ARTICLE OPEN ACCESS

Next-generation sequencing approach tohyperCKemiaA 2-year cohort study

Anna Rubegni MD Alessandro Malandrini MD Claudia Dosi MD Guja Astrea MD Jacopo Baldacci BS

Carla Battisti MD Giulia Bertocci BSc M Alice Donati MD M Teresa Dotti MD Antonio Federico MD

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 20195e352 doi101212NXG0000000000000352

Correspondence

Dr Santorelli

filippo3364gmailco

AbstractObjectiveNext-generation sequencing (NGS) was applied in molecularly undiagnosed asymptomatic orpaucisymptomatic hyperCKemia to investigate whether this techniquemight allow detection ofthe genetic basis of the condition

MethodsSixty-six patients with undiagnosed asymptomatic or paucisymptomatic hyperCKemia referredto tertiary neuromuscular centers over an approximately 2-year period were analyzed usinga customized targeted sequencing panel able to investigate the coding exons and flankingintronic regions of 78 genes associated with limb-girdle muscular dystrophies rhabdomyolysisand metabolic and distal myopathies

ResultsA molecular diagnosis was reached in 33 cases corresponding to a positive diagnostic yield of50 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 mildproximal muscle weakness (found in 27) and myalgia (in 24) Fourteen patients witha molecular diagnosis andmild myopathic features onmuscle biopsy remained asymptomatic ata 24-month follow-up

ConclusionsThis study of patients with undiagnosed hyperCKemia highlighting the advantages of NGSused as a first-tier diagnostic approach in genetically heterogeneous conditions illustrates theongoing 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 carefulsurveillance

From the IRCCS Fondazione Stella Maris (AR GA JB GB SL FMS DC) Pisa Italy Department of Medicine (AM CB MTD AF FG SS NV) Surgery and NeurosciencesUniversity of Siena Department of Clinical and Experimental Medicine (CD GS DT) University of Pisa Metabolic Disease Unit (MAD MS) AOU Meyer Children HospitalFlorence Department of Molecular and Developmental Medicine (SG) University of Siena Siena Pediatric Neurology (RG FM) AOU Meyer Children Hospital FlorenceNeurophysiopathology Multiple Sclerosis Center Hospital Binaghi (MAM) Cagliari Pediatric Neurology and Nemo Clinical Centre (EM) Fondazione Policlinico Universitario ldquoAGemelli IRCSSrdquo Universita Cattolica del Sacro Cuore Rome and Department of Neurosciences (PT) Biomedicine and Movement Sciences University of Verona Italy

Go to NeurologyorgNG 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 40 (CC BY-NC-ND) which permits downloadingand 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 copy 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Creatine kinase (CK) levels can be mildly and transientlyincreased as a result of muscle injury or exercise even inhealthy individuals Persistent elevation of serum CK (termedhyperCKemia) is defined according to European Federationof Neurological Societies (EFNS) guidelines (eanorgGuideline-Reference-Center26990html) as the presence ofserum CK values beyond 15 times the upper limit of normal(ULN) in least 2 measurements1 and it is a common reasonfor referrals to specialized neuromuscular centers

Increased serum CK can be present in the absence of obviousclinical signs2 In individuals with a normal neurologic ex-amination the condition is termed asymptomatic or isolatedhyperCKemia and it can signal the presence of several un-suspected metabolic cardiac rheumatic or endocrine con-ditions When no underlying cause is found it is referred to asidiopathic hyperCKemia The clinical management of idio-pathic hyperCKemia is unclear although clinically asymp-tomatic affected patients are potentially susceptible tomalignant hyperthermia3

In asymptomatic hyperCKemia definition of the correct di-agnosis may be time consuming and success is not guaran-teed Next-generation sequencing (NGS) has recently beenproposed as a cost-effective strategy for the molecular di-agnosis of inherited neuromuscular disorders4 The efforts todefine the molecular etiology in hyperCKemia come from theneed to refine follow-up avoiding unnecessary examinationsand to improve counseling in the family We set out to explorewhether NGS might allow detection of the molecular basis ofhyperCKemia addressing this question in a consecutive seriesof adults and children recruited at 7 Italian tertiary neuro-muscular centers over an approximately 2-year period

MethodsStandard protocol approvals registrationsand patient consentsThis study was approved by the Tuscany Regional PediatricEthics committee All the procedures complied with theHelsinki Declaration of 1975 Genetic studies and musclebiopsies were performed with written informed consent Allparticipants (including parents or legal guardians in case ofminor patients) were provided pre- and post-test geneticcounseling as routine in our neurogenetic clinics

Patients and study designOver an approximately 2-year period (May 2016ndashAugust2018) 66 patients presenting with hyperCKemia (meeting

the EFNS criteria) were consecutively referred to the neu-rology pediatric or neuropediatric units of 7 Italian tertiaryneuromuscular centers for clinical and diagnostic purposesAll 66 met the inclusion criteria for our study (1) persistentserum CK elevation at rest and (2) values higher than twicethe ULN on at least 2 occasions after refraining frommuscularexercise for at least 72 hours before CK measurement Foreach patient we collected clinical and laboratory data and theresults of familial segregation analyses and previous genetictests MRI scans of thigh and calf muscles were performed in26 patients EMG in 26 and muscle biopsy in 57 in all casesusing routine clinical methods5 Before this study we hadperformed multiplex ligation-dependent probe amplifica-tion analysis in all the patients to exclude multiexon rear-rangements in the DMD gene and tested the levels of acidalpha-glucosidase from dried blood spots6 to detect possibleundiagnosed late-onset Pompe disease Patients with bor-derline enzyme values or biallelic mutations in the GAAgene were not included in this study DNA samples wereanalyzed at a single center

