BACKGROUND:
Mutations in ryanodine receptor 1 gene (RYR1) are frequent causes of myopathies. They classically present with central core disease; however, clinical variability and histopathologic overlap are being increasingly recognized.

PATIENTS:
Patient 1 is a 15-year-old girl with mild proximal, four-limb weakness from age 5, presenting with a progressive scoliosis starting at age 10. Patient 2 is an 18-year-old girl with progressively worsening muscle hypotrophy and mild proximal, four-limb weakness. She developed a rapidly progressive scoliosis from age 11 and needed surgical treatment at age 14 years. Patient 3 is an 11-year-old boy with moderate proximal limb weakness and progressive neck flexor weakness, first noticed at age 2. Muscle biopsies revealed type 1 fiber predominance (Patients 1 and 2) or abnormal type 1 fiber uniformity (Patient 3). Different RYR1 variants were identified in all patients. In Patients 1 and 3, these changes were validated as being pathogenic.

CONCLUSIONS:
These patients illustrate early-onset, progressive myopathies with predominant axial involvement. Histopathologic findings were abnormal but not specific for a diagnosis, particularly central core myopathy. Genetic testing helped broaden the range of phenotypes included in the RYR1-related myopathies. Our patients reinforce the need to recognize the broad histopathologic variability of RYR1-related myopathies and sometimes lack of pathognomonic findings that may reduce the diagnostic threshold of this disease. We suggest that the predominance of type 1 fibers and involvement of axial muscles may be an important element to consider the RYR1 gene as candidate

