THE JOURNAL OF PEDIATRICS • www.jpeds.com WORKSHOP/SYMPOSIUM SUMMARY Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne Muscular Dystrophy Annemieke Aartsma-Rus, PhD1, Madhuri Hegde, PhD, FACMG2, Tawfeg Ben-Omran, MD, FRCPS, FCCMG, FACMG3, Filippo Buccella, PharmD4, Alessandra Ferlini, MD, PhD5, Pia Gallano, PhD6, R. Rodney Howell, MD7, France Leturcq, PharmD8, Ann S. Martin, MS, CGC9, Anna Potulska-Chromik, MD, PhD10, Jonas A. Saute, MD, PhD11, Wolfgang M. Schmidt, PhD12, Thomas Sejersen, MD, PhD13, Sylvie Tuffery-Giraud, PhD14, Zehra Oya Uyguner, PhD15, Luci A. Witcomb, PhD16, Shu Yau, PhD, FRCPath17, and Stanley F. Nelson, MD18 D uchenne muscular dystrophy (DMD) is a severe, pro- For example, although 100% of delegates understood the gressive, X-linked, recessive neuromuscular disease that importance of genetic testing for DMD, more than 10% did affects approximately 1 in 5000 live male births.1 Pa- not perform additional genetic tests if deletion/duplication tients with DMD experience progressive muscle weakness, testing was negative.11 Survey results from the 2017 and 2018 owing to the absence of functional dystrophin protein, and typi- TREAT-NMD Expert Masterclasses on DMD, attended by more cally experience delayed walking, difficulty running or climb- than 100 delegates combined (primarily from pediatrics and ing stairs, and frequent falls.2,3 However, several nonmotor signs neurology backgrounds from 27 and 20 countries, respec- and symptoms (eg, behavioral issues, neurocognitive defi- tively), showed that some delegates experienced difficulties in- cits, and speech delay) also can be associated with the disease.3 terpreting DMD genetic test results (Table I; available at Boys with DMD are generally diagnosed between 4 and 5 years www.jpeds.com) and subsequently were not always aware of of age, when their physical development begins to diverge more whether patients were eligible for treatment with mutation- clearly from that of their peers,3-6 and, with corticosteroid treat- specific therapies. Together, these issues highlight the need for ment, typically lose ambulation at a median age of about 12.0- shorter times to diagnosis for patients with DMD and clearer 14.0 years.7 However, this can vary by country. recommendations for DMD genetic testing to ensure com- The accurate and early diagnosis of DMD plays a crucial role plete genetic assessment is performed to reach an accurate in the effective management of patients: it has the potential genetic diagnosis. This review, supported by a systematic lit- to lead to earlier intervention; appropriate genetic counsel- erature search, presents expert consensus on ways of reduc- ing; treatment with mutation-specific therapies (where appli- ing the time to diagnosis of DMD. cable); and appropriate assignment to clinical trials. However, reports indicate that significant delays in diagnosis of DMD persist.4-6,8 It has been shown that in the absence of a family Methods history of DMD, there is a delay of approximately 1 year from onset of earliest symptoms to the first assessment by a health- The Delphi Consensus Initiative presented here is focused on care professional.4 A further delay of approximately 1 year from how to reduce the time to diagnosis of DMD. The develop- this first assessment to referral to a neurologist or a neuro- ment process for this initiative is summarized in Figure 1. The muscular specialist is observed (mean age at diagnosis ± SD: steering committee comprised 3 experts in the field of human 4.9 ± 1.7 years).4 Findings by Vry et al6 suggest that this could genetics, specializing in the diagnosis of DMD and/or inter- be shorter, with a mean delay of just over 1 year from first pretation of genetic mutations: S. F. Nelson (nonvoting chair), symptoms to diagnosis (mean delay ± SD: 1.3 ± 1.8 years). A. Aartsma-Rus (voting co-chair), and M. Hegde (voting co- Overall, these findings suggest that, on average, there has been chair). After an initial meeting of the steering committee, 14 little to no reduction in the age at diagnosis for patients with experts in the field were invited to form the expert voting panel. DMD over the last 30 years.9,10 The panel comprised primarily medical geneticists specializ- Once DMD is suspected, genetic testing is required to obtain ing in the diagnosis of patients with DMD; however, 2 child a complete diagnosis.3,11,12 However, results from a recent survey neurologists, 1 patient advocate, and 1 genetic counselor also of 41 delegates from Europe, Turkey, and India (primarily child were invited. All members of the expert voting panel and the neurologists and clinical/molecular geneticists) revealed that 2 voting co-chairs voted anonymously on the statements to there may be issues relating to the genetic diagnosis for DMD. reach consensus. A systematic literature review was also per- formed to support development of the consensus statements BMD Becker muscular dystrophy CK Creatine kinase DMD Duchenne muscular dystrophy Detailed affiliations, funding, and conflicts of interest disclosure available at www.jpeds.com MLPA Multiplex ligation-dependent probe amplification NBS Newborn screening 0022-3476/$ - see front matter. © 2018 The Authors. Published by Elsevier Inc. This is an open access NGS Next-generation sequencing article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). https://doi.org10.1016/j.jpeds.2018.10.043 305 THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 • January 2019 Figure 1. Delphi Consensus Initiative development process following the steps outlined by Rosenfeld et al13 and summary of the Grading of Recommendations Assessment, Development, and Evaluation process,14-17 including voting options. (PRISMA flow diagram; Figure 2 and Table II [available at and that this finding also should require a high index of sus- www.jpeds.com]). The evidence was graded using the Grading picion from physicians. The consensus group agreed on a of Recommendations Assessment, Development, and Evalu- number of nonmotor signs and symptoms that may also act ation system (Figure 1). Further information regarding the de- as indicators of DMD (autism spectrum disorder, delayed velopment process is provided in the Appendix (available at speech/cognitive deficits, and gross motor delay; Statement 2) www.jpeds.com). The grading of evidence for the statements but are not always associated with the disease and may need was reviewed and agreed on by the expert voting panel wider clinical assessment. As such, these symptoms may some- (Table III). times be overlooked, and as a result, patients could be re- ferred incorrectly, for example to a physical, occupational, or speech therapist.4 As a result, the time to diagnosis for these Discussion patients can be delayed. The consensus group therefore agreed that for patients presenting with cognitive or developmental Consensus Statements deficits, DMD should be considered as part of the differen- The 15 consensus statements are presented in Table III. A tial diagnosis. summary of the supporting information is provided herein, Patients presenting with the motor and nonmotor signs and with a full discussion included in the Appendix. Several other symptoms of DMD as per Statements 1 and 2 should imme- more general statements were discussed, but only those most diately have their serum creatine kinase (CK) levels tested and pertinent to reducing the time to diagnosis of DMD are pre- be referred to a child neurologist or a neuromuscular specialist.3 sented here. A marked increase in serum CK, defined as >2000 IU/L,31 should prompt further investigation for DMD. However, el- Section 1: Reducing the Time to Diagnosis of DMD evated CK levels within the range of approximately 500- Signs and symptoms of DMD (motor and nonmotor) are pre- 1200 U/L (1.5 times the upper limit of normal for men), even sented in the 2018 care guidelines for DMD.3 The consensus if asymptomatic, may be indicative of other neuromuscular group agreed on a series of motor signs and symptoms disorders that require further assessment.103 It has been shown (Table III) that are typically observed in patients with DMD that once a patient has had a serum CK test, the time to reach (supported by the literature; Statement 1). It also should be a complete diagnosis of DMD is relatively short (mean age ± SD noted that patients are sometimes referred because of el- [range] at first CK test, 4.7 ± 1.7 [0.3-8.6] years, n = 151; and evated alanine transaminase or aspartate transaminase levels,3 at complete diagnosis, 4.9 ± 1.7 [0.3-8.8] years, n = 154).4 306 Aartsma-Rus et al Muscular Dystrophy Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne January 2019 Table III. Summary of statements and recommendations for reducing the time to diagnosis of DMD Final Consensus Strength of Level of GRADE Key supporting Consensus statements vote recommendation evidence* score† evidence Section 1: Reducing the time to diagnosis of DMD Statement 1: The following signs, symptoms, and characteristics should be considered typical indicators of A + = 93%; A = 7% Strongly in favor Moderate 1B 3,4,8,18-22 DMD: calf hypertrophy (pseudohypertrophy); delayed walking; difficulty climbing/descending stairs; difficulty rising from the floor; difficulty running/walking; elevated serum CK levels (including elevated ALT and AST); a family history of DMD; frequent falls; Gowers' sign; male sex; and muscle weakness. Statement 2: Autism spectrum disorder, delayed speech/cognitive deficits, and gross motor delay can be A + = 93%; A = 7% Strongly in favor Moderate 1B 3,4,8,18,21,23-30 indicators of DMD but are not always associated with the disease. Statement 3: After initial presentation, patients with suspected DMD should have their serum CK levels tested A + = 93%; A = 7% Strongly in favor Moderate 1B 3,4,8,18,31-33 and be referred to a specialist (a child neurologist or neuromuscular specialist). Statement 4: A lack of awareness of DMD and associated symptoms by the healthcare professional and long A + = 67%; A = 33% Strongly in favor Low 1C 4,8 waiting times to see a specialist are the primary factors leading to a delay in initial diagnosis. Statement 5: Genetic testing is crucial for obtaining a complete diagnosis of DMD, and should be considered A + = 94%; A = 6% Strongly in favor Low 1C 3,11,18 and expert opinion the gold standard. Statement 6: In the majority of cases, a complete genetic diagnosis can be made using MLPA or CGH to detect A + = 88%; A = 6%; Strongly in favor Moderate 1B 3,11,12,18,34-44 and expert opinion deletions or duplications in the DMD gene. N = 6% Statement 7: If exon-level deletions/duplications in the DMD gene are not identified, small-scale mutations (by A + = 93%; A = 7% Strongly in favor Low 1C 3,11,35 and expert opinion sequencing of exons and flanking regions) should be tested for as the next step. Statement 8: Muscle biopsies with dystrophin staining are generally not needed to obtain a complete diagnosis A + = 73%; A = 27% Strongly in favor Low 1C 3,11,12,35,43,45-54 and expert opinion of DMD, unless DNA testing is negative. Statement 9: Delays in the initial clinical diagnosis/referral to a specialist, the sequential nature of the genetic A + = 93%; A = 7% Strongly in favor Low 1C 4,8,11,19 and expert opinion testing process, and incomplete or nonexhaustive genetic testing should be addressed in order to prevent delays in reaching a complete genetic diagnosis for patients with DMD. Section 2: Recommendations for next steps following a suspected DMD diagnosis Statement 10: Patients with signs and symptoms of DMD and elevated serum CK levels should be referred for A + = 100% Strongly in favor Low 1C 3,55,56 and expert opinion genetic testing to either a clinical geneticist or a neuromuscular specialist. Statement 11: A medical/clinical geneticist, a child neurologist, or a neuromuscular specialist should request A + = 100% Strongly in favor Low 1C 12,57 and expert opinion the genetic test and should provide clinical information relevant to the diagnosis as part of the sample submission to the clinical genetics laboratory, and the genetic diagnostic test should be performed by an accredited laboratory. Statement 12: Educational meetings for physicians and laboratory specialists on topics relating to the genetic A + = 93%; A = 7% Strongly in favor Low 1C 58-62 and expert opinion WORKSHOP/SYMPOSIUM SUMMARY diagnosis of DMD would help to improve the understanding of genetic test reports and the interpretation of genetic test results. Statement 13: Genetic testing is necessary to inform carrier testing, family planning, genetic counseling, A + = 100% Strongly in favor Low 1C Carrier testing/family planning11,63-73; prognosis and optimal management strategies, natural history data gathering, and prenatal diagnosis. genetic counseling71,74; prognosis/management23,75-78; natural history7,79,80; prenatal diagnosis12,31; and expert opinion Statement 14: After a patient receives a complete genetic diagnosis of DMD, it is mandatory that carrier testing A + = 100% Strongly in favor Moderate 1B 11,12 ; carrier testing64-66,68-70,73,81-90; of the mother and other at-risk female family members be offered with appropriate pre- and postgenetic germline mosaicism/de novo counseling (information regarding germline mosaicism and de novo mutations should also be offered). mutations91-95; family planning/genetic Similarly, testing of other at-risk male family members should also be offered. counseling71,74; and expert opinion Statement 15: When a family history is present, pre-symptomatic CK testing and prenatal testing can lead to A + = 87.5%; Strongly in favor Low 1C 1,12,31,96-102 and expert opinion earlier detection and thus management of DMD, which is important for family planning.‡ A = 12.5% ALT, alanine aminotransferase; AST, aspartate aminotransferase; CGH, comparative genome hybridization; CK, creatine kinase; DMD, Duchenne muscular dystrophy; GRADE, Grading of Recommendations Assessment, Development, and Evaluation. *The level of evidence for most of the statements was graded as either low or moderate, owing to the fact that most of the studies included here are observational in nature rather than randomized controlled trials (due to the nature of this initiative). When there were multiple corroborative supporting observational studies, we have selected “moderate” for quality of evidence. †Consensus: A + = strongly agree; A = agree; N = neither agree nor disagree; D = disagree; D + = strongly disagree. Grade of recommendation: 1A = strong recommendation, high-quality evidence; 1B = strong recommendation, moderate-quality evidence; 1C = strong recommendation, low-quality or very low-quality evidence; 2A = weak recommendation, high-quality evidence; 2B = weak recommendation, moderate-quality evidence; 2C = weak recommendation, low-quality or very low-quality evidence. 307 ‡Where applicable depending on country-specific legislation on presymptomatic testing of patients aged ≤18 years. THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 Indeed, patients who are identified by an incidental finding of dystrophin staining confirms dystrophinopathy, mRNA analy- elevated CK (before onset of signs and symptoms) can be di- sis should be performed to identify any impact on mRNA agnosed earlier.8 The consensus group agreed that a lack of splicing11,12,45,113 that escapes detection by both MLPA and DNA awareness of the signs and symptoms of DMD and delays in sequencing. RNA analysis is also crucial to determine the con- the time taken to see a specialist are the primary factors con- sequence of the mutation on the mRNA and can be consid- tributing to a delay in the initial diagnosis. In support of this, ered for discordant phenotypes. Muscle biopsy remains a a retrospective chart review of 156 patients without a family relevant diagnostic tool, especially for mutation types in which history of DMD using the Muscular Dystrophy Surveillance, the mutation is poorly predictive of disease progression (for Tracking and Research Network (MD STARnet) showed, in a instance, a deletion of exons 3-7 can cause both BMD and subset of patients (n = 127), that 63.8% (81/127) were seen by DMD phenotypes), or for determining dystrophin expres- a pediatrician/family practitioner at their first evaluation and sion in boys with unexpectedly mild disease progression.11 We that a CK test was ordered as a result of the first evaluation note that there are several databases that can help physicians in only 34.6% (44/127) of cases.4 This finding indicates a need to determine the severity of individual and rare causal muta- to increase awareness of DMD among front-line healthcare tions in the DMD gene (Appendix). In addition to consult- professionals. ing these databases, physicians entering new data on genotype The consensus group agreed that genetic testing is crucial and phenotype are warranted to help inform future diagnoses. to obtain a complete diagnosis of DMD. The DMD gene is one The consensus group agreed that delays in the initial clini- of the largest known human genes (2.2 Mb), containing 79 cal diagnosis or referral to a specialist, the sequential nature exons34,104 with a relatively high mutation rate (~30% of cases of the genetic testing process, and incomplete or nonexhaustive are caused by a de novo mutation).11,45,105 The approximate dis- genetic testing should be addressed to prevent delays in diag- tribution of mutations in the DMD gene is as follows: dele- nosis. In addition, if the ordering physician is not sufficiently tion of 1 or more exons, 68%; duplication of 1 or more exons, informed or trained to understand the hierarchy of tests needed 11%; small-scale mutations, 20% (small-scale deletions, 5%; to provide a complete genetic diagnosis for DMD, the testing small-scale insertions, 2%; splice-site, 3%; nonsense, 10%; mis- process could be terminated prematurely (as shown sense, 0.4%); and deep intronic mutations, 0.3%.35 This dis- elsewhere).11 It would therefore be helpful if the responsibil- tribution is supported by a number of studies.23,36,37,46,106-110 It ity for these decisions was integrated with the primary testing is therefore practical to test for DMD gene mutations in order laboratory’s operating procedures. The laboratory would thus of frequency. The consensus group agreed on 3 statements that be obligated to contact the ordering physician to discuss the outline the recommended steps needed to reach a complete next level of testing required either to obtain a complete genetic genetic diagnosis of DMD (Figure 3).4,35,46 First, multiplex diagnosis of DMD or to exclude DMD from the diagnosis. ligation-dependent probe amplification (MLPA) or compara- tive genome hybridization should be used to screen for dele- Section 2: Recommendations for Next Steps tions or duplications in the DMD gene. These tests are Following a Suspected DMD Diagnosis recommended because they can screen all 79 exons of the DMD A patient with signs and symptoms of DMD and elevated CK gene; however, it should be noted that there are several limi- levels should be referred to a clinical geneticist/neuromuscular tations associated with MLPA. For example, point mutations specialist during the genetic testing process, because these in- or polymorphisms along the probe hybridization region can dividuals are best placed to provide accurate interpretation of present as single-exon deletions when using this method, and genetic test results55 and can help to avoid diagnostic delay thus a second confirmatory test (usually Sanger sequencing) (Table III).3 is required.11,12,31 However, if the mutation is identified and cor- In 2010, best practice guidelines for the molecular diagno- relates with the severity of symptoms (eg, DMD or Becker mus- sis of DMD/BMD were developed using a consensus-building cular dystrophy [BMD]), no further genetic testing is typically approach.12 More recently, general standards and guidelines for required. If exonic deletions/duplications in the DMD gene are the interpretation of sequence variants have been published not identified, small-scale mutations should be tested for by by the American College of Medical Genetics and Genomics sequencing exons and flanking intronic regions. If clinical signs and the Association for Molecular Pathology.57 and genetic testing are conclusive, in most cases, muscle bi- The consensus group agreed that a clinical geneticist usually opsies are not needed. Nevertheless, if no mutation is de- is responsible for the interpretation of the genetic test results tected after DNA analysis, it is possible that the patient may but that the process should be shared between the clinical ge- have a large rearrangement, such as an inversion (or translo- neticist, the physician who ordered the test (typically a child cation in females11), a deep intronic mutation that affects splic- neurologist or neuromuscular specialist), and/or a genetic ing, or an alternative diagnosis.12 Inversions and translocations counselor. are challenging to detect with most conventional genetic analy- Although improving the clarity of genetic test reports and ses, because they do not affect copy number.111 Deep intronic directing physicians to the appropriate specialists for assis- mutations often can be detected by next-generation sequenc- tance would ultimately ensure a quicker and more accurate ing if the full genomic sequence of the gene is available37,112; genetic diagnosis for patients, it is also important for physi- however, it can be difficult to predict the impact of intronic cians (nongeneticists) to understand genetic test results, because mutations (or variations) on mRNA. If a muscle biopsy with this will have a direct impact on patient management. The 308 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY Figure 3. Diagnostic steps for reaching a complete diagnosis of DMD. ALT, alanine aminotransferase; AST, aspartate amino- transferase; NGS, next-generation sequencing. *Elevated CK levels within the range of approximately 500-1200 U/L (1.5 times the upper limit of normal for men), even if asymptomatic, may be indicative of other neuromuscular disorders that require further assessment.103 †Point mutations or polymorphisms along the probe hybridization region can present as single-exon deletions when using MLPA and thus a second confirmatory test (typically Sanger sequencing) may be required.11,12,31 ‡Testing of at-risk (asymptomatic) male relatives will be dependent on country-specific legislation. Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 309 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 consensus group therefore agreed that educational meetings Conclusions for physicians and laboratory specialists on topics relating to the genetic diagnosis of DMD would help to improve physi- Delays in the diagnosis of DMD have remained relatively un- cians’ understanding of genetic tests and the interpretation of changed over the last 30 years,9,10 despite advances in our un- the results. derstanding of the natural history and improvements in genetic The consensus group also agreed on 3 statements to high- testing. Delays occur early in the diagnostic pathway, because light the next steps that should be taken after a complete di- of a lack of awareness of DMD and its signs and symptoms agnosis has been reached. These statements highlight the among families, and, more pertinently, among front-line health- importance of carrier testing, genetic counseling, family- care professionals.4 Issues relating to the understanding of planning decisions, and prenatal diagnosis. It is estimated that genetic testing required to obtain a complete diagnosis of DMD approximately one-third of patients with DMD develop the have been highlighted (Table I).11 Patients presenting with the disease owing to de novo mutations and that the remaining typical motor signs and symptoms of DMD, as well as the less two-thirds inherit the mutation from carrier mothers.11,63,64 If well-recognized neurocognitive deficits, developmental delays, the mutation identified in the affected patient is not carried and elevated liver enzymes, should be immediately referred to by the mother in her somatic cell line, the mother may be a a specialist (child neurologist or neuromuscular specialist) and germline mosaic and should be provided with counseling about should have their serum CK levels measured. Patients with a the risk of having a second son with the disease12 or having a marked elevation in serum CK should be referred to a clini- daughter that is a carrier.11 Female carriers are typically as- cal geneticist as soon as possible, and the full range of sequen- ymptomatic because DMD is an X-linked inherited disease; tial genetic tests offered to provide a complete diagnosis. After however, carriers can develop mild clinical symptoms, includ- diagnosis, it is mandatory to offer carrier testing to mothers ing muscle weakness and scoliosis, 65,66 and are at an in- and other at-risk female relatives. By highlighting these issues creased risk of cardiomyopathy.67-69 Despite this, approximately and providing an in-depth discussion of the DMD diagnos- one-third of potential carriers are likely unaware of their carrier tic pathway, we hope that patients will be diagnosed earlier, status. 64,70 Genetic testing is thus important for family- care provided as soon as possible, and personalized interven- planning decisions and counseling of other family members.71,74 tion provided for eligible patients. ■ Genetic information also is used for genotype–phenotype correlations,7,75,76,79,114 so that patients can be provided with prog- This process was conducted and managed by an independent third- nostic information and be offered appropriate medical party agency (PharmaGenesis London, London, United Kingdom), and management.11,23,77,78 we acknowledge the support of Sally Janani, MSc, and Nicola Baines, BSc (PharmaGenesis London) for their support with the Delphi con- As per the 2010 best practice guidelines for the molecular sensus process. Both were funded by PTC Therapeutics Inc. diagnosis of DMD/BMD, prenatal testing is advised to be carried out only for at-risk male pregnancies (those with a Submitted for publication Jun 27, 2018; last revision received Sep 27, 2018; family history of the disease).12 Prenatal screening is not cur- accepted Oct 24, 2018 rently recommended for female fetuses, because it is not yet Reprint requests: Stanley F. Nelson, MD, Department of Human Genetics, University of California, Box 957088, 5506A Gonda Center, Los Angeles, CA possible to determine whether a female heterozygote for a DMD 90095. E-mail: snelson@ucla.edu mutation will exhibit any signs of disease.12 However, the con- sensus group agreed that when a family history is present, pre- natal testing leads to earlier detection of DMD and is thus Disclaimer important for family-planning decisions. Recommendations regarding prenatal diagnosis will depend on country-specific The Delphi Consensus statements presented here are based on legislation.12,31 The consensus group also agreed that in the pres- the opinions of carefully selected experts in the field and are ence of a family history of disease, presymptomatic CK testing for information and educational purposes only. The state- would lead to earlier detection and thus earlier management ments may reflect gaps in current knowledge, but, where pos- of patients with DMD, but would be dependent on country- sible, have been supported by relevant literature. These specific legislation. statements do not reflect clinical practice guidelines or legal The 195th European Neuromuscular Centre International standards of care and, as such, do not include all potential di- Workshop (2012) report presented discussions on newborn agnostic or management steps. The responsible physician, in screening (NBS) for DMD; the meeting was attended by 21 light of all of the circumstances presented by the individual experts from 7 countries. It was discussed that the introduc- patient, must determine the appropriate treatment, diagno- tion of NBS for DMD using CK testing would help to detect sis, and management. the disease earlier in patients and reduce the risk of having additional children with DMD.1 A pilot study in the US Endorsements demonstrated the feasibility of a 2-tier NBS system for DMD using dried blood spots to test CK levels, followed by DMD Endorsed by the European Paediatric Neurology Society gene testing96; however, there are many complexities involved (EPNS), the Muscular Dystrophy Association (MDA), with NBS programs, some of which are discussed in the Duchenne Parent Project Italy, Parent Project Muscular Dys- Appendix. trophy (PPMD), and TREAT-NMD. 310 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY References 21. 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Dastur RS, Kachwala MY, Khadilkar SV, Hegde MR, Gaitonde PS. 59. Greb AE, Brennan S, McParlane L, Page R, Bridge PD. Retention of Identification of deletions and duplications in the Duchenne muscular medical genetics knowledge and skills by medical students. Genet Med dystrophy gene and female carrier status in western India using 2009;11:365-70. combined methods of multiplex polymerase chain reaction and mul- 60. Klitzman R, Chung W, Marder K, Shanmugham A, Chin LJ, Stark M, tiplex ligation-dependent probe amplification. Neurol India 2011;59:803- et al. Attitudes and practices among internists concerning genetic testing. 9. J Genet Couns 2013;22:90-100. 42. Bovolenta M, Neri M, Fini S, Fabris M, Trabanelli C, Venturoli A, et al. 61. Burke S, Stone A, Bedward J, Thomas H, Farndon P. A “neglected part A novel custom high density-comparative genomic hybridization array of the curriculum” or “of limited use”? 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Evidence-based path to newborn screening for Duchenne ternational Neuromuscular Research Group Duchenne natural history muscular dystrophy. Ann Neurol 2012;71:304-13. study. Ann Neurol 2015;77:684-96. Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 rounds, (2) the collection, analysis and interpretation of data Detailed affiliations or the development of the consensus statements, (3) the writing of the report, or (4) the decision to submit the paper From the 1Department of Human Genetics, Leiden for publication. The consensus process was administered by University Medical Center, Leiden, The Netherlands; PharmaGenesis London, a third-party agency, which was 2 Department of Human Genetics, Emory University funded by PTC Therapeutics Inc under the guidance of the School of Medicine/School of Biological Sciences, Georgia steering committee. No honoraria were provided to the Institute of Technology/Perkin Elmer Genetics, Atlanta, steering committee or the expert voting panel for participat- GA; ing in this initiative. 3 A.A-R. is employed by Leiden University Medical Center Clinical and Metabolic Genetics, Department of Pediatrics, (LUMC), which has patents on exon skipping technology, some Hamad Medical Corporation, Doha, Qatar; of which have been licensed to BioMarin and subsequently 4 Duchenne Parent Project Italy, Rome; sublicensed to Sarepta Therapeutics. As co-inventor of some 5 of these patents, A.A-R. is entitled to a share of royalties. Re- Unit of Medical Genetics, University of Ferrara, Ferrara, muneration for consultancy is paid to LUMC. LUMC also Italy; received speaker honoraria from BioMarin Pharmaceuticals 6 U705 CIBERER, Servei de Genetica, Hospital de Sant Pau, and PTC Therapeutics. M.H. is employed by PerkinElmer Ge- Barcelona, Spain; netics and receives royalty payments from Agilent Technolo- 7 gies and Oxford Gene Technologies for next-generation Miller School of Medicine, University of Miami, Miami, sequencing products and has received speaker honoraria from FL; BioMarin, Genzyme, Pharmaceuticals, and PTC Therapeu- 8 Department of Genetics and Molecular Biology, tics. F.B. is a community pharmacy owner, has acted as a Hospitalier Universitaire Paris Centre, Cochin Hospital, consultant for PTC Therapeutics and Santhera Pharmaceuti- Paris, France; cals, and has received honoraria related to advisory board par- 9 ticipation or speaker activities. A.F. received honoraria via the Parent Project Muscular Dystrophy, Hackensack, NJ; Ferrara Hospital for participation in clinical trials and also 10 Department of Neurology, Medical University of Warsaw, served as a PTC Diagnostic Board member and a Sarepta Eu- Warsaw, Poland; ropean Scientific Board member, and received honoraria related 11 to the board participation or speaker activities. R.H. receives Medical Genetics and Neurology Services, Hospital de speaking fees from Sanofi-Genzyme. F.L. served on a scien- Clinicas de Porto Alegre/Internal Medicine Department, tific advisory board for PTC Therapeutics Inc in France and Universidade Federal do Rio Grande do Sul, Porto Alegre, also received honoraria related to speaker activities. A.M. re- Brazil; ceived speaker honoraria from BioMarin and Sarepta Thera- 12 Neuromuscular Research Department, Medical University peutics. A.P-C. is employed by the Medical University of Warsaw of Vienna, Vienna, Austria; and received honoraria for lectures or consultancy from 13 Allergan, Biogen, Kedrion, Novo-Nordisk, PTC Therapeutics Department of Women’s and Children’s Health, Karolinska (clinical trial investigator), Sanofi-Genzyme, and Teva Phar- Institute/Astrid Lindgrens Barnsjukhus, Karolinska maceutical Industries. J.S. received research funding from PTC University Hospital, Stockholm, Sweden; Therapeutics. W.S. received honoraria for serving as a con- 14 Laboratory of Rare Genetic Diseases (LGMR), University sultant from PTC Therapeutics and Santhera Pharmaceuti- of Montpellier, Montpellier, France; cals. T.S. received research grants from Sanofi-Genzyme, and 15 honoraria for lectures or consultancy from Biogen, BioMarin, Department of Medical Genetics, Istanbul Medical Faculty, PTC Therapeutics, and Sanofi-Genzyme. S.T-G. served on a Istanbul University, Istanbul, Turkey; scientific advisory board for PTC Therapeutics Inc in France, 16 PharmaGenesis London; and remuneration for this activity was paid to the University 17 of Montpellier. The University of Montpellier also received Viapath Analytics, Guy’s Hospital, London, United speaker honoraria from PTC Therapeutics Inc. Z.U. received Kingdom; and honoraria from PTC Therapeutics Inc. L.W. is an employee 18 Department of Human Genetics, University of California, of PharmaGenesis London, London, United Kingdom, and Los Angeles, CA was funded by PTC Therapeutics Inc to provide medical writing and editorial support (including to perform the systematic review of the literature) for this initiative under the guidance Funding and Conflicts of Interest Disclosure of the steering committee. S.N. is employed by UCLA and received honoraria from PTC Therapeutics, Sanofi-Genzyme, This initiative was funded by PTC Therapeutics Inc; however, Sarepta Therapeutics, and Solid Biosciences for DMD- neither PTC Therapeutics Inc nor any other commercial related advisory panels. T.B-O, P.G., and S.Y. declare no con- entity was involved in (1) the study design or the voting flicts of interest. 313.e1 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY during round 2 (using a ranking or 5-point Likert scale). Appendix Voting rounds 1 and 2 were conducted using SurveyMonkey (https://www.surveymonkey.co.uk/). Introduction Voting round 3 was developed in advance of a 1-day con- Example survey results from the 2017 and 2018 TREAT- sensus meeting held in May 2017 in Copenhagen, Denmark, NMD Expert Masterclasses regarding the genetic diagnosis of and the draft statements were circulated to the consensus group patients with DMD are presented in Table I, available at before the meeting. This round comprised 24 statements for www.jpeds.com. which the consensus group had to rate their level of agree- ment using a 5-point Likert scale (“strongly agree” [A+]; “agree” Methods [A]; “neither agree nor disagree” [N]; “disagree” [D]; or “strongly Systematic Literature Searches. Search terms were identi- disagree” [D+]; Figure 1.1-4) using anonymized electronic fied by the steering committee and literature searches per- keypads. Consensus was defined a priori as at least 75% agree- formed in Ovid (2017 Ovid Technologies, Inc, New York, New ment (either “strongly agree” [A+] or “agree” [A]). Each ques- York) by PharmaGenesis London, London, United Kingdom, tion was introduced by the nonvoting chair. If consensus was to screen the MEDLINE and EMBASE databases. Search terms not reached after the first round of voting, alterations to the (title/abstract) were: Duchenne muscular dystrophy, DMD or statement were made based on discussions before the next Duchenne AND any of the following search terms: sign; round of voting was conducted. symptom; creatine kinase; transaminase; diagnos*; gene*; genetic The 3 members of the steering committee then graded the test*; genetic report*; genetic counsel*; delay*; screen*; carrier; level of evidence and strength of recommendations support- mutation; sequenc*; comparative genome hybridi?ation; CGH; ing each statement using the Grading of Recommendations multiplex ligation-dependent probe amplification; MLPA; mul- Assessment, Development, and Evaluation system (Figure 1). tiplex polymerase chain reaction; multiplex PCR; biops*; pre- The grading was reviewed and agreed on by the expert voting natal; neonatal; female; family planning; germline mosaicism; panel (Table III). de novo; practitioner; p?ediatrician; p?ediatric neurologist; neu- romuscular specialist. The “?” and “*” functions searched for Additional Supporting Information for the Consensus spelling variations and variations of the word ending, respec- Statements. Additional supporting information from the sys- tively. Search results were limited to: articles published tematic literature review for each consensus statement is de- from January 1, 2010, to April 8, 2018; English; studies of tailed below. humans; and full journal articles. The full electronic search Section 1: Reducing the Time to Diagnosis of DMD strategy is provided in Table II, available at www.jpeds.com. (Table III). PharmaGenesis London exported the initial raw results and highlighted articles of potential relevance using a traffic light Statement 1. The following signs, symptoms, and character- system (red—unlikely to be relevant; amber—potentially rel- istics should be considered typical indicators of DMD: calf hy- evant; green—likely to be relevant). The 3 members of the pertrophy (pseudohypertrophy); delayed walking; difficulty steering committee then reviewed the relevance of the sup- climbing/descending stairs; difficulty rising from the floor; dif- porting literature. Any articles included by the authors that ficulty running/walking; elevated serum CK level (including were not identified by the systematic literature review were elevated alanine transaminase and aspartate transaminase); a categorized as ad hoc in the PRISMA flow diagram in Figure 2, family history of DMD; frequent falls; Gowers’ sign; male sex; available at www.jpeds.com. Data were then extracted by and muscle weakness. PharmaGenesis London, and used, under the guidance of the steering committee, to draft questions to aid the develop- ment of the Consensus Statements. Vote: A + = 93%; A = 7%; grade of recommendation: 1B Development of the Consensus Statements via Iterative Discussion Voting. Voting round 1 included 29 questions drafted by the In support of this statement (Table III), a retrospective chart steering committee based on initial review of the literature, to review of 156 boys with DMD (no family history of disease) which the consensus group provided free-text responses. The using the Muscular Dystrophy Surveillance, Tracking, and Re- results were collated by PharmaGenesis London to ensure voter search Network (MD STARnet) assessed the range of signs and anonymity, and the anonymized results were reviewed by the symptoms first reported to healthcare providers by patients who nonvoting chair. During each voting round, the consensus group were later diagnosed with DMD (n = 111).5 In patients aged was encouraged to comment on the wording and content of 3 to <5 years, the signs and symptoms included calf hyper- the questions, themes, and statements. After each round, the trophy, 7.1%; difficulty rising from the floor, 23.8%; diffi- steering committee made changes to the statements to reflect culty climbing, 31.0%; frequent falls/clumsiness, 33.3%; the feedback received. difficulty running/walking, 38.1%; and muscle weakness, Voting round 2 included 20 questions formulated using 40.5%.5 These symptoms, with the exception of muscle weak- the responses from round 1. The consensus group then rated ness, difficulty running/walking, and difficulty climbing, were the importance of, or agreement with, the themes identified the most frequently reported within this age range compared Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e2 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 with younger or older patients with DMD.5 Muscle weakness to have an intellectual disability. Of 87 patients who com- and difficulty climbing were reported more frequently in older pleted the full neurodevelopmental assessment, 21% (18/87) patients (≥5 years old), and difficulty running/walking was more scored in the autistic spectrum disorder range, 24% (21/87) frequently reported in patients aged 1.5 to <3 years and pa- showed clinical hyperactivity, and 44% (38/87) had severe dif- tients aged ≥5 years, as the symptom(s) first reported to health- ficulties with inattention.11 Similarly, a study in Germany of care providers.5 263 patients with DMD showed that 30% of patients experi- These findings are supported by several other studies. First, enced a delay in gross motor development and that approxi- a mixed-methods study of parents’ and patients’ experiences mately 40% of patients showed cognitive impairment (learning of the diagnosis of DMD in Australia reported that the initial difficulty, 26%; intellectual disability, 17%).12 A smaller case symptoms noted by parents (n = 62) (by >20% of parents) were note review of 20 boys with DMD (without a family history calf hypertrophy, 42%; complaining of tired legs, 26%; diffi- of disease) in the United Kingdom showed that speech delay culties with walking, 35%; frequent falls, 44%; tiring easily, 37%; was a presenting feature in 25% of the cohort and that this difficulties running, 65%; and difficulties climbing stairs, 61%.6 was identified in 45% of the patients at the time of diagnosis.7 Similarly, a case note review of 20 boys with DMD in the United A study of patients with DMD from eastern India showed that Kingdom showed that 20% (8/20) of boys reported “diffi- approximately one-third of boys with DMD had mild intel- culty with steps,” and 20% (8/20) of boys experienced “falls” lectual disability (IQ 38-63).9 A recent study of 209 caregiv- as part of their initial symptom profile. Three of the 20 boys ers of boys with DMD identified through MD STARnet showed experienced delayed first walking (later than the 18-month mile- that cognitive deficits were reported in 38.4% of boys.13 Fur- stone), and 4 of the boys were diagnosed from an incidental thermore, a chart review study of 179 boys with DMD (1989- finding of elevated serum CK level.7 A retrospective review of 2012) showed that delayed walking and cognitive impairment 24 patients with DMD in Korea reported that their initial symp- were correlated (P ≤ .0001).14 It also has been shown that boys toms were: difficulty rising from the floor, 16.7%; lower- with DMD have a reduced rate of implicit learning com- extremity muscle weakness, 90.0%; family history of DMD, pared with boys with typical development, even in the absence 29.2%; and calf hypertrophy, 87.5%. Mean serum CK levels of global intellectual disability.15 also were elevated (14 144 IU/L).8 In addition, a study of pa- The location of the DMD gene mutation also has been shown tients with DMD from eastern India showed that of the 81 pa- to correlate with the severity of cognitive impairment. For tients assessed, 100% had lower-limb weakness, 97.5% had example, an observational study of 47 Italian boys with DMD neck-muscle weakness, 93.8% had calf hypertrophy, 70.4% ex- showed that Full-Scale IQ scores correlated with the location hibited Gowers’ sign, and 27.1% had a family history of DMD.9 of the dystrophin gene mutation; mutations in the distal region Lastly, a retrospective study of medical records for 540 pa- of the DMD gene were associated with more severe cognitive tients in the US with DMD showed that patients with a family deficits.16 Patients with point mutations in the DMD gene ex- history of disease are typically seen and diagnosed at an earlier hibited a higher degree of cognitive impairment than those with age than those without a family history (with family history deletions or duplications (P = .005). In addition, patients with vs without family history: age at initial evaluation, 30.8 months mutations in the distal region of the DMD gene had lower IQ vs 56.8 months; P < .001; age at CK measurement, 35.1 months levels than those who had mutations in the proximal region.17 vs 64.0 months; P < .001).10 Age at genetic testing did not differ Additional information is presented in a recent review of the significantly between the 2 groups (56.2 months vs 64.3 months, literature examining neuropsychological and neurobehavioral respectively). functioning in patients with DMD.18 Commentary These neurocognitive deficits