None of the 66 individuals had a medical history of anesthesia-related complications or a family history of cardiovascularevents Any family history of neuromuscular disorders or mildmuscle complaints was carefully recorded Occasional causes ofhyperCKemia such as malignancies drug and alcohol abuserheumatic thyroid and parathyroid disorders infections andhematologic diseases were all excluded All patients underwentroutine serum chemistry including serum myoglobin mea-surement No participant was under statin treatment or takingother drugs potentially capable of inducing hyperCKemia

NGS workflow and sequencing analysesWe used the SureSelect technology (Agilent Santa ClaraCA) and SureDesign software (earraychemagilentcomsuredesign) to design amultiexon amplicon panel containinga total of 78 genes known to be associated with limb-girdlemuscular dystrophies (LGMDs) rhabdomyolysis and meta-bolic and distal myopathies7 the panel spanned more than259 Mbp with gene coverage gt99 To analyze the dataobtained from our study we used a routine bioinformaticpipeline8 that adopts the Ingenuity Variant analysis suite(Qiagen appsingenuitycom) To assign pathogenicity weset up a precise Alissa (Agilent) pipeline using the followingcriteria a sequence quality score greater than 30 a read depthgreater than 30 and rare occurrence in publicly availablepolymorphic data sets (with a minor allele frequency lt001for autosomal dominant and lt01 for autosomal recessivegenes) with less than 1 occurrence in homozygosity in

GlossaryCK = creatine kinase EFNS = European Federation of Neurologic Societies HPO = Human Phenotype Ontology LGMD =limb-girdle muscular dystrophyNGS = next-generation sequencingULN = upper limit of normal VUS = variants of unknownsignificance

2 Neurology Genetics | Volume 5 Number 5 | October 2019 NeurologyorgNG

gnomADv21 (gnomadbroadinstituteorg macarthurlaborg20181017gnomad-v2-1) As reported previously wedetermined predictably or probably deleterious scores usingan in silico pipeline using a set of 10 prediction softwarepackages8 Putatively deleterious variants were validated byPCR-based standard capillary Sanger sequencing both inpatients and in relatives whose DNA was available for segre-gation studies also to determine inheritance and phases ofmultiple gene variants and to establish whether variants hadoccurred de novo Segregation in affected and unaffectedrelatives made it possible to better define pathogenic variantsonce we had identified those more likely to be diseasecausative

Routine morphology and immunofluorescence analysis ofmuscle proteins were performed on vastus lateralis biopsysamples according to standard protocols

Data availabilityThe data set used and analyzed during the current study isavailable from the corresponding author on reasonable request

ResultsWe evaluated 66 patients with hyperCKemia (44 men and22 women age range 5ndash73 years) referred over an ap-proximately 2-year period to tertiary neuromuscular centers

Most of the patients were Italian adults (ge16 years) and ap-parently sporadic cases Although none of them had obviousneuromuscular disorders 16 had a positive family history ofmuscle weakness or fatigue compatible with autosomal domi-nant inheritance in 13 and a recessive pattern of inheritance in3 Their mean age at onset (ie at the time of the first docu-mented evidence of hyperCKemia) was 267 plusmn 192 years whenthey had serum CK levels ranging from 300 to 103000 UIL(normal lt190 UIL) Their disease duration ranged from 3months to 30 years Clinical examination revealed mild limb-girdle muscle weakness (median Medical Research Councilscale score of 5 in upper girdle and 4+ in lower girdlemuscle) in19 patients (figures 1ndash2) The distribution of the muscleweakness in these symptomatic patients is presented in figure 2using the Human Phenotype Ontology (HPO) ID codes andnomenclature9 Thirteen participants reported occasional ex-ercise intolerance andmyalgia but showed no clear evidence ofmuscle weakness Thirty-four patients remained completelyasymptomatic over a 2-year observational period (figure 1)The patientsrsquo serum CK levels at their latest neurologic ex-amination ranged from 150 to 64610 UIL

Cardiac examination performed at some point during thecourse of the clinical follow-up was unremarkable in all thepatients except 4 whose ECG showed arrhythmias of un-certain significance Pulmonary and functional respiratorytests were within normal limits in all the patients andremained so throughout the study

Figure 1 Flowchart of patientsrsquo enrollment and results of genetic testing

VUS = variants of unknown significance

NeurologyorgNG Neurology Genetics | Volume 5 Number 5 | October 2019 3

EMG performed on average 1 year after hyperCKemia onsetshowed myopathic changes in 17 patients Few patients un-derwent repeated neurophysiologic follow-up and none ofthese showed substantial modifications Twenty-six patientsunderwent muscle MRI scans at least once during the studyand these scans were unremarkable in 17 and not informativein the other 9 Fifty-seven patients underwent a skeletalmuscle biopsy In 8 cases (14) this showed clear dystrophicchanges with fat and connective tissue replacement suggest-ing a possible diagnosis of muscular dystrophy whereas thevast majority (3857 67) showed only mild nonspecificmyopathic changes with normal muscle protein staining andno abnormal metabolic features (figure 3 and supplementaryfigure e-1 linkslwwcomNXGA175) Muscle biopsy wasnormal in 11 patients