Melo Pires, M

Oliveira, J

Rocha, J

Santos, M

Santos, R

Taipa, R

English

Institutional repository of Hospital de Braga

lable at ScienceDirectPediatric Neurology 51 (2014) 275e278Contents lists avaiPediatric Neurologyjournal homepage: www.elsevier .com/locate/pnuClinical ObservationsRyanodine Myopathies Without Central CoresdClinical,Histopathologic, and Genetic Description of Three CasesJoão Rocha MDa,*, Ricardo Taipa MDb, Manuel Melo Pires PhDb,Jorge Oliveira MSc c, Rosário Santos MSc c, Manuela Santos MDdaDepartment of Neurology, Hospital de Braga, Braga, PortugalbNeuropathology Unit, Centro Hospitalar do Porto, Porto, PortugalcMolecular Genetics Unit, Centro de Genética Médica Dr. Jacinto Magalhães, Centro Hospitalar do Porto, Porto, PortugaldNeuromuscular Diseases Unit, Department of Pediatric Neurology, Centro Hospitalar do Porto, Porto, PortugalArticle HistReceived F* CommuNeurology;Portugal.E-mail a0887-8994/$http://dx.doiabstractBACKGROUND: Mutations in ryanodine receptor 1 gene (RYR1) are frequent causes of myopathies. They classicallypresent with central core disease; however, clinical variability and histopathologic overlap are being increasinglyrecognized. PATIENTS: Patient 1 is a 15-year-old girl with mild proximal, four-limb weakness from age 5, presentingwith a progressive scoliosis starting at age 10. Patient 2 is an 18-year-old girl with progressively worsening musclehypotrophy and mild proximal, four-limb weakness. She developed a rapidly progressive scoliosis from age 11 andneeded surgical treatment at age 14 years. Patient 3 is an 11-year-old boy with moderate proximal limb weaknessand progressive neck flexor weakness, first noticed at age 2. Muscle biopsies revealed type 1 fiber predominance(Patients 1 and 2) or abnormal type 1 fiber uniformity (Patient 3). Different RYR1 variants were identified in allpatients. In Patients 1 and 3, these changes were validated as being pathogenic. CONCLUSIONS: These patientsillustrate early-onset, progressive myopathies with predominant axial involvement. Histopathologic findingswere abnormal but not specific for a diagnosis, particularly central core myopathy. Genetic testing helpedbroaden the range of phenotypes included in the RYR1-related myopathies. Our patients reinforce the need torecognize the broad histopathologic variability of RYR1-related myopathies and sometimes lack of pathogno-monic findings that may reduce the diagnostic threshold of this disease. We suggest that the predominance oftype 1 fibers and involvement of axial muscles may be an important element to consider the RYR1 gene ascandidate.Keywords: ryanodine receptor, RYR1, myopathy, muscle biopsyPediatr Neurol 2014; 51: 275-278 2014 Elsevier Inc. All rights reserved.IntroductionThe ryanodine receptor 1 gene (RYR1) encodes the mainCa2þ channel in the skeletal muscle’s sarcoplasmic reticu-lum, having a crucial role in muscle excitation-contractionmechanisms.1 More than 300 distinct mutations in RYR1(19q13.1) have been reported.2 Classically, they have beenory:ebruary 10, 2014; Accepted in final form April 24, 2014nications should be addressed to: Dr. Rocha; Department ofHospital de Braga; Sete Fontes, S. Victor; 4710-243 Braga,ddress: joaomrocha@gmail.com- see front matter  2014 Elsevier Inc. All rights reserved..org/10.1016/j.pediatrneurol.2014.04.024associated with autosomal dominant central core disease(MIM #117000) and malignant hyperthermia susceptibility.Clinical features have been described as globally nonpro-gressive and include congenital hypotonia and minorweakness with proximal lower limb predominance (pelvicgirdle). Sometimes, axial involvement without respiratorycompromise, motor milestones delay, or skeletal malfor-mations (hip dislocation, scoliosis, and foot deformities)may be observed.3 Throughout the years, other phenotypeshave been attributed to RYR1 mutations, including KingDenborough syndrome, limb-girdle myopathy, core-rodmyopathy, bent spine myopathy, exertional or drug-associated myalgia, and rhabdomyolysis and asymptom-atic creatine kinase elevation. A marked interpersonal andJ. Rocha et al. / Pediatric Neurology 51 (2014) 275e278276even intrafamilial variation in phenotype has been evidentin previous reports.3-6 Typical muscle histopathology in-cludes type 1muscle fibers with central amorphous areas ofsarcomeric disorganization characterized by the absence ofmitochondria and of oxidative enzymatic activity (“cores”).7In recent years, there has been an increased awareness of amarked clinical variability and histopathologic overlap withother myopathies, precluding a direct diagnosis.3,8 Thesephenoptypes include myopathies with multiminicores andatypical cores, centrally placed nuclei, cores and nemalinerods, congenital fiber-type disproportion, and marked pre-dominance or uniformity of type 1 fibers.7,8Although RYR1 gene analysis was initially restricted tohotspot regions because of its large size (106 exons),sequencing all the exonic regions of the gene is now rec-ommended, especially when patients have atypical musclephenotypes, which are difficult to correlate with the geno-type, or in sporadic cases. The broader analysis of RYR1 genehas brought the molecular diagnosis of these patients to anew level of complexity because several new sequencevariants are often found, frequently described as unclassi-fied variants, making the genetic diagnosis problematic.We report three independent pediatric patients withearly-onset myopathies with nonspecific histopathologyand highlight some clinical characteristics that can lead tothe diagnosis; in two instances, mutations were identifiedin RYR1 gene.Patient DescriptionsPatient 1A 15-year-old girl with a family history of “lower limb weakness”(affecting her mother, maternal uncle, and grandfather) was firstobserved in the Neuropediatrics Department at age 11 with a slight delayin motor milestones but stable development up to 10 years of age. At thispoint, she started developing a scoliosis that has progressed to a severeform. Surgical treatment has been proposed but not yet performed.Physical examination revealed a right ptosis, bilateral facial palsy, andarched palate. She had considerable axial weakness with neck flexorinvolvement, dorso-lumbar scoliosis (Fig 1A), and a moderate, proximal,nonprogressive upper and lower limb weakness. A muscle biopsy per-formed when she was 6 years old revealed normal muscle fiber sizevariability and preserved internal structure but marked type 1 fiberpredominance (Fig 2, P1). Molecular study of the RYR1 gene revealed theFIGURE 1.Pattern with marked axial involvement: (A) Patient 1 vertebral column x-rarevealing severe dorso-lombar scoliosis; (C) Patient 3 lateral profile with narropresence of a heterozygous mutation c.14677C>T (p.Arg4893Trp) inexon 102 (hotspot 3) (Fig 3). This mutation was first identified byMonnier et al. in two distinct families with autosomal dominant (AD)transmission. Similarly, the mutation was also found in our patient’smother and uncle suggesting an AD inheritance pattern.Patient 2An 18-year-old girl with an unremarkable family history had beenmonitored by the Neuropediatrics Department from 12 years of age. Shewas described as hypotonic since birth and had mildly delayed motormilestones. Progressive fatigue and weakness after physical activity wasevident since early childhood, and at age 11, she began developing arapidly progressive, severe scoliosis requiring surgical correction andnoninvasive ventilation by age 14. On examination, she had globalmuscle hypotrophy and low weight and stature. She also has an archedpalate and axial weakness with involvement of neck flexors, narrowthorax, and dorso-lombar scoliosis (Fig 1B). Low-grade proximal andslowly progressive upper and lower limbweakness with joint retractionshas been observed during follow-up. Muscle biopsy at 12 years of agerevealed increased variability in fiber diameter with frequent fiber at-rophy and occasional fiber hypertrophy. There was some endomysialfibrosis and adipose substitution but with preserved internal structure(Fig. 2, P2). Marked type 1 fiber predominance was also noted. Molecularstudy of the RYR1 gene revealed a heterozygous missense variant withunknown significance c.7843C>T (p.Arg2615Cys) in exon 49 (hotspot 2)(Fig 3). This previously unreported variant affects a highly conservedresidue. RYR1 exonic regions were fully sequenced, and her asymptom-atic father was found to carry the same variant. The muscle comple-mentary DNA study, restricted to the RYR1 transcript region where thevariant is located, demonstrated biallelic expression because thec.7843C>T was detected in heterozygosity.Patient 3An 11-year-old boy with an unremarkable family history, firstobserved in the Neuropediatrics Department at age 10, exhibited aminordelay in motor milestones from 2 years of age. On physical examination,he presented with a bilateral facial palsy, arched palate, and axialweakness with neck flexor weakness and a narrow thorax (Fig 1C). Hehad a moderate-grade proximal and nonprogressive upper and lowerlimb weakness. His muscle biopsy, performed at 10 years of age hadnormal fiber size variability with global preservation of internal struc-ture and only slight central dimming on oxidative stain but no cores. Hehad abnormal type 1 fiber uniformity (Fig 2, P3). Molecular RYR1revealed three sequence variants: c.4711A>G (p.Ile1571Val) in exon 33;c.10097G>A (p.Arg3366His in exon 67; c.11798G>A; p.Tyr3933Cys) inexon 86. These variants have been previously described in the sameallele.3 A heterozygous mutation c.14537C>T (p.Ala4846Val) in exon 101y revealing dorso-lombar scoliosis; (B) Patient 2 vertebral column x-rayw thorax.FIGURE 2.Muscle biopsy histopathologic features. (A) Hematoxylin and eosin stain; (B) Nicotinamide adenine dinucleotide stain; and (C) Adenosine triphosphatase 4,35stain. P1: (A)normalmusclefiber size variability; (B andC) internal structure is normal, butmarked type1fiberpredominance is observed;P2: (A) someatrophyandoccasionalfiberhypertrophyandendomysialfibrosiswith scantyadipose replacement; (B andC)normal internal structurewith type1fiberpredominance;P3: (A) normal fiber size variability; (B and C) internal structure is normal and slight central dimming on oxidative stain, but no cores are observed, and type 1fiber uniformity. Scale bar: 100 mm.J. Rocha et al. / Pediatric Neurology 51 (2014) 275e278 277(hotspot 3) was also present, previously reported as pathologic9 (Fig 3).The parents are awaiting screening to assess the possibility of autosomalrecessive inheritance.All analytical studies were normal in the three cases, includingmuscle creatine kinase.DiscussionOur patients illustrate early-onset myopathies withprominent progressive axial involvement, variable clinicalFIGURE 3.Molecular study of RYR1 gene: image reveals sequences’ electropherograms coseverity, and progressive course. The first two patientsrapidly evolved to severe scoliosis during the second decadeof life. Patient 3 has not developed scoliosis, having onlymarked neck flexor weakness, but he is the youngest of thethree individuals, having only started the second decade oflife. Histopathologic examination revealed changes sug-gestive of but not classical for RYR1-related myopathy.All patients had marked type 1 fiber predominancewithout typical “cores,” a feature recently recognized to bentaining mutations (all variants are heterozygous).J. Rocha et al. / Pediatric Neurology 51 (2014) 275e278278associated with RYR1 myopathies.3,10 Noncore RYR1-relatedmyopathies may be more frequent than initially thoughtand are probably underdiagnosed. Amburgey et al.4 statedthat 49% of recessive cases in their study (n ¼ 106) werenoncore myopathies. In these patients, histopathologicpresentation evolves during the course of the disease andthe typical central coresmay only appear in later stages.4,7,11Patient 3 may be an example of such an evolving coursewhere there seems to be a central dimming on oxidativestains, without a clear core formation. Endomysial fibrosisand adipose substitution, as found in the biopsy of Patient 2,are characteristics more commonly associated with dys-trophies and are less specific traits, but they have also beendescribed in RYR1-related myopathies.7,12Concerning molecular analysis, Patients 1 and 3 haveRYR1 variants previously recognized as pathogenic. How-ever, Patient 2, the most severely affected of our three cases,has a heterozygous missense variant of unknown signifi-cance (p.Arg2615Cys) in hotspot 2 of the ryanodine receptorgene. This variant, previously unreported in the literatureand in sequence variant databases, affects a highlyconserved residue and is of paternal origin. RYR1 exonicregions were fully sequenced, and no other mutation wasfound. Although the patient’s father is an asymptomaticcarrier of the samemutation, we cannot completely excludeits pathologic role, because high inter- and intra-familiarphenotype variability, ranging from asymptomatic toseverely affected individuals, has been described with othermutations in this disease.13 Epigenetic allele silencing of theRYR1 gene due to imprinting could also provide an expla-nation for our case.14 However, this hypothesis wasexcluded because we have detected biallelic expression ofRYR1 gene in complementary DNA obtained from muscle,demonstrating heterozygous expression. In the first case,transmission is compatible with an autosomal dominantinheritance, in contrast with Patient 3, where autosomalrecessive inheritance is likely. This assumption was madebecause this sporadic case has four missense variants, threeof which have been previously described in the same allele,and where p.Ile1571Val and p.Tyr3933Cys were consideredas mutations.3Our report highlights the phenotypic variability of RYR1myopathies, which may lack pathognomonic histopatho-logic findings, or these may overlap with other non-RYR1myopathies. This is a serious obstacle in the diagnostic pro-cess, wheremuscle biopsy is considered the “gold standard”.Our cases illustrate the paradigmof somepatients that in thepast would go years without a specific diagnosis, until theydevelopedhistopathologic pathognomonic features.Genetictesting has helped to solvemany of these difficult diagnoses.However, in the case of RYR1 genetic analysis, there are stