the genotypes and (c.550delA; p.[Thr184ArgfsTer36]) over eight times more types of variants is shown in Fig. 3a, b. The 865 variants were (p = 0.00028), and the European founder ANO5 variant accounted for by 520 distinct variants (Table S4); of these, 119 (c.191dupA; p.[Asn63fs]) almost five times more (p = had been submitted to ClinVar previously, while the 0.00016). This suggests that these variants are likely to play 1482 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE ARTICLE a b Inframe Intitiation lost Intronic Hemizygous 2% 1% 0.1% 5% Splice region Stop lost 4% 0.1% Double heterozygous Essential 34% splice site Heterozygous 8% 29% Nonsense 11% Missense 52% Frameshift 22% Homozygous 32% c 1000 d 450 900 400 800 350 Recurrent (116) In ClinVar 700 (306) 300 600 Number of variants Number of variants 250 500 In ClinVar (119) 200 400 150 Unique 300 (285) Novel (559) 100 Recurrent Novel 200 (49) (401) 50 100 Unique (70) 0 0 Total Distinct In ClinVar Novel Fig. 3 Breakdown of the suspected pathogenic variants and genotypes in the MYO-SEQ cohort. (a) Zygosity of the solved patients’ variants (n = 506; 489 patients, 20 with an additional gene to report). (b) Type of variants suspected to be pathogenic. Initiation and stop loss occurred twice each (n = 865). (c) The 865 occurrences were accounted for by 520 distinct variants, of which 119 were reported as pathogenic in ClinVar and 401 were novel in their association to disease at the time of the analysis. (d) Of the 119 distinct variants reported in ClinVar, 70 were detected in only one individual (unique) while 49 were detected in multiple patients. Of the 401 distinct variants that were novel in their association to disease, 285 were detected in only individual cases, while 116 were detected in multiple families. GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1483 ARTICLE a role in disease manifestation and that a second cryptic and R20 ISPD-related, were absent from our cohort. No SNVs disease-causing variant might not have been detected by our and only one CNV were identified in the SGCD gene, in analysis; we therefore interrogated these carriers further by keeping with other sequencing projects from China (n = looking into the noncoding regions present in their exome 756),24 Italy (n = 504),6 and the United States (n = 4656)7 data. We thus identified one novel 3′ UTR and two rare where no SGCD variants were found. Interestingly, LGMD R6 CAPN3 intronic variants, one of which (c.1746–20C>G) δ-sarcoglycan–related patients accounted for >1% of the occurred in three cases. Histopathological analysis showed diagnosed cases in a South American cohort (n = 2103),25 markedly reduced calpain3 immunostaining, suggesting that highlighting that the geography-specific prevalence of some this noncoding variant may affect splicing or protein stability. diseases should be considered during clinical work-ups. Per In addition, we identified an intronic deletion sex bias, we corroborated previous findings that ANO5 (c.585–31_585–24delTCTGCTGA) in one of the SGCA diagnosis is more frequent in males.15 This is likely due to carriers, which results in partial expression of the α- the milder, sometimes asymptomatic, presentation of LGMD sarcoglycan protein.21 Conversely, none of the reported R12 anoctamin5-related in females; because of this, females pathogenic intronic variants in DYSF13 were found in the can present to clinic much later in life, or not at all, skewing DYSF carrier. the diagnostic rate in favor of males. The relatively small benefit observed when extending the Many patients harbored suspected pathogenic variants in gene list from 169 to 429 genes is in line with a panel study of genes associated with treatable or manageable conditions 700 NMD genes where exome sequencing of the remaining Our study identified 64 patients (6.5%) that might benefit undiagnosed cases did not improve the pick-up rate for known from treatment or specific management options related to NMD genes.26 However, having exome data available, such as their diagnosis. For example, variants in COLQ, DOK7, in our study, allows for both gene discovery and data analysis