Through multigene targeted NGS analysis we identified anaverage of 50 rare variants in each patient To facilitate ourldquoneedle in a haystackrdquo search we used a stringent set of bio-informatics filters including effects on the protein scores forpredicting pathogenicity allele frequencies in public and

internal databases and annotation as disease-associated var-iants with Alissa a customizable pipeline tool10 Presence ofthe specific variant in the HumanGenomeMutationDatabase(hgmdcfacuk) was not considered a mandatory criterion forattributing pathogenic significance Bioinformatics data werecombined and critically reevaluated taking into considerationclinical presentation with the relative HPO definition age atonset and segregation studies in familial cases Moreover incases submitted to muscle biopsy we integrated histologicand immunofluorescence features

On the basis of these procedural steps and criteria alreadyused by us in previous research7 we assigned a confirmatorygenetic diagnosis to 19 patients presenting pathogenicdisease-associated (n = 13) or likely pathogenic (n = 6)variants We also identified 14 patients harboring changespredicted to affect function in genes corresponding to theclinical suspicions in these cases thereby increasing group ofindividuals with a molecular diagnosis to 33 patients Weconfirmed the molecular diagnosis using segregation analysisor immunofluorescence or Western blotting on muscle

Figure 2 Clinical features of symptomatic patients presented using HPO ID codes and nomenclature

(A) Muscle weakness pattern frequenciesexpressed as percentage rates Clinicalphenotypes are indicated using the HPO IDcodes HP0003325 = limb-girdle muscleweakness HP0008994 = proximal muscleweakness in lower limbs HP0003749 =pelvic girdle muscle weakness (B) Fre-quencies of clinical phenotypes associatedwith hyperCKemia expressed as percent-age rates Clinical phenotypes are in-dicated using HPO codes andnomenclature HPO = human phenotypeontology

4 Neurology Genetics | Volume 5 Number 5 | October 2019 NeurologyorgNG

biopsies or their combination Detailed information on thesepatients including clinical characteristics CK levels musclebiopsy and gene mutations is shown in tables 1 and 2 whereasthe genetic data are summarized in figure 4 and further detailedin supplementary web data (tables e-1 to e-4 linkslwwcomNXGA176 linkslwwcomNXGA177 linkslwwcomNXGA178 and linkslwwcomNXGA179)

Of the aforementioned 33 patients 9 showedmild limb-girdlemuscle weakness 10 showed myalgia andor occasional ex-ercise intolerance and 1 case rhabdomyolysis whereas 14were asymptomatic despite having muscle biopsies showinglow immunofluorescence for specific proteins (Figure 1 fig-ure e-1 linkslwwcomNXGA175) Immunofluorescenceanalysis was performed to corroborate loss-of-function var-iants in 7 cases With regard to the genetic analyses patho-genic variants in RYR1 were found in 11 patients in ANO5 in4 Four patients harbored pathogenic variants in genesencoding sarcoglycans whereas 3 inCAPN3 (figure 4) SerumCK levels in the group of molecularly defined patients rangedfrom 600 to 67490 UIL whereas muscle biopsy (performedin 3133 individuals) showed evidence of dystrophic pro-cesses in 6 (figure 3 figure e-1) mild nonspecific musclechanges in 21 and no noteworthy alterations in 4 patients

Variants of unknown significance (VUS) with a potential caus-ative role were found in 17 additional patients (supplementarytable e-3 linkslwwcomNXGA178) In these cases either

there was only a partial correspondence with the clinical phe-notype or we discovered a single truncating variant (or a knowndisease-associated variant) in a recessive candidate gene MostVUS occurred in 3 genes namely CAPN3 CPT2 and ANO5(figure 1 and supplementary table e-3 linkslwwcomNXGA178) Although it is possible that the low number of genes inour panel limited its informativeness on the patientsrsquo genotypesit is also conceivable that in the VUS subgroup the second allelemay carry a deep intronic change leading to a mRNA splicingdefect or large scale gene deletionsduplications or a variation ingenomic regulatory regions not covered in our study Our studyexplored none of these possibilities

Sixteen patients harbored molecular findings not compatiblewith their phenotypes Although we cannot exclude a possiblerole for some of the variants detected in this subgroup otheradditional and undetected genetic changes could well be re-sponsible for their phenotypes

DiscussionBoth the EFNS guidelines1 and the authors of a more recentreview article35 recommend conducting further investigations inindividuals with apparently idiopathic hyperCKemia if the CKlevel is repeatedly more than 3 times higher than the ULN EMGreveals myopathic changes and the patient is younger than 25years However it is anticipated that advancing knowledge in the

Figure 3 Myopathologic changes in patients with limb-girdle muscle weakness (top row AndashC) myalgia and exerciseintolerance (middle row DndashF) and in asymptomatic patients (bottom row GndashI)