and developmental delays can The consensus group agreed that patients were sometimes sometimes be overlooked; as a result, patients with DMD can identified inadvertently as a result of high serum CK levels, be referred to the incorrect specialist. This was exemplified in detected as part of routine screening or assessments unre- a retrospective chart review of 156 boys with DMD using MD lated to a diagnosis of DMD. It also was discussed that a family STARnet, which showed that although 16.5% (21/127) of pa- history of DMD would act as a strong indicator of DMD, par- tients were correctly referred to either a neurologist or a neu- ticularly in a male patient presenting with signs and symp- romuscular specialist, 15.7% (20/127) were referred to a toms of the disease. physical, occupational, or speech therapist as a result of their first evaluation.5 Statement 2. Autism spectrum disorder, delayed speech/ Commentary cognitive deficits, and gross motor delay can be indicators of No additional supporting information is included. DMD but are not always associated with the disease. Statement 3. After initial presentation, patients with sus- pected DMD should have their serum CK tested and be re- Vote: A + = 93%; A = 7%; grade of recommendation: 1B ferred to a specialist (a pediatric neurologist or neuromuscular specialist). Discussion In support of this statement, a European study of 4 centers found that 26% (34/130) of patients with DMD were reported Vote: A + = 93%; A = 7%; grade of recommendation: 1B 313.e3 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY Discussion DMD (with a family history of the disease) in the United Serum CK level was first recognized as a marker of mus- Kingdom showed that in 19 of the boys, the serum CK level cular dystrophy approximately 60 years ago.19 Levels of serum test was performed in secondary care, thus delaying the time CK are elevated in patients with muscular dystrophy; this is to diagnosis (10-50 months, n = 15; <10 months, n = 4). The caused by leakage of cytoplasmic CK from damaged muscle 1 boy whose serum CK level test was requested by a general fiber cells into the blood circulation.20 Findings from a retro- practitioner had a minimal delay in the time to diagnosis (<10 spective case note review of 20 boys with DMD (with a family months),7 highlighting the importance of recognizing symp- history of the disease) in the United Kingdom showed that a toms early. Furthermore, an Australian mixed-methods study delay in requesting a serum CK level test accounted for the ma- has shown that parents visit a range of healthcare profession- jority of the delay experienced by patients (mean [range] di- als in their search for a diagnosis, and 51% of the parents sur- agnostic delay from first visit with a healthcare professional veyed (29/57) felt that their child could have been diagnosed to diagnosis of DMD, 8.8 [0-50] months; delay in CK level test, earlier.6 Findings from the largest cross-sectional survey of Eu- 7.2 [0-49] months; and time from CK test result available to ropean patients with DMD21 also showed a delay of approxi- genetic diagnosis, 1.6 [0-4] months).7 The mean (range) age mately 1 year from report of first symptoms to diagnosis (mean at diagnosis for these patients was 51.7 (10-91) months; delay ± SD: 1.3 ± 1.8 years); however, this varied by country. however, in 4 boys who had been diagnosed based on inci- Commentary dental serum CK level findings as part of an unrelated illness, When asked for their opinion, most consensus group the age at diagnosis was greatly reduced (35.3 [10-57] months).7 members (93% [13/14]) agreed that a “wait-and-see” ap- Commentary proach by healthcare professionals can lead to a delay in the The consensus group agreed that a serum CK level test plays initial diagnosis of DMD. When asked if a “wait-and-see” ap- a pivotal role in the diagnostic process for DMD and that this proach by the family also could lead to a delay in the initial should be requested immediately or as soon as possible by pri- diagnosis, the responses were more varied: 50% answered “yes”; mary care physicians, along with referral to a specialist (a pe- 43% answered “not sure”; and 7% answered “no.” An initial diatric neurologist or a neuromuscular specialist), for any patient delay of approximately 1 year from when the first signs or presenting with associated signs and symptoms of the disease. symptoms are noted in the child (usually by the family, a care- Overall, 87% of consensus group members (13/15) indicated giver, or a school teacher) to the child’s first evaluation by a that the request for a serum CK level test should be made “im- healthcare provider also has been reported.5 Furthermore, a mediately” or “as soon as possible” after the patient presents mixed-methods study of parents’ and patients’ experiences re- with initial symptoms; the remaining 13% (2/15) indicated that ported that factors affecting a parent’s decision to seek medical this should be done within 1 week. Similarly, the majority (75% help include lack of self-confidence; being a first-time parent; [9/12]) of consensus group members also agreed that referral reassurance from others; and the broad range of normal de- to a specialist should occur immediately or as soon as possible velopment seen in children.6 These findings highlight that after receiving a positive CK test result; the remaining 25% (3/ awareness of DMD needs to be improved generally (not just 12) indicated that this should occur within 1 week. for front-line healthcare professionals); however, this is beyond the scope of this initiative. Statement 4. A lack of awareness of DMD and associated During the meeting, the consensus group members also were symptoms by the healthcare professional and long waiting times asked whether a 4-week wait to see a specialist for additional to see a specialist are the primary factors leading to a delay in testing (ie, after an initial clinical diagnosis) was appropriate. initial diagnosis. Of the 15 consensus group members who responded, 87% (13/ 15) answered “yes”, and 13% (2/15) answered “no.” The con- sensus group members were then asked if their patients are Vote: A + = 67%; A = 33%; grade of recommendation: 1C seen within 4 weeks for this additional testing. Of the 15 who responded, 47% (7/15) answered “not always”; 27% (4/15) an- Discussion swered “unsure”; 20% (3/15) answered “yes”; and 7% (1/15) In support of this statement, a retrospective chart review of answered “no,” demonstrating that delays in seeing a special- 156 patients without a family history of DMD using MD ist are experienced. STARnet showed that there is a delay of approximately 1 year from first evaluation by the healthcare professional to refer- Statement 5. Genetic testing is crucial for obtaining a com- ral to a neurologist or neuromuscular specialist (mean age of plete diagnosis of DMD and should be considered the gold patient ± SD [range]: first evaluation, 3.6 ± 1.7 [0.2-8.0] years; standard. first neurology/neuromuscular specialist visit, 4.6 ± 1.7 [0.3- 8.6] years).5 This highlights a significant delay in the time from first assessment to seeing a specialist. Several patients also were Vote: A + = 94%; A = 6%; grade of recommendation: 1C referred incorrectly (eg, to a speech therapist),5 demonstrat- ing a lack of awareness of some of the signs and symptoms Discussion associated with DMD among front-line healthcare profession- Full characterization of the mutation affecting the DMD als. Similarly, a retrospective case note review of 20 boys with gene is required to determine its predicted effect on the Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e4 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 reading frame. This is important because mutations that PCR; these tests can also be used for carrier testing in cause a frame-shift, or that change an amino acid codon into females.22,29 a stop codon, generally result in a more severe phenotypic presentation of the disease (ie, DMD), owing to the prema- Multiplex Ligation-Dependent Probe Amplification. Lalic et al30 ture termination of translation. This results in truncated, designed and validated the MLPA assay to screen all 79 exons nonfunctional dystrophin protein.22 In contrast, mutations of the DMD gene for deletions and duplications. The MLPA that do not disrupt the reading frame typically result in a less assay was then used to test samples from 123 unrelated pa- severe form of the disease (ie, BMD), owing to the produc- tients with DMD or BMD who had already been screened by tion of a shorter but partly functional version of the dystrophin multiplex PCR. The study showed that MLPA was able to detect protein.22 all previously identified deletions, as well as a number of new At this time, there are 2 mutation-specific therapies with a mutations that were previously not detected (new deletions, form of approval. Ataluren has received conditional approval n = 7; new duplications, n = 9; new point mutation, n = 1 [note: from the European Medicines Agency for the treatment of point mutations are detected by MLPA only when the muta- ambulatory patients with nonsense mutation DMD.23 Eteplirsen tion lies within the binding site of the MLPA probe]).30 MLPA has received accelerated approval from the US Food and also was able to determine the precise genetic rearrange- Drug Administration for the treatment of patients with ment, which is important for determining the effect on the DMD who have a mutation in the DMD gene amenable to reading frame.30 mRNA reframing by exclusion of exon 51 from mature Ultimately, the study showed that MLPA outperformed mRNA.24,25 the Beggs and Chamberlain multiplex PCR test (detecting Commentary ~13% more mutations). In light of this, the authors recom- No additional supporting information is included. mended that MLPA be considered the method of choice for initial DNA analysis of patients with suspected DMD or BMD.30 Statement 6. In the majority of cases, a complete genetic di- The improved detection rate of MLPA compared with mul- agnosis can be made using MLPA or comparative genome hy- tiplex PCR is supported by a number of other studies.31-33 bridization (CGH) to detect deletions or duplications in the However, the MLPA assay has a number of limitations: it is DMD gene. unable to provide information regarding the location of intronic breakpoints22,34 and point mutations or nonpathogenic poly- Vote: A + = 88%; A = 6%, N = 6%; grade of recommendation: 1B morphisms in the probe binding site can present as single- exon deletions when using this method.29,35,36 A second Discussion confirmatory test, generally Sanger sequencing of the in- There are several databases that can be accessed to obtain volved exons, should therefore be performed for these false- information on DMD gene mutations: positive test results.22,29,34,37 In addition, false-negative test results may occur if there is a partial exonic deletion that is not • The Leiden Open Variation Database is an online open source coincident with the ligation site of the MLPA probe.36 Despite that currently contains more than 10 000 DMD gene mu- this, MLPA is a commercially available test and is currently tations (www.dmd.nl).26 • The UMD-TREAT-NMD database was recently set up as part the most widely used.29 of an international effort to provide up-to-date informa- Comparative Genome Hybridization. Soon after the develop- tion