GFPT1, and RAPSN—all associated with congenital myasthe- reiteration when new disease genes are characterized, neither nic syndrome (CMS)—were identified in eight patients (<1%). of which are possible even with a large NGS panel approach. We also identified ten patients (1%) with compound This was the case for the BVES, POGLUT1, and PYROXD1 heterozygous variants in GAA, associated with Pompe patients who were missed in the first round of analysis, as the disease.22 Nine patients (1%) harbored variants in either genes had not yet been described as disease-causing. By SCN4A or CLCN1 and thus were suspected to have ion interrogating the whole exome of the unsolved patients, we channel disorders that could benefit from selective drug identified and have functionally characterized two novel genes, interventions. We identified 13 female patients (1%) who one associated with a rare secondary dystroglycanopathy and were likely to be manifesting carriers of Duchenne muscular the other, POPDC3, found in three unrelated individuals, dystrophy, an X-linked disorder that often escapes clinical associated with a typical LGMD phenotype.27 Two additional diagnosis in females. Finally, we identified 24 patients (2%) novel candidate genes are under investigation and we expect with heterozygous suspected pathogenic variants in RYR1, that this will only increase as the exome data of unsolved which can confer susceptibility to malignant hyperthermia. patients are more intricately examined. It is possible that further causative variants have not been DISCUSSION assigned pathogenicity due to the limitations of proband- We sought to improve the diagnostic pathway of patients with only analysis or that they have not been detected due to limb-girdle muscle diseases by implementing TES as a first- nonuniform coverage and/or poorly designed capture baits pass diagnostic strategy. By focusing on 429 NMD genes that might not target relevant tissue-specific exons. For within the exome data, we solved 49% of our cohort, example, repeat regions in NEB or TTN are known to be comparable with equivalent NGS panel studies.6,7 A small difficult to map to the genome and therefore variants in number of common NMD genes, namely CAPN3, DYSF, these regions are poorly called.28 Somatic mosaicisms,29 ANO5, DMD, RYR1, TTN, COL6A2, and SGCA accounted for only detected by deep coverage sequencing, would also have more than half of the solved cases. Our finding that LGMD been missed. A further proportion of the remaining R1 calpain3-related was the most common LGMD in our unsolved cases might be caused by genetic changes in cohort is in agreement with other European studies.6 Standard intergenic, intronic, or regulatory regions not covered by ES. calpain3 immunoblot testing can only achieve a low The overrepresentation in our disease cohort of carriers of sensitivity,23 which could explain why a tenth of the MYO- known pathogenic variants in recessive genes likely implies SEQ cohort was not diagnosed with this LGMD prior to that a second cryptic variant is yet to be detected. In fact, enrollment. Moreover, many of the calpain3-related patients when interrogating these cases further we identified were referred from centers without the technical infrastruc- recurrent suspected pathogenic intronic variants and CNVs. ture to prescreen their patients. Otherwise, these pathogenic heterozygous variants might Given the considerable phenotypic overlap and the rarity of act as disease modifiers, contributing to the disease severity some of these muscle conditions, it is almost impossible for or progression.30 clinicians, however expert, to individually diagnose every Using modified pipelines, we have identified several disease- disease. Some of the LGMDs, such as R15 POMGnT1-related causing CNVs, accounting for 3% of the diagnoses. However, 1484 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE ARTICLE more complex structural variants such as translocations and clinic and/or private health providers. Rather than imple- inversions, would not have been detected.31 In a similar menting small NGS panel approaches, led by phenotypic clues manner, tri- and tetranucleotide expansion repeats, such as that can often be misleading due to the high clinical the cause of some adult onset neuromuscular conditions (e.g., heterogeneity of muscle diseases, patients should be directly myotonic dystrophy), are typically missed in standard short- referred for exome sequencing. The exome sequencing data of read NGS data. Interestingly, four unsolved cases were NMD patients of European and Middle Eastern origin should reported to present with myotonia, percussion myotonia, or first be promptly analyzed for pathogenic variants in the eight myotonic discharges. Other novel late-onset myopathies genes most commonly associated with muscle disease in our might be caused by similar mechanisms and long-read NGS cohort—CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, would be needed to identify them. Digenic or non-Mendelian and SGCA. This is expected to diagnose over a quarter of inheritance, such as genomic imprinting, may also account for individuals. The analysis pipeline should then be extended to a proportion of the undiagnosed patients32,33 and will need a include additional NMD genes, enabling a diagnosis in a different methodological and analytical strategy. further quarter of patients. For those patients who remain It is also possible that some of the unsolved patients may undiagnosed, their ES data can be further interrogated. have acquired forms of muscle disease, such as acquired Importantly, exome data can be retrospectively and repeatedly immune, inflammatory, noninflammatory, or even statin- analyzed for pathogenic variants in novel disease genes as new induced myopathies. In fact, during the project four patients discoveries are made, making it a much more cost-effective (<1%) obtained a confirmed diagnosis of inclusion body approach. myositis. Regular screening for anti-NT5c1A and anti- HMGCoR should provide a more realistic indication of the prevalence of these diseases in the affected population. SUPPLEMENTARY INFORMATION Over 6% of our patients received a genetic outcome that The online version of this article (https://doi.org/10.1038/s41436- resulted in specific monitoring and tailored treatments. For 020-0840-3) contains supplementary material, which is available example, for the CMS patients, genetic diagnoses were vital to authorized users. to provide adequate treatment, as this depends on the disease subtype and causative gene. Indeed, the referring ACKNOWLEDGEMENTS clinicians reported that these patients all showed marked We thank the patients for donating their tissue samples. MYO- improvement after appropriate treatments were com- SEQ was funded by Sanofi Genzyme, Ultragenyx, LGMD2I menced. The pathogenicity of the RYR1 variants could not Research Fund, Samantha J. Brazzo Foundation, LGMD2D always be fully confirmed, yet it was prudent to return these Foundation and Kurt+Peter Foundation, Muscular Dystrophy potential diagnoses to physicians to manage possible risk of UK, and Coalition to Cure Calpain 3. Analysis was provided by the malignant hyperthermia. For most of the solved cases, Broad Institute of MIT and Harvard Center for Mendelian however, who might not benefit from specific treatment, Genomics (Broad CMG) and was funded by the National Human their genetic diagnosis will nevertheless allow appropriate Genome Research Institute, the National Eye Institute, and the family planning, counseling, and monitoring of their National Heart, Lung, and Blood Institute grant UM1 HG008900, diseases. and in part by National Human Genome Research Institute grant MYO-SEQ was a worldwide partnership between acade- R01 HG009141. mia, industry, and patient organizations. Frequent commu- nication with the referring physicians was essential for the success of the project; to reach a meaningful diagnosis, DISCLOSURE interpretation of the genetic data was always in the context D.G.M. is a founder with equity in Goldfinch Bio, and has received of clinical, histopathological, and MRI data. The participat- research support from AbbVie, Astellas, Biogen, BioMarin, Eisai, ing centers provided consent for data sharing facilitated by Merck, Pfizer, and Sanofi Genzyme. V.S. is or has been a principal the European Genome-Phenome Archive (EGA) and RD- investigator for trials sponsored by Sanofi Genzyme, GSK, Connect (https://platform.rd-connect.eu/), and we advocate Prosensa/Biomarin, IonisPharmceuticals, and Sarepta. He has that adopting such an approach will enable future received speaker honoraria from Sanofi Genzyme. He is or has matchmaking between extremely rare cases, such as BVES- been on advisory boards for Acceleron Pharma, Audentes related myopathy34 or LGMD R21 POGLUT1-related.35 In Therapeutics, Biomarin, Bristol-Myer Squibb, Italfarmaco S.p.A., addition, thanks to the large cohort size and standardized Nicox, Pfizer, Sanofi Genzyme, Santhera, Sarepta Therapeutics, deep phenotypic data, we were able to expand the clinical Summit Therapeutics, Tivorsan, and TrophyNOD. The other and mutational spectrum of known causative genes, authors declare no conflicts of interest. such as TRIM32,36 POMK,37 DPM3,38 POMT2,39 and other dystroglycanopathies.40 Publisher’s note Springer Nature remains neutral with regard Based on our findings from this large-scale international to jurisdictional claims in published maps and institutional collaboration, we suggest a new diagnostic approach in the affiliations. GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1485 ARTICLE REFERENCES 24. 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Variants in the oxidoreductase License, which permits any non-commercial use, sharing, distribution and PYROXD1 cause early-onset myopathy with internalized nuclei and reproduction in any medium or format, as long as you give appropriate credit to myofibrillar disorganization. Am J Hum Genet. 2016;99:1086–1105. the original author(s) and the source, and provide a link to the Creative 21. Gonzalez-Quereda L, Gallardo E, Topf A, et al. A new mutation of the Commons license. You do not have permission under this license to share SCGA gene is the cause of a late onset mild phenotype limb girdle adapted material derived from this article or parts of it. The images or other third muscular dystrophy type 2D with axial involvement. Neuromuscul Disord. party material in this article are included in the article’s Creative Commons 2018;28:633–638. license, unless indicated otherwise in a credit line to the material. If material is not 22. Johnson K, Topf A, Bertoli M, et al. Identification of GAA variants through included in the article’s Creative Commons license and your intended use is not whole exome sequencing targeted to a cohort of 606 patients with permitted by statutory regulation or exceeds the permitted use, you will need to unexplained limb-girdle muscle weakness. Orphanet J Rare Dis. 2017; obtain permission directly from the copyright holder. To view a copy of this 12:173. license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. 23. Fanin M, Angelini C. Protein and genetic diagnosis of limb girdle muscular dystrophy type 2A: the yield and the pitfalls. Muscle Nerve. 2015;52:163–173. © The Author(s) 2020 1486 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE ARTICLE Ana Töpf, PhD 1, Katherine Johnson, PhD1,67, Adam Bates, MSc1, Lauren Phillips, BSc1, Katherine R. Chao, BSc2,3, Eleina M. England, MSc2,3, Kristen M. Laricchia, MSc2,3, Thomas Mullen, PhD2,3, Elise Valkanas, BA2,3, Liwen Xu, MD, MPhil2,3, Marta Bertoli, MD1,68, Alison Blain, PhD1, Ana B. Casasús, MSc1, Jennifer Duff, PhD1, Magdalena Mroczek, MD1, Sabine Specht, MD1, Monkol Lek, PhD2,3,69, Monica Ensini, PhD1,70, Daniel G. MacArthur, PhD2,3,71,72, the MYO-SEQ consortium and Volker Straub, MD1 1 John Walton Muscular Dystrophy Research Centre, Translational and Clinical Research Institute, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK; 2Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; 3Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; 67Present address: Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK; 68Present address: Northern Genetics Service, Newcastle upon Tyne NHS Foundation Trust, Newcastle upon Tyne, UK; 69Present address: Department of Genetics, Yale University School of Medicine, New Haven, CT, USA; 