(A and B) Hematoxylin and eosin (HE)staining demonstrating marked vari-ation in fiber size several central nu-clei and increased connective tissuestaining in P4 and P16 respectively(C) HE staining showing severaldegenerating fibers in P29 (D) HEstaining revealing slight variation infiber size and some central nuclei inP3 (E) Gomori trichrome stainingshowing some hypotrophic fibers andincreased internal nuclei in P28 (F)NADH-TR staining showing moth-eaten fibers in P33 (G) HE stainingdemonstrating mild variation in fibersize and a few central nuclei in P7 (Hand I) Gomori trichrome stainingshowing slight variation in fiber sizeand some internal nuclei in P19 andP22 respectively Magnification 200times

NeurologyorgNG Neurology Genetics | Volume 5 Number 5 | October 2019 5

Table 1 Clinical and genetic features in 13 patients with pathogenic variants

IDSexage (y)

Serum CK(UIL) Clinical findings HPO ID code

Musclebiopsy

Mutatedgene Mutation cDNA level Mutation protein level Inheritance Reference

P9 F54 y 1310 Asymptomatic NA NA ANO5b c1733TgtCc2295CgtG pPhe578SerpTyr765 AR [11]

P10 F61 y 2000 Limb-girdle muscle weakness HP0003325 Mildmyopathicsigns

CAPN3b c633GgtTc1537insCCCCATCTCTCAG

pLys211AsnMet513Thrfs68

AR [12]

P13 M32 y 1800 Myalgia and exercise intolerance HP0003326 HP0009020

Normal TCAPc c33_35del- pGlu12del- AD This work

P15 M7 y 7348 Asymptomatic NA NA CAPN3b c549delAc2115+2TgtA pThr184Argfs35splicesitea

AR [1314]

P16 M14 y 2800 Limb-girdle muscle weakness HP0003325 Dystrophicsigns

SGCGc c195+1GgtAc195+1GgtA Splice siteasplice sitea AR This work

P22 F48 y 2000 Asymptomatic NA Mildmyopathicsigns

ANO5b c1356CgtGc1356CgtG pTyr452pTyr452 AR This work

P25 M11 y 19000 Exercise intolerance and pelvic girdlemuscle weakness

HP0009020 HP0003749

Dystrophicsigns

DMDc c151_153del pLeu51del XL This work

P27 F67 y 900 Pelvic girdle muscle weakness HP0003749 Mildmyopathicsigns

PYGMc c2262delAc406GgtA pLys754Asnfs49pGly136Ser

AR [14]

P28 F32 y 4500 Exercise intolerance and myalgia HP0009020 HP0003326

Mildmyopathicsigns

SGCAc c739GgtAc850CgtT pVal247MetpArg284Cys AR [15 16]

P29 F6 y 16340 Limb-girdle muscle weakness HP0003325 Dystrophicsigns

SGCGc c521delTc521delT pLeu174Leufs21pLeu174Leufs21

AR This work

P30 M66 y 1800 Asymptomatic NA Mildmyopathicsigns

ANO5b c629CgtTc726dupT pSer210LeupSer243Phefs2

AR [17]

P32 M53 y 780 Myalgia and exercise intolerance HP0003326 HP0009020

Mildmyopathicsigns

PYGMCPT2b

PYGM c[148CgtT] CPT2c[1348AgtT]

PYGMp[Arg50] CPT2p[Arg450]

NA [18ndash20]

P33 M18 2937 Myalgia and exercise intolerance HP0003326 HP0009020

Mild myopathicsigns

MYH7b c3790delG- pGlu1264Argfs34- AD This work

Abbreviations AD = autosomal dominant AR = autosomal recessive HPO = Human Phenotype Ontology NA = not available XL = X-linkeda Predicting splice site loss and exon skipping (fruitflyorgseq_toolssplicehtml) Functional validation was derived by bimmunofluorescenceprotein studies combined with predictive early protein truncation or cimmuno-fluorescence studies only

6NeurologyG

enetics

|Vo

lume5N

umber

5|

Octob

er2019

NeurologyorgN

G

Table 2 Clinical and genetic features in 20 patients with ldquolikelyrdquo pathogenic variants

ID

Sexage(y)

SerumCK (UIL)

Clinicalfindings

HPO IDcode

Musclebiopsy

Mutatedgene

MutationcDNA level

Mutationprotein level Inheritance Reference

P1 M19 y

3000 Asymptomatic NA Mildmyopathicsigns

RYR1 c8888TgtC- pLeu2963Pro- AD [21]

P2 M56 y

800 Asymptomatic NA Mildmyopathicsigns

RYR1b c7373GgtA- pArg2458His- AD [22]

P3 M68 y

816 Exerciseintolerance

HP0009020

Mildmyopathicsigns

RYR1 c1163CgtT- pSer388Leu- NA This work

P4d M48 y

1044 Limb-girdlemuscleweakness

HP0003325

Dystrophicsigns

GMPPBb c95CgtTc727CgtT

pPro32LeupArg243Trp

AR [8 23]

P5 F65y

600 Myalgia HP0003326

Mildmyopathicsigns

MYH7 c2009TgtC- pVal670Ala- NA This work

P6 M17 y

865 Asymptomatic NA Mildmyopathicsigns

RYR1b c7291GgtA- pAsp2431Asn- NA [24]

P7 M32 y

1000 Asymptomatic NA Mildmyopathicsigns

RYR1b c5036GgtA- pArg1679His- AD [25]

P8 M40 y

1000 Myalgia HP0003326

Mildmyopathicsigns

RYR1b c7048GgtA- pAla2350Thr- AD [26]

P11d F51y

3000 Limb-girdlemuscleweakness

HP0003325

Dystrophicsigns

DYSFc c862GgtTc2875CgtT

pAsp288TyrpArg959Trp

AR [27]

P12 M11 y

728 Myalgia andexerciseintolerance

HP0003326HP0009020

Mildmyopathicsigns

RYR1b c6599CgtT- pAla2200Val- AD [28]

P14 F65y

3300 Limb-girdlemuscleweakness

HP0003325

Dystrophicsigns

ANO5 c580CgtTc2219CgtT

pArg194TrppSer740Phe

AR This work

P17d M5y

67490 Rhabdomyolysis HP0003201

Mildmyopathicsigns

CPT2b c338CgtTc338CgtT

pSer113LeupSer113Leu

AR [29]

P18 M29 y

600 Asymptomatic NA Normal RYR1b c[4711AgtG10097GgtA]c11798A gt G

p[(Ile1571ValArg3366His)]pTyr3933Cys

NA [30ndash32]

P19 M39 y

2000 Asymptomatic NA Normal CAPN3 c[1395_1397del2257GgtA]1453AgtG

p[(Leu465_Glu466delAsp753Asn)]pMet485Val

AR [12 32 33]

P20d M24 y

12000 Asymptomatic NA Mildmyopathicsigns

SGCA c242GgtAc739GgtA

pArg81HispVal247Met

NA [15]

P21 F53y

500 Limb-girdlemuscleweakness

HP0003325

Mildmyopathicsigns

RYR1 c10923-8CgtA-

Splice sitea- NA This work

P23 M64 y

1000 Asymptomatic NA Mildmyopathicsigns

RYR1 c14812AgtG- pIle4938Val- NA This work

P24d M67 y

1103 Myalgia HP0003326

Mildmyopathicsigns

MYOT c179CgtT- pSer60Phe- AD [34]

Continued

NeurologyorgNG Neurology Genetics | Volume 5 Number 5 | October 2019 7

field ofmuscle disorderswill lead to the identification of previouslyunrecognized causes of hyperCKemia thereby reducing the pro-portion of idiopathic cases This would be an important de-velopment as it would allowmore precise clinical monitoring andfollow-up and help to clarify the natural history of the differentforms

Studies conducted before NGS entered the clinical arena consid-ered the diagnostic impact of muscle biopsy in the evaluation ofpatients with asymptomatic hyperCKemia2336ndash38 and the relativefrequency of common genes such as DMD38 In recent years

NGSwhich has involved the development of different gene panelsand seen the focus switch to peculiar muscle phenotypes hastransformed the approach to the study of neuromuscular disorders(the relevant data are for the most part reviewed in reference 4)Some studies have also addressed among other aspects the ge-notype of patients with isolated hyperCKemia reporting a limiteddiagnostic yield (21) in fewer than 40 patients3940

Herein we report the results obtained in a cohort of 66patients with hyperCKemia half of whom were asymptomaticon evaluation Our use of massive parallel sequencing with

Figure 4 Diagnostic rates and molecular results

(A) Pathogenic or likely pathogenic variants were found in 50 of the patients whereas 257 showed variants that required further characterization and243 did not present pathogenic variants (B) Type of causative mutations identified in diagnosed patients missense (67) small indels (18) splice sitevariants (6) and nonsense mutations (9) (C) Bar chart showing number of patients with ldquopathogenicrdquo (red) ldquolikely pathogenicrdquo variants (yellow) andvariants of unknown significance (green) for each gene

Table 2 Clinical and genetic features in 20 patients with ldquolikelyrdquo pathogenic variants (continued)

ID

Sexage(y)

SerumCK (UIL)

Clinicalfindings

HPO IDcode

Musclebiopsy

Mutatedgene

MutationcDNA level

Mutationprotein level Inheritance Reference

P26d M21 y

1929 Asymptomatic NA Normal POMT2c c[1733GgtA239CgtT]c707TgtG

p[(Arg578HisPro80Leu)]pLeu236Arg

AR This work

P31 M26 y

40000 Asymptomatic NA Mildmyopathicsigns

RYR1c c5288CgtTc7681CgtT

pPro1763LeupLeu2561Phe

NA This work

Abbreviations AD = autosomal dominant AR = autosomal recessive HPO = human phenotype ontology NA = not availableaPredicting splice site loss and exon skipping (fruitflyorgseq_toolssplicehtml)bFunctional validation derived by immunofluorescenceprotein studies combined with predictive early protein truncationprevious published work withevidences of pathogenicitycFunctional validation derived through immunofluorescence studies onlyd Patients with ldquolikelyrdquo pathogenic variants confirmed by immunofluorescence analysis or previously published evidence of pathogenicity

8 Neurology Genetics | Volume 5 Number 5 | October 2019 NeurologyorgNG

a multigene panel combined with the application of stringentbioinformaticsfilters and integration ofmuscle phenotypes (bothclinical and morphological) produced a 50 diagnostic yielda rate higher than those previously reported3940 This difference islikely related to the combination of genotype with integratedclinico-morphological phenotype data in the present study to itsmore homogeneous population (mostly adults in our cohort asopposed to themixed populations studied by others) to technicalimprovements implicit in more recent gene panel technologiesor to a combination of these factors There were no straightfor-ward differences between the solved and unsolved participantswith clinical features and serum CK values being roughly thesame in the 2 subgroups Our data confirm that main challengesin NGS data analysis are the clinical interpretation of molecularfindings and the distinction of causative mutations from theplethora of not clinically significant DNA variations The lowfrequency of variants or their absence in reference polymorphicdatabases is recognized as a necessary but not sufficient criterionto assign pathogenicity Moreover in silico predictions presentoften unanticipated discordance between different predictingalgorithms4 All this make more complex to reach a final mo-lecular diagnosis Considering our results overall there emergeseveral points worth underlining First of the 33 patients whoreceived a molecular diagnosis 11 harbored mutations in RYR1and all of these presented normal muscle MRI scans and subtlemuscle involvement on skeletal muscle biopsy Only 1 presentedepisodes of rhabdomyolysis 2 presented myalgia and 1 showedmild limb-girdle muscle weakness Conversely 7 patients werefully asymptomatic with a normal clinical evaluation associatedwith serum steady CK levels ranging from 500 to 1000 UILAlthough we did not seek to further corroborate our genomicfindings by evaluating their functional impact on intracellularcalcium homeostasis through complex studies or by performingimmunoblotting with commercial antibodies all the detectedRYR1 variants were rare ones namely those inherited segregatedin familial cases with clinical manifestations and all are likely toperturb secondary ryanodine-1 receptor structure in proteinmodeling (not shown) Overall our data imply that mutations inRYR1may be a common in hyperCKemia even when CK levelsare lt1000 UIL Of interest we observed that the RYR1 par-ticipants presented pseudometabolic features more frequentlythan patients harboring other genotypes but did not stand up asa clinically different subgroup Second we identified biallelicmutations in ANO5 in 4 patients and mutations in SGCA in 2adults with long-lasting hyperCKemia without muscle weaknessThese data support previous impressions suggesting that analysisof known LGMD genes is justified even when isolated hyper-CKemia or minimal muscle weakness is the sole clinicalmanifestation4142 Third we frequently observed mild non-specific muscle abnormalities consisting of variation in fiber sizenuclear internalization or these 2 features combined thesealterationswere always unrelated to serumCK levels age at onsetclinical features disease duration and mutated gene Neverthe-less muscle biopsy remains an important part of the diagnosticprocess in hyperCKemia as it can also fulfill a ldquofunctionalrdquo roleassisting in the clarification of uncertain cases This is illustratedby case P28 in our study where 2 changes in SGCA were

substantiated by reduced α-sarcoglycan labeling in skeletal muscle(figure e-1 linkslwwcomNXGA175 A-B) Fourth 26 of thepatients were found to harbor VUS Nonetheless we frequentlyidentified single deleterious variants in CAPN3 and CPT2 2autosomal recessive genes where manifesting heterozygosity re-portedly occurs43ndash45 The existence in hyperCKemia of symp-tomatic carriers various degrees of clinical severity and largeintrafamilial heterogeneity of the phenotype (ranging fromasymptomatic to fatal) could be explained by factors other thanthe genotype In the case of CPT2-related myopathy for exam-ple one might consider different degrees of exposure totriggeringenvironmental factors (eg temperature nutritionfitness level) capable of increasing dependency on fat oxidationand further impairing exercise performance46 Taking these fac-tors into consideration as well as the possibility of synergisticheterozygosity34 (as in our case P32) and digenic inheritance it isimperative to combine thorough expert clinical and myopatho-logic evaluation with NGS as a prelude to a higher diagnostic ratein patients with paucisymptomatic hyperCKemia Finally iso-lated hyperCKemia can be the sole feature alerting to a pro-gressive muscular disorder requiring careful surveillance and mayopen new prospective in future therapeutic opportunities

AcknowledgmentThe authors thank Catherine J Wrenn who provided experteditorial assistance

Study fundingFunding partially provided by Telethon Foundation grantsGUP13004 to GA) and Regione Toscana FAS SALUTE 2014(CUP 404216092014066000060 to FMS)

DisclosureDisclosures available NeurologyorgNG

Publication historyReceived by Neurology GeneticsMarch 21 2019 Accepted in final formJune 21 2019

Appendix Authors

Name Location Role Contribution

AnnaRubegni MD

IRCCSStellaMaris Pisa

Author Designed and conceptualizedthe study analyzed the dataand drafted the manuscript forintellectual content

AlessandroMalandriniMD

Universityof Siena

Author Major role in the acquisition ofdata and drafted themanuscript for intellectualcontent

Claudia DosiMD

Universityof Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Guja AstreaMD

IRCCSStellaMaris Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Continued

NeurologyorgNG Neurology Genetics | Volume 5 Number 5 | October 2019 9

References1 Kyriakides T Angelini C Schaefer J et al European Federation of Neurological

Societies EFNS guidelines on the diagnostic approach to pauci- or asymptomatichyperCKemia Eur J Neurol 201017767ndash773

2 Silvestri NJ Wolfe GI Asymptomaticpauci-symptomatic creatine kinase elevations(hyperckemia) Muscle Nerve 201347805ndash815

3 Malandrini A Orrico A Gaudiano C et al Muscle biopsy and in vitro contracture testin subjects with idiopathic HyperCKemia Anesthesiology 2008109625ndash628

4 Nigro V Savarese M Next-generation sequencing approaches for the diagnosis ofskeletal muscle disorders Curr Opin Neurol 201629621ndash627

5 Astrea G Petrucci A Cassandrini D et al Myoimaging in the NGS era the discoveryof a novel mutation in MYH7 in a family with distal myopathy and core-likefeaturesmdasha case report BMC Med Genet 20161725

6 Musumeci O la Marca G SpadaM et al LOPED study looking for an early diagnosisin a late-onset Pompe disease high-risk population J Neurol Neurosurg Psychiatry2016875ndash11

7 Savarese M Di Fruscio G Mutarelli M et al MotorPlex provides accurate variantdetection across large muscle genes both in single myopathic patients and in pools ofDNA samples Acta Neuropathol Commun 20142100

8 Astrea G Romano A Angelini C et al Broad phenotypic spectrum and genotype-phenotype correlations in GMPPB-related dystroglycanopathies an Italian cross-sectional study Orphanet J Rare Dis 201813170

9 Thompson R Straub V Limb-girdle muscular dystrophiesmdashinternational collabo-rations for translational research Nat Rev Neurol 201612294ndash309

10 DrsquoAmore A Tessa A Casali C et al Next generation revolution inmolecular diagnosisof hereditary spastic paraplegias a large Italian cross-sectional study Front Neurol20189981

11 Hicks D Sarkozy A Muelas N et al A founder mutation in anoctamin 5 is a majorcause of limb-girdle muscular dystrophy Brain 2011134171ndash182

12 Piluso G Politano L Aurino S et al Extensive scanning of the calpain-3 genebroadens the spectrum of LGMD2A phenotypes J Med Genet 200542686ndash693

13 Richard I Broux O Allamand V et al Mutations in the proteolytic enzyme calpain 3cause limb-girdle muscular dystrophy type 2A Cell 19958127ndash40

14 Kubisch C Wicklein EM Jentsch TJ Molecular diagnosis of McArdle disease revisedgenomic structure of the myophosphorylase gene and identification of a novel mu-tation Hum Mutat 19981227ndash32

15 Piccolo F Roberds SL Jeanpierre M et al Primary adhalinopathy a common cause ofautosomal recessivemuscular dystrophy of variable severity Nat Genet 199510243ndash245

16 Duggan DJ Gorospe JR Fanin M Hoffman EP Angelini C Mutations in the sar-coglycan genes in patients with myopathy N Engl J Med 1997336618ndash624

17 Savarese M Di Fruscio G Tasca G et al Next generation sequencing on patients withLGMD and nonspecific myopathies findings associated with ANO5 mutationsNeuromuscul Disord 201525533ndash541

Appendix (continued)

Name Location Role Contribution

JacopoBaldacci BS

IRCCSStellaMaris Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

CarlaBattisti MD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

GiuliaBertocci BSc

IRCCSStellaMaris Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

M AliceDonati MD

AOUMeyerFlorence

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

M TeresaDotti MD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

AntonioFedericoMD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

FabioGiannini MD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

SalvatoreGrosso MD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

RenzoGuerrini MD

Universityof Florence

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Sara LenziMD

IRCCSStellaMaris Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Maria AMaioli MD

HospitalBinaghiCagliari

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

FedericoMelani MD

AOUMeyerFlorence

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

EugenioMercuri MD

CatholicUniversityRome

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

MicheleSacchini MD

AOUMeyerFlorence

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

SimonaSalvatoreMD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Appendix (continued)

Name Location Role Contribution

GabrieleSiciliano MD

Universityof Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

DeborahTolomeoMD

Universityof Pisa

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Paola ToninMD

Universityof Verona

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Nila VolpiMD

Universityof Siena

Author Acquisition analysis andinterpretation of data andcritical revision of themanuscript

Filippo MSantorelliMD

IRCCSStellaMaris Pisa

Author Interpreted the data revisedthe manuscript for intellectualcontent and provided funds forthe study

DeniseCassandriniPhD

IRCCSStellaMaris Pisa

Author Interpreted the data andrevised the manuscript forintellectual content

10 Neurology Genetics | Volume 5 Number 5 | October 2019 NeurologyorgNG

18 Tsujino S Shanske S DiMauro S Molecular genetic heterogeneity of myophos-phorylase deficiency (McArdlersquos disease) N Engl J Med 1993329241ndash245

19 Anichini A Fanin M Vianey-Saban C et al Genotype-phenotype correlations in a largeseries of patients with muscle type CPT II deficiency Neurol Res 20113324ndash32

20 Vockley J Rinaldo P BennettMJMatern D Vladutiu GD Synergistic heterozygositydisease resulting frommultiple partial defects in one or more metabolic pathways MolGenet Metab 20007110ndash18