about DMD gene mutations worldwide (http://umd.be/ TREAT_DMD/); data from more than 30 national regis- ment of MLPA, a novel DMD-CGH array covering the full tries are collated here.27 genomic region of the DMD gene was tested.38 The assay was • The Human Gene Mutation Database (HGMD) is a data- able to detect all previously identified deletions and duplica- base for human inherited diseases and is maintained by the tions (4/4) and was able to provide the location of intronic Institute of Medical Genetics in Cardiff, United Kingdom breakpoints for these patients. The CGH assay also was able (http://www.hgmd.cf.ac.uk/ac/index).28 to identify the causative mutations in 3 of 8 of the patients with DMD who had previously tested negative by MLPA.38 CGH Owing to the fact that approximately 80% of all DMD mu- provides a high-resolution assay that has enabled the detec- tations are either large deletions or duplications, it is practi- tion of complex genomic rearrangements and intronic cal to screen for these mutations as the first step toward reaching breakpoints29,39,40 and therefore has a slightly greater muta- a complete genetic diagnosis of DMD.22 Multiplex poly- tion detection rate than MLPA. This is supported by several merase chain reaction (PCR) using the Chamberlain and col- other studies.33,41 leagues, and Beggs and colleagues primer sets, along with more Ultimately, if deletion/duplication testing is positive, and the recent primer sets, can be used to detect approximately 98% mutation is fully characterized and correlates with the sever- of all DMD gene deletions; however, these assays do not char- ity of symptoms (eg, DMD or BMD), then no further genetic acterize the boundaries of these mutations, do not screen all testing is required.42,43 In some cases, patients with a BMD phe- 79 exons of the DMD gene, and are unable to detect notype have been found to have an out-of-frame DMD gene duplications.22,29 MLPA and CGH are quantitative assays and deletion according to MLPA analysis. An explanation for this can characterize all of the mutations not detected by multiplex discrepancy is that with MLPA, only a portion of the exon is 313.e5 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY analyzed, and there have been some rare cases reported where codons.22 Intronic mutations detected by NGS have been re- the deletion extends to the splice site of the adjacent exon but ported in a number of cases of DMD,51-54 and muscle biop- not the MLPA target site. sies also have been used to confirm the presence of aberrant Commentary dystrophin mRNA in patients with DMD.36,39,55-57 In addition In the near future, newer assays such as next-generation se- to cases of deep intronic mutations, muscle biopsies may also quencing (NGS) will be able to evaluate deletions, duplica- be considered if the patient presents with a discordant phe- tions, and point mutations in a single assay.44,45 However, notype (ie, the genotype would predict DMD, but the patient because routine NGS is not yet widely available, it was not in- presents with BMD, or vice versa)22,58,59; however, obtaining this cluded as part of this consensus statement. information will not ultimately change the disease course or how the patient is managed.22 Statement 7. If exon-level deletions/duplications in the DMD Commentary gene are not identified, small-scale mutations (by sequenc- For novel or ultra-rare mutations not previously associ- ing of exons and flanking regions) should be tested for as the ated with dystrophinopathy, family-based segregation analy- next step. sis should be recommended (it should be noted that depending on the diagnostic strategies in different countries, segrega- tion analysis is systematically performed). Vote: A + = 93%; A = 7%; grade of recommendation: 1C Statement 9. Delays in the initial clinical diagnosis/referral to Discussion a specialist, the sequential nature of the genetic testing process, If deletion/duplication testing is negative, it is possible that and incomplete or nonexhaustive genetic testing should be ad- the patient may have a small-scale mutation in the DMD gene dressed to prevent delays in reaching a complete genetic di- (these account for ~20% of all DMD gene mutations).22,46 agnosis for patients with DMD. Despite these findings, a recent survey of 41 individuals (pri- marily pediatric neurologists and clinical geneticists) from Europe, Turkey, and India at the 2015 TREAT-NMD Expert Vote: A + = 93%; A = 7%; grade of recommendation: 1C Masterclass on DMD showed that >10% of respondents did not perform additional tests if deletion/duplication testing was Discussion negative.22 The reasons for this included the cost of Sanger se- Despite improvements in genetic testing, the sequential quencing and the need for shipping of samples to other labo- nature of the genetic testing process has the potential to lead ratories for analysis.22 This was further supported by survey to delays in diagnosis, particularly for patients with rare mu- results from the 2017 and 2018 TREAT-NMD Expert tations (ie, those who will be diagnosed at the very end of the Masterclasses on DMD (Table I, available at www.jpeds.com), diagnostic pathway). However, the availability of NGS will ul- in which 18.8% and 11.1% of the delegates, respectively, did timately provide a single-platform test to detect the majority not know the correct next steps following a negative MLPA of DMD gene mutations and should significantly reduce the test result. time to and cost of diagnosis.45,47,49 Small-scale mutations (single-nucleotide variants) typi- As discussed, incomplete or nonexhaustive genetic testing cally are identified using Sanger sequencing of all individual can result in patients not receiving a complete or definitive di- exons22; however, because it is becoming more cost- and agnosis. A survey of 41 healthcare professionals (primarily pe- time-effective,47 it is likely that in the near future, this meth- diatric neurologists and clinical geneticists) showed that odology will be replaced by next-generation whole-exome although 100% understood the importance of genetic testing, sequencing.22,44,45,48-50 more than 10% would not request further tests if deletions or Commentary duplications were not identified.22 Furthermore, results from No additional supporting information is included. the 2017 and 2018 TREAT-NMD Expert Masterclasses on DMD revealed a lack of understanding of DMD genetics (ie, the se- Statement 8. Muscle biopsies with dystrophin staining are gen- quential steps needed to obtain a complete genetic diagnosis erally not needed to obtain a complete diagnosis of DMD, for the patient as shown in Figure 3) and difficulties with the unless DNA testing is negative. interpretation of genetic test results (Table I, available at www.jpeds.com). Ultimately, this lack of understanding could result in pa- Vote: A + = 73%; A = 27%; grade of recommendation: 1C tients receiving an incomplete or even incorrect genetic diag- nosis and prevent them from receiving a mutation-specific Discussion therapy for which they are eligible. A deep intronic mutation can cause part of an intron to be Commentary incorporated into the dystrophin mRNA if it is recognized as When asked how long it should take from a specialist or- an exon by the splice-site machinery (such sequences are known dering the genetic test to the patient receiving a complete genetic as cryptic or pseudo-exons). The inclusion of intronic se- diagnosis, the majority of consensus group members indi- quences can disrupt the reading frame and generate stop cated that this should take no longer than 8 weeks (77% [10/13]). Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e6 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 Section 2: Recommendations for next steps following a sus- in the US showed that 73.7% and 87.1% of internists rated their pected DMD diagnosis (Table III). knowledge of (1) genetics and (2) genetics guidelines as poor, respectively. The internists also acknowledged that they re- Statement 10. Patients with signs and symptoms of DMD and quired further training on particular topics (eg, when to order elevated serum CK levels should be referred for genetic testing tests, 79.0%; how to counsel patients, 82.0%; how to inter- to either a clinical geneticist or a neuromuscular specialist. pret results, 77.3%).63 Commentary No additional supporting information is included. Vote: A + = 100%; grade of recommendation: 1C Statement 13. Genetic testing is necessary to inform carrier Discussion testing, family planning, genetic counseling, prognosis and No additional supporting information is included. optimal management strategies, natural history data gather- Commentary ing, and prenatal diagnosis. No additional supporting information is included. Statement 11. A medical/clinical geneticist, a pediatric neu- Vote: A + = 100%; grade of recommendation: 1C rologist, or a neuromuscular specialist should request the genetic test and should provide clinical information relevant to the di- Discussion agnosis as part of the sample submission to the clinical ge- Genetic testing is important to inform carrier testing, family netics laboratory, and the genetic diagnostic test should be planning, and genetic counseling for patients and their fami- performed by an accredited laboratory. lies. A qualitative study examined the impact of genetic testing on extended family members. Thirteen grandmothers in fami- lies with individuals showing fragile X syndrome or DMD were Vote: A + = 100%; grade of recommendation: 1C interviewed. The interviews showed that most grandmothers Discussion expressed feelings of guilt or responsibility. This highlighted Laboratories should be formally accredited or certified by that although genetic counseling is generally focused on family organizations such as the International Laboratory Accredi- planning, it also should be offered to extended family members tation Cooperation,29,60 or be covered by such programs as the because of the psychosocial impact.64 Similarly, a qualitative Clinical Laboratory Improvement Amendments program.61 study on parent communication with siblings of children af- Commentary fected by inherited conditions also highlighted the impor- The consensus group members were asked who performs tance of genetic counseling for unaffected siblings, who are at the genetic testing in their respective countries. Of the 16 risk of being carriers.65 members, 14 indicated that this is performed by a molecular Genetic information is also important for natural history genetics laboratory; however, only 4 of these 14 specified that data gathering, because such studies can help to provide in- the laboratory had to be accredited. It was therefore felt that formation on prognosis and risk of complications; for example, this should be specified in the statement, because it is recom- it has been shown that patients with deletions flanking exon mended by current guidelines.29,61 44 or a deletion of exons 3-7 lose ambulation later than those with other out-of-frame deletions (hazard ratio [95% CI]: de- Statement 12. Educational meetings for physicians and letion amenable to exon 44 skipping, 0.34 [0.15-0.74]; P = .007; laboratory specialists on topics relating to the genetic diag- exons 3-7 deletion: 0.24 [0.07-0.82]; P = .02).66-68 nosis of DMD would help to improve the understanding of Commentary genetic test reports and the interpretation of genetic test No additional supporting information is included. results. Statement 14. After a patient receives a complete genetic di- agnosis of DMD, it is mandatory that carrier testing of the Vote: A + = 93%; A = 7%; grade of recommendation: 1C mother and other at-risk female family members be offered with appropriate pre- and postgenetic counseling (informa- Discussion tion regarding germline mosaicism and de novo mutations In support of this statement, a study of views on genetics should also be offered). Similarly, testing of other at-risk male training for nongenetic specialists in the United Kingdom family members also should be offered. showed that of 90 general practitioners surveyed, 90% (81/ 90) felt that genetics was increasingly important and should be given more attention in their training; 83% (75/90) felt that Vote: A + = 100%; grade of recommendation: 1B they did not know all they needed to know about genetics; and 71% (64/90) felt that the training they had received had been Discussion insufficient to prepare them for their current role.62 Simi- As per the 2010 best practice guidelines, if carrier status has larly, a study of 220 internists from 2 academic medical centers been confirmed in an individual, genetic counseling should be 313.e7 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY offered to that individual and to other at-risk family members. Commentary Prenatal screening also may be offered, depending on local During the consensus meeting, 93% (14/15) of group practices/legislation.29 If prenatal testing is offered and returns members indicated that, at their institute, routine carrier testing a positive result, the genetic test report should clearly indi- was offered to those affected; however, there are geographical cate that the male fetus is predicted to have DMD or BMD. If differences regarding coverage. Members from the US (n = 4) the mutation identified in the affected patient is not carried all indicated that they had experienced problems or had heard by the mother in her somatic cell line, the mother may be a of problems when requesting carrier testing, either because in- germline mosaic and should be provided with counseling about surance companies would not cover the cost of the test or the risk of having a second son with the disease and that daugh- because the physician requesting the test had to provide suf- ters are at risk of being a carrier.22,29,69 If the mother is found ficient rationale to obtain funding to perform it. In contrast, not to be a carrier, the presence of a de novo mutation should the majority of members from Western Europe and the region be explained; de novo mutations are thought to occur in ap- of Central and Eastern Europe, Middle East, and Africa indi- proximately one-third of isolated cases, and these have been cated that they did not experience difficulties when request- reported throughout the literature.70-73 ing carrier tests to be performed. Symptoms of female carriers can include calf pseudohy- Owing to the difficulties associated with lack of insurance pertrophy; cardiomyopathy; cramps; frequent falls; Gowers’ sign; or insufficient insurance coverage, charities and pharmaceu- muscle weakness; myalgia74-77; and in some cases, intellectual tical companies offer or have offered free or subsidized genetic and mental health problems.75,78,79 A recent study of 36 female testing for patients and at-risk female relatives; one such ini- DMD and BMD carriers showed that 47% (17/36) had at least tiative is the Decode Duchenne program, set up by Parent 1 pathologic finding on cardiac magnetic resonance imaging, Project Muscular Dystrophy. Decode Duchenne offers free including left ventricular ejection fraction <55% (n = 5 [DMD, genetic testing for individuals with a diagnosis or symptoms n = 4; BMD, n = 1]) and presence of late gadolinium enhance- of DMD and reduced-rate carrier testing for at-risk female ment (n = 16 [DMD, n = 13; BMD, n = 3]).80 Similarly, a ret- relatives.92 Based on the findings in the literature and the dis- rospective US observational study of 22 female DMD carriers cussions at the consensus meeting, it was felt that it should be who underwent cardiac magnetic resonance analysis also mandatory to offer carrier testing of mothers and other at- showed the presence of cardiac involvement: 18% (4/22) had risk female relatives (first- and second-degree female rela- left ventricular ejection fraction <55%, and 35% (7/20) were tives, which include the mother of the individual with DMD, found to have late gadolinium enhancement.81 These find- along with the individual’s female siblings, female offspring, ings are supported by a series of case studies and a prospec- maternal grandmothers, maternal aunts, and their offspring), tive study of 15 confirmed female carriers.82-87 to provide appropriate medical management. Despite these issues, it is thought that approximately one-third of potential carriers could be unaware of their Statement 15. When a family history is present, presympto- carrier status88,89 and may need to be reassessed for risk of matic CK testing and prenatal testing can lead to earlier de- carrier status.90 This is supported by a number of studies. tection and thus management of DMD, which is important First, a study in the United Kingdom examined levels of for family planning.* carrier testing from 1971 to 2008. In Western Scotland, 843 potential carriers (from 195 families) were tested: of these, Vote: A + = 87.5%; A = 12.5%; grade of recommendation: 1C 16% and 48% of first-degree and second-degree/more distant relatives had not been tested, respectively. In England, 1223 *Where applicable, depending on country-specific legisla- potential carriers (from 349 families) were tested: of these, tion on presymptomatic testing patients aged ≤18 years. 49% of first-degree and 65% of second-degree/more distant relatives had not been tested to determine their carrier Discussion status.91 One study in the Netherlands also showed that of Although indirectly related, NBS and presymptomatic CK patients registered up to July 1, 2009, 33.7% (35/104) of testing have some common discussion points. A report from adult sisters/maternal aunts of patients with DMD who had the 195th European Neuromuscular Centre International Work- a 50% risk of being a carrier had not been tested by DNA shop (2012) presented discussions on NBS for DMD; the analysis. This percentage was similar (30.4% [45/148]) for meeting was attended by 21 experts from 7 countries. The adult sisters with a lower-risk carrier status (4.3% risk).88 In report summarized that although (at the time of the study) the US, a recent study by Bogue et al also estimated that 37% there had been 17 pilot NBS programs, no country currently of carriers who had an increased risk of cardiomyopathy had had a national screening program for DMD.93 The workshop never had an echocardiogram and cited that the most com- group advised that because of the risk of false-positive and false- monly identified barrier to carrier testing in the US was the negative results with the blood-spot CK test, a 2-tier system cost of the genetic tests.89 should be employed. This approach would use an initial CK As previously discussed, genetic counseling also should be test followed by DMD gene testing for those with elevated CK offered for siblings of affected patients, and extended family levels.93-95 The feasibility of this approach was demonstrated members (such as grandmothers), to manage the psychoso- by Mendell et al.95,96 Benefits of an NBS program included earlier cial impact.64,65 diagnosis and recognition of previously overlooked symptoms. Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e8 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 During the Canadian NBS program, boys who were diag- The consensus group also was asked if NBS for DMD should nosed with DMD were monitored afterwards. In a number of be performed: 47% (7/15) members answered “yes”; 47% (7/ these boys, neurocognitive motor developmental delay was iden- 15) answered “not sure”; and one answered “no.” The reason tified at a much earlier age (12-18 months) than would be for 47% answering “not sure” was primarily related to the anticipated.93 The report also highlighted that physiotherapy absence of available treatments for patients younger than 5 years and corticosteroid treatment could be implemented earlier as of age. However, the earlier treatment of patients with corti- a result of NBS programs.93 Although only pilot studies have costeroids has been shown to be beneficial.103,104 Lastly, the con- been performed thus far, a NBS program for DMD is being sensus group members were asked if they felt that CK-based initiated in the Zhejiang Province in China.97 NBS would lead to an earlier genetic diagnosis and improve Stress and anxiety often are cited as the reasons for not per- standards of care, natural history studies, genetic counseling, forming NBS for DMD; however, a recent survey of parents and family planning: 75% of members (12/16) answered “yes.” of children with DMD, BMD, or spinal muscular atrophy The consensus group members were asked whether they felt showed that the level of support for NBS for these condi- that prenatal testing for DMD should be offered in the absence tions was >90%.98 Furthermore, a recent Internet-administered of a family history of disease. Of the 16 members, 56% (9/ study of 2991 adults showed that participants were more likely 16) answered “no,” 31% (5/16) answered “not sure,” and 13% to select an optional DMD NBS program when information (2/16) answered “yes.” about this option was presented alongside a mandatory DMD NBS program,99 highlighting the importance of how these Further Reading schemes are presented to parents and carers. A publication by Kwon et al presented a proposed sched- 1. Guyatt GH, Cook DJ, Sackett DL, Eckman M, Pauker S. Grades of rec- ule of follow-up visits (and their objectives) for patients iden- ommendation for antithrombotic agents. Chest 1998;114:441S-444S. tified as having DMD by NBS.96 The aim of this publication 2. Guyatt GH, Oxman AD, Kunz R, Falck-Ytter Y, Vist GE, Liberati A, et al. was to provide anticipatory guidance for healthcare profes- GRADE: going from evidence to recommendations. BMJ 2008;336:1049- sionals, the patients, and the families/guardians to provide ef- 51. 3. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso- fective care for the patient’s lifespan after a diagnosis as a result Coello P, et al. GRADE: an emerging consensus on rating quality of evi- of NBS. dence and strength of recommendations. BMJ 2008;336:924-6. After a genetic diagnosis has been received, prenatal testing 4. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Schunemann also can be performed (depending on local practices) for any HJ. GRADE: what is “quality of evidence” and why is it important to clinicians? BMJ 2008;336:995-8. at-risk male fetuses29,100; this can help to inform any family- 5. Ciafaloni E, Fox DJ, Pandya S, Westfield CP, Puzhankara S, Romitti PA, planning decisions. Preimplantation screening also is offered et al. Delayed diagnosis in Duchenne muscular dystrophy: data from the at a number of specialist centers.29 Muscular Dystrophy Surveillance, Tracking, and Research Network (MD A study from The Netherlands examined the impact of 26 STARnet). J Pediatr 2009;155:380-5. years of prenatal testing for DMD.101 The study showed that 6. Wong SH, McClaren BJ, Archibald AD, Weeks A, Langmaid T, Ryan MM, during the period 1984-2009, 635 prenatal diagnoses were made; et al. A mixed methods study of age at diagnosis and diagnostic odyssey for Duchenne muscular dystrophy. Eur J Hum Genet 2015;23:1294- of these, 51% were male, and 46% of these male fetuses either 300. were affected by DMD or had an increased risk of the disease. 7. van Ruiten HJ, Straub V, Bushby K, Guglieri M. Improving recogni- As a result of this prenatal testing, 145 male fetuses were tion of Duchenne muscular dystrophy: a retrospective case note review. aborted, and 174 continued to full term unaffected by DMD. Arch Dis Child 2014;99:1074-7. 8. Na SJ, Kim WJ, Kim SM, Lee KO, Yoon B, Choi YC. Clinical, immuno- However, in the cohort of boys during 1961-1974 (n = 397), histochemical, Western blot, and genetic analysis in dystrophinopathy. 62% of the boys did not have an affected relative, suggesting J Clin Neurosci 2013;20:1099-105. that they were the first affected individual in their family. During 9. Dey S, Senapati AK, Pandit A, Biswas A, Guin DS, Joardar A, et al. Genetic the period 1993-2002, 88% of boys in the cohort were the first and clinical profile of patients of Duchenne muscular dystrophy: expe- affected with DMD in their family. The fact that current policy rience from a tertiary care center in Eastern India. Indian Pediatr 2015;52:481-4. recommends not to test female fetuses, and that many female 10. Holtzer C, Meaney FJ, Andrews J, Ciafaloni E, Fox DJ, James KA, et al. members are not tested for their carrier status (in this study, Disparities in the diagnostic process of Duchenne and Becker muscu- 78% of girls ≥16 years old who were at risk of being a carrier lar dystrophy. Genet Med 2011;13:942-7. had not been tested), has likely contributed to the increased 11. Ricotti V, Mandy WP, Scoto M, Pane M, Deconinck N, Messina S, et al. incidence of first affected boys being born. Neurodevelopmental, emotional, and behavioural problems in Duchenne muscular dystrophy in relation to underlying dystrophin gene muta- Commentary tions. Dev Med Child Neurol 2016;58:77-84. When asked whether CK testing should be offered to younger 12. Sarrazin E, von der Hagen M, Schara U, von Au K, Kaindl AM. Growth asymptomatic or presymptomatic male siblings at risk of DMD, and psychomotor development of patients with Duchenne muscular dys- 100% of consensus group members who responded an- trophy. 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A new de novo mutation in a 2016;2:e103. 313.e11 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY 91. McGowan R, Challoner BR, Ross S, Holloway S, Joss S, Wilcox D, et al. 98. Wood MF, Hughes SC, Hache LP, Naylor EW, Abdel-Hamid HZ, Results of Duchenne muscular dystrophy family screening in practice: Barmada MM, et al. Parental attitudes toward newborn screening for leaks rather than cascades? Clin Genet 2013;83:187-90. Duchenne/Becker muscular dystrophy and spinal muscular atrophy. 92. DuchenneConnect. DuchenneConnect: DecodeDuchenne—frequently Muscle Nerve 2014;49:822-8. asked questions. https://www.duchenneconnect.org/component/content/ 99. Lillie SE, Tarini BA, Janz NK, Zikmund-Fisher BJ. Framing optional article/114-website-content/decodeprogram/961-faq.html. Accessed No- genetic testing in the context of mandatory newborn screening tests. BMC vember 26, 2018. Med Inform Decis Mak 2015;15:50. 93. Ellis JA, Vroom E, Muntoni F. 195th ENMC International Workshop: 100. Wang H, Xu Y, Liu X, Wang L, Jiang W, Xiao B, et al. Prenatal diagno- newborn screening for Duchenne muscular dystrophy December 14- sis of Duchenne muscular dystrophy in 131 Chinese families with 16, 2012, Naarden, The Netherlands. Neuromuscul Disord 2013;23:682- dystrophinopathy. Prenat Diagn 2017;37:356-64. 9. 101. Helderman-van den Enden AT, Madan K, Breuning MH, van der Hout 94. Moat SJ, Bradley DM, Salmon R, Clarke A, Hartley L. Newborn bloodspot AH, Bakker E, de Die-Smulders CE, et al. An urgent need for a change screening for Duchenne muscular dystrophy: 21 years experience in Wales in policy revealed by a study on prenatal testing for Duchenne muscu- (UK). Eur J Hum Genet 2013;21:1049-53. lar dystrophy. Eur J Hum Genet 2013;21:21-6. 95. Mendell JR, Shilling C, Leslie ND, Flanigan KM, al-Dahhak R, Gastier- 102. ESHG. Genetic testing in asymptomatic minors: recommendations of Foster J, et al. Evidence-based path to newborn screening for Duchenne the European Society of Human Genetics. Eur J Hum Genet 2008;17:720- muscular dystrophy. Ann Neurol 2012;71:304-13. 1. 96. Kwon JM, Abdel-Hamid HZ, Al-Zaidy SA, Mendell JR, Kennedy A, 103. Merlini L, Cicognani A, Malaspina E, Gennari M, Gnudi S, Talim B, et al. Kinnett K, et al. Clinical follow-up for Duchenne muscular dystrophy Early prednisone treatment in Duchenne muscular dystrophy. Muscle newborn screening: a proposal. Muscle Nerve 2016;54:186-91. Nerve 2003;27:222-7. 97. Ke Q, Zhao ZY, Griggs R, Wiley V, Connolly A, Kwon J, et al. Newborn 104. Merlini L, Gennari M, Malaspina E, Cecconi I, Armaroli A, Gnudi S, et al. screening for Duchenne muscular dystrophy in China: follow-up diag- Early corticosteroid treatment in 4 Duchenne muscular dystrophy pa- nosis and subsequent treatment. World J Pediatr 2017;13:197-201. tients: 14-year follow-up. Muscle Nerve 2012;45:796-802. Table I. Example survey results from the 2017 and 2018 TREAT-NMD*,† Expert Masterclasses on DMD regarding the genetic diagnosis of patients with DMD Question Correct TREAT- Total Number who Number Number who answer NMD number of answered who answered “I Expert responses, correctly, answered do not know,” Masterclass N n (%) incorrectly, n (%) n (%) MLPA analysis is negative for a patient with suspected Sequencing 2017 64 51 (79.7) 12 (18.8)‡ 1 (1.6) DMD. What is your next step? (1) Multiplex PCR, (2) of all exons Consider another differential diagnosis, (3) Sequencing of all and flanking 2018 45 40 (88.9) 5 (11.1)§ 0 (0.0) exons and flanking sequences, (4) Muscle biopsy and sequences dystrophin analysis, (5) I do not know. MLPA analysis reveals a deletion of exon 20. What is your Perform a 2017 64 31 (48.4) 31 (48.4) 2 (3.1) next step? second test (1) Multiplex PCR, (2) Perform a second test to confirm the to confirm 2018 45 12 (26.7) 31 (68.9) 2 (4.4) finding, (3) Nothing, the diagnosis is confirmed, (4) Muscle the finding biopsy and dystrophin analysis, (5) I do not know. A patient with a deletion of exon 51 is eligible for which No therapy 2017 65 18 (27.7) 39 (60.0) 8 (12.3) therapy? (1) Ataluren/stop-codon readthrough, yet available (2) Eteplirsen/exon-51 skipping, (3) No therapy yet available, (4) I do not know. 2018 49 21 (42.9) 20 (40.8) 8 (16.3) Multiplex analysis reveals the presence of exons 44, 45, and I do not 2017 57 14 (24.6) 43 (75.4) NA 51, and the absence of exon 50. Is this patient eligible for know 2018 46 12 (26.1) 34 (73.9) NA exon 51 skipping? (1) Yes, (2) No, (3) I do not know. CGH, comparative genome hybridization; DMD, Duchenne muscular dystrophy; MLPA, multiplex ligation-dependent probe amplification; NA, not applicable; PCR, polymerase chain reaction. Questions answered incorrectly by at least 25% of respondents have been highlighted. *2017 TREAT-NMD Expert Masterclass, May 18-19, 2017, Lisbon, Portugal (TREAT-NMD received an unrestricted educational grant from PTC Therapeutics International, Ltd, for the organization of this meeting). 2018 TREAT-NMD Expert Masterclass organized and funded by PTC Therapeutics International, Ltd, in collaboration with TREAT-NMD, May 2-3, 2018, Madrid, Spain. †2017 TREAT-NMD Expert Masterclass: 61 attendees provided information on their job roles (child neurologist, n = 24; other, n = 11; trainee doctor, n = 7; neurologist, n = 5; physiotherapist, n = 5; neurology nurse, n = 4; pediatrician, n = 2; geneticist, n = 2, and pediatric nurse, n = 1). 2018 TREAT-NMD Expert Masterclass: 39 attendees provided information on their job roles (child neu- rologist, n = 15; other, n = 14; pediatrician, n = 5; neurologist, n = 4; and pediatric nurse, n = 1). ‡Seven (10.9%) selected muscle biopsy. §Four (8.9%) selected muscle biopsy. Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e12 Muscular Dystrophy THE JOURNAL OF PEDIATRICS • www.jpeds.com Volume 204 Figure 2. PRISMA flow diagram of systematic literature searches. The searches were conducted using Ovid to screen the EMBASE and MEDLINE databases. PCR, polymerase chain reaction. *Disease search terms (limited to title/abstract): Duchenne mus- cular dystrophy, DMD, or Duchenne AND any of the following search terms (limited to title/abstract): sign; symptom; creatine kinase; transaminase; diagnos*; gene*; genetic test*; genetic report*; genetic counsel*; delay*; screen*; carrier; mutation; sequenc*; comparative genome hybridi?ation; CGH; multiplex ligation-dependent probe amplification; MLPA; multiplex polymerase chain reaction; multiplex PCR; biops*; prenatal; neonatal; female; family planning; germline mosaicism; de novo; practitioner; p?e- diatrician; p?ediatric neurologist; neuromuscular specialist. The “?” function was included to search for variations of the spell- ing; the “*” function was included to search for variations of the word; and search terms were limited to the title or abstract of articles only. The full electronic search strategy is presented in Table II. †Search criteria: articles published from January 1, 2010, to April 8, 2018; English language; studies of humans; full journal articles. ‡Relevant articles older than 2010 (if no newer reference could be found) were identified ad hoc by the steering committee and expert voting panel. 313.e13 Aartsma-Rus et al January 2019 WORKSHOP/SYMPOSIUM SUMMARY Table II. Electronic search strategy for MEDLINE and EMBASE databases, performed in Ovid (2017 Ovid Tech- nologies, Inc) Lines Search 1 Duchenne muscular dystrophy.tw 2 DMD.tw 3 Duchenne.tw 4 1 or 2 or 3 5 sign.tw 6 symptom.tw 7 creatine kinase.tw 8 transaminase.tw 9 diagnos*.tw 10 gene*.tw 11 genetic test.tw 12 genetic report.tw 13 genetic counsel*.tw 14 delay*.tw 15 screen*.tw 16 carrier.tw 17 mutation.tw 18 sequenc*.tw 19 comparative genome hybridi?ation.tw 20 CGH.tw 21 multiplex ligation-dependent probe amplification.tw 22 MLPA.tw 23 multiplex polymerase chain reaction.tw 24 multiplex PCR.tw 25 biops*.tw 26 prenatal.tw 27 neonatal.tw 28 female.tw 29 family planning.tw 30 germline mosaicism.tw 31 de novo.tw 32 practitioner.tw 33 p?ediatrician.tw 34 p?ediatric neurologist.tw 35 neuromuscular specialist.tw 36 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or 35 37 4 and 36 38* limit 37 to dd=20100101-20170621 [Limit not valid in Ovid MEDLINE(R),Ovid MEDLINE(R) Daily Update,Ovid MEDLINE(R) In-Process,Ovid MEDLINE(R) Publisher; records were retained] 39* limit 38 to ed=“20100101-20170621” [Limit not valid in Embase; records were retained] 40 limit 39 to English language 41 limit 40 to human 42 Limit 41 to humans 43 Remove duplicates from 42 *The first search was performed from January 1, 2010, to June 21, 2017; an update was then performed from June 22, 2017, to April 8, 2018, using the same strategy. Search results were then exported, and full journal articles selected, using Excel's filter function. Evidence-Based Consensus and Systematic Review on Reducing the Time to Diagnosis of Duchenne 313.e14 Muscular Dystrophy