70Present address: Directorate-General for Research and Innovation, Directorate E, Unit E2 Combatting Diseases, Brussels, Belgium; 71Present address: Centre for Population Genomics, Garvan Institute of Medical Research, Sydney, Australia; 72Present address: Centre for Population Genomics, Murdoch Children’s Research Institute, Melbourne, Australia The MYO-SEQ consortium Ela Akay4,73, Jorge Alonso-Pérez5, Jonathan Baets6,7, Nina Barisic8, Alexandra Bastian9,10, Sabine Borell11, Teodora Chamova12, Kristl Claeys13, Jaume Colomer14, Sandra Coppens15,16, Nicolas Deconinck17,18, Willem de Ridder6,7, Jordi Díaz-Manera5,19, Cristina Domínguez-González19,20, Alexis Duncan21, Hacer Durmus22, Nagia A. Fahmy23, Maria Elena Farrugia24, Roberto Fernández-Torrón25,26,27, Lidia Gonzalez-Quereda19,28, Jana Haberlova29, Maja von der Hagen30, Andreas Hahn31, Antonia Jakovčević32, Ivonne Jerico Pascual33, Solange Kapetanovic34, Viktorija Kenina35, Janbernd Kirschner11, Andrea Klein36,37, Heike Kölbel38, Anna Kostera-Pruszczyk39, Richa Kulshrestha40, Jaana Lähdetie41, Mahsa Layegh42, Cheryl Longman21, Adolfo López de Munain25,26,27,43, Wolfgang Loscher44, Anna Lusakowska39, Paul Maddison4, Armelle Magot45, Anirban Majumdar46, Pilar Martí19,47,48, Amaia Martínez Arroyo49, Radim Mazanec29, Sandra Mercier45, Tiziana Mongini50, Nuria Muelas19,47,48, Andrés Nascimento14, Shahriar Nafissi42, Shirin Omidi42, Carlos Ortez14, Stéphanie Paquay51, Yann Pereon45, Stojan Perić52, Valentina Ponzalino50, Vidosava Rakočević Stojanović 52, Gauthier Remiche53, Aida Rodríguez Sainz49, Sabine Rudnik54, Iciar Sanchez Albisua55, Manuela Santos56, Ulrike Schara38, Andriy Shatillo57, Jadranka Sertić58, Ulrich Stephani59, Sonja Strang-Karlsson60,61, Yves Sznajer62, Ani Tanev12, Ivailo Tournev12,63, Peter Van den Bergh51, Vinciane Van Parijs51, Juan Vílchez19,47,48, Katharina Vill64, John Vissing65, Carina Wallgren-Pettersson60,66, Julia Wanschitz44, Tracey Willis40, Nanna Witting65 and Miren Zulaica25,26 4 Queen’s Medical Centre, Nottingham University Hospitals NHS Trust, Nottingham, UK; 5Neuromuscular Disorders Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 6Neuromuscular Reference Centre, Department of Neurology, Antwerp University Hospital, Antwerpen, Belgium; 7Laboratory of Neuromuscular Pathology, Institute Born-Bunge, University of Antwerp, Antwerpen, Belgium; 8Clinical Medical Centre Zagreb, University of Zagreb, Zagreb Medical School, Zagreb, Republic of Croatia; 9Colentina University Hospital-CDPC-CCC, Bucharest, Romania; 10University of Medicine and Pharmacy Carol Davila Bucharest, Faculty of Dental Medicine, Bucharest, Romania; 11Department of Neuropediatrics, Faculty of Medicine, University of Freiburg, Freiburg, Germany; 12Department of Neurology, UMHAT, Alexandrovska Medical University, Sofia, Bulgaria; 13 Department of Neurology, University Hospitals Leuven, and Laboratory for Muscle Diseases and Neuropathies, KU Leuven, Leuven, Belgium; 14Unitat de Patologia Neuromuscular, Servei de Neurologia, Hospital Sant Joan de Deu, ISCIII, CIBERER, Barcelona, Spain; 15Department of Pediatric Neurology, Neuromuscular Reference Center, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium; 16Department of Pediatric Neurology, Neuromuscular Reference Center, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgium; 17Neuromuscular Reference Center, Universitair Ziekenhuis Gent (UZ Gent), Ghent, Belgium; 18Neuromuscular Reference Center, Hôpital Universitaire des Enfants Reine Fabiola (HUDERF), ULB, Brussels, Belgium; 19Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Madrid, Spain; 20Instituto de Investigación Hospital 12 de Octubre Centro de Actividades Ambulatorias, Madrid, Spain; 21West of Scotland Centre for Genomic Medicine, GENETICS in MEDICINE | Volume 22 | Number 9 | September 2020 1487 ARTICLE Queen Elizabeth University Hospital, Glasgow, UK; 22Department of Neurology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey; 23Neuromuscular Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt; 24Institute of Neurological Sciences, Queen Elizabeth University Hospital, Glasgow, UK; 25Neuroscience Area, Biodonostia Research Institute, San Sebastián, Gipuzkoa, Spain; 26Department of Neurology, Hospital Universitario Donostia, OSAKIDETZA, San Sebastián, Gipuzkoa, Spain; 27 CIBERNED, Instituto de Salud Carlos III, Ministry of Science, Innovation and Universities, Madrid, Spain; 28Genetics Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 29Department of Neurology, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic; 30Department of Neuropediatrics, Medical Faculty Carl Gustav Carus, Technische Universitat Dresden, Dresden, Germany; 31Department of Child Neurology, Justus-Liebig-University Giessen, Gießen, Germany; 32Department of Pathology and Cytology, University Hospital Center Zagreb, School of Medicine Zagreb, Zagreb, Republic of Croatia; 33Department of Neurology, Complejo Hospitalario de Navarra, IdisNa, Pamplona, Spain; 34Neuromuscular Consult Unit, Hospital Universitario Basurto, Bilbao, Spain; 35Riga East University Hospital, Rare Disease Unit, Riga, Latvia; 36Department of Neuropediatrics, University Childrens Hospital Basel, Basel, Switzerland; 37Department of Neuropediatrics Inselspital Bern, Bern, Switzerland; 38Department of Neuropediatrics, Centre for Neuromuscular Disorders in Children and Adolescents, Children’s Hospital University Clinic Essen, University of Duisburg, Essen, Germany; 39Department of Neurology, Medical University of Warsaw, Warsaw, Poland; 40The Muscle Team, RJAH Orthopaedic Hospital NHS Foundation Trust, Oswestry, UK; 41Deptartment Pediatric Neurology, University of Turku and Turku University Central Hospital, Turku, Finland; 42Department of Neurology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran; 43Department of Neurosciences, University of the Basque Country UPV-EHU, San Sebastián, Spain; 44 Department of Neurology, Medical University of Innsbruck, Innsbruck, Austria; 45Centre de Référence Maladies Neuromusculaires AOC, Hôtel-Dieu, University Hospital, Nantes, France; 46Department of Paediatric Neurology, Bristol Children’s Hospital, Bristol, UK; 47 Neuromuscular Reference Centre, Neurology Department, Hospital Universitari i Politècnic La Fe, Valencia, Spain; 48 Neuromuscular Disorders Unit, Instituto de Investigación Sanitaria La Fe, Valencia, Spain; 49Departamento de Neurología, Hospital Galdakao, Galdakao, Spain; 50Neuromuscular Unit, Department of Neurosciences, University of Turin, Turin, Italy; 51 Cliniques Universitaires St-Luc, Centre de Référence Neuromusculaire, Université de Louvain, Brussels, Belgium; 52Faculty of Medicine, University of Belgrade, Neurology Clinic, Clinical Center of Serbia, Belgrade, Serbia; 53Centre de Référence Neuromusculaire Erasme-HUDERF, Department of Neurology, CUB Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium; 54 Institute of Human Genetics, Medical University Innsbruck, Innsbruck, Austria; 55Department of Child Neurology, Children’s Hospital, University of Tübingen, Tübingen, Germany; 56Centro Hospitalar Universitário Porto, Porto, Portugal; 57Institute of Neurology, Psychiatry and Narcology of NAMS of Ukraine, Kharkiv, Ukraine; 58Department of Laboratory Diagnostics, Clinical Medical Centre Zagreb, University of Zagreb, Zagreb Medical School, Zagreb, Republic of Croatia; 59Klinik für Neuropädiatrie, University Medical Center Schleswig-Holstein, Kiel, Germany; 60Folkhaelsan Department of Medical Genetics, Helsinki, Finland; 61 Department of Clinical Genetics, HUSLAB, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 62Cliniques Universitaires St-Luc, Centre de Génétique Humaine, Université de Louvain, Brussels, Belgium; 63Department of Cognitive Science and Psychology, New Bulgarian University, Sofia, Bulgaria; 64Dr. v. Hauner Children’s Hospital, Department of Pediatric Neurology and Developmental Medicine, LMU—University of Munich, Munich, Germany; 65Copenhagen Neuromuscular Center Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; 66Department of Medical Genetics, University of Helsinki, Helsinki, Finland; 73Present address: Department of Neurology, Royal Victoria Infirmary, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. 1488 Volume 22 | Number 9 | September 2020 | GENETICS in MEDICINE