21 Bohm J Vasli N Malfatti E et al An integrated diagnosis strategy for congenitalmyopathies PLoS One 20138e67527

22 Manning BM Quane KA Lynch PJ et al Novel mutations at a CpG dinucleotide inthe ryanodine receptor in malignant hyperthermia Hum Mutat 19981145ndash50

23 Carss KJ Stevens E Foley AR et al Mutations in GDP-mannose pyrophosphorylaseB cause congenital and limb-girdle muscular dystrophies associated with hypo-glycosylation of α-dystroglycan Am J Hum Genet 20139329ndash41

24 Sambuughin N Sei Y Gallagher KL et al North American malignant hyperthermiapopulation screening of the ryanodine receptor gene and identification of novelmutations Anesthesiology 200195594ndash599

25 Vukcevic M Broman M Islander G et al Functional properties of RYR1 mutationsidentified in Swedish patients with malignant hyperthermia and central core diseaseAnesth Analg 2010111185ndash190

26 SambuughinNNelsonTE Jankovic J et al Identification and functional characterization ofa novel ryanodine receptor mutation causing malignant hyperthermia in North Americanand South American families Neuromuscul Disord 200111530ndash537

27 Cagliani R Fortunato F Giorda R et al Molecular analysis of LGMD-2B and MMpatients identification of novel DYSF mutations and possible founder effect in theItalian population Neuromuscul Disord 200313788ndash795

28 SambuughinNHolleyHMuldoon S et al Screening of the entire ryanodine receptor type1 coding region for sequence variants associated with malignant hyperthermia susceptibilityin the north american population Anesthesiology 2005102515ndash521

29 Taroni F Verderio E Dworzak F Willems PJ Cavadini P DiDonato S Identificationof a common mutation in the carnitine palmitoyltransferase II gene in familial re-current myoglobinuria patients Nat Genet 19934314ndash320

30 Tammaro A Di Martino A Bracco A et al Novel missense mutations and unexpectedmultiple changes of RYR1 gene in 75 malignant hyperthermia families Clin Genet201179438ndash447

31 Duarte ST Oliveira J Santos R et al Dominant and recessive RYR1 mutations inadults with core lesions and mild muscle symptoms Muscle Nerve 201144102ndash108

32 Gillies RL Bjorksten AR Davis M Du Sart D Identification of genetic mutations inAustralian malignant hyperthermia families using sequencing of RYR1 hotspotsAnaesth Intensive Care 200836391ndash403

33 FaninM Fulizio L Nascimbeni AC et al Molecular diagnosis in LGMD2A mutationanalysis or protein testing Hum Mutat 20042452ndash62

34 Selcen D Engel AG Mutations in myotilin cause myofibrillar myopathy Neurology2004621363ndash1371

35 Venance SL Approach to the patient with HyperCKemia Continuum (MinneapMN) 2016221803ndash1814

36 Filosto M Tonin P Vattemi G et al The role of muscle biopsy in investigatingisolated muscle pain Neurology 200768181ndash186

37 Simmons Z Peterlin BL Boyer PJ Towfighi J Muscle biopsy in the evaluation ofpatients with modestly elevated creatine kinase levels Muscle Nerve 200327242ndash244

38 Prelle A Tancredi L Sciacco M et al Retrospective study of a large population ofpatients with asymptomatic or minimally symptomatic raised serum creatine kinaselevels J Neurol 2002249305ndash311

39 Savarese M Di Fruscio G Torella A et al The genetic basis of undiagnosed musculardystrophies and myopathies results from 504 patients Neurology 20168771ndash76

40 Wu L Brady L Shoffner J Tarnopolsky MA Next-generation sequencing to diagnosemuscular dystrophy rhabdomyolysis and hyperCKemia Can J Neurol Sci 201845262ndash268

41 Papadopoulos C LaforEt P Nectoux J et al Hyperckemia and myalgia are commonpresentations of anoctamin-5-related myopathy in French patients Muscle Nerve2017 561096ndash1100

42 Tarnopolsky M Hoffman E Giri M Shoffner J Brady L Alpha-sarcoglycanopathypresenting as exercise intolerance and rhabdomyolysis in two adults NeuromusculDisord 201525952ndash954

43 Zatz M Starling A Calpains and disease N Engl J Med 20053522413ndash242344 Vissing J Barresi R Witting N et al A heterozygous 21-bp deletion in CAPN3 causes

dominantly inherited limb girdle muscular dystrophy Brain 20161392154ndash216345 Fanin M Anichini A Cassandrini D et al Allelic and phenotypic heterogeneity in 49

Italian patients with the muscle form of CPT-II deficiency Clin Genet 201282232ndash239

46 Fontaine M Kim I Dessein AF et al Fluxomic assay-assisted diagnosis orientation ina cohort of 11 patients with myopathic form of CPT2 deficiency Mol Genet Metab2018123441ndash448

NeurologyorgNG Neurology Genetics | Volume 5 Number 5 | October 2019 11

DOI 101212NXG000000000000035220195 Neurol Genet

Anna Rubegni Alessandro Malandrini Claudia Dosi et al Next-generation sequencing approach to hyperCKemia A 2-year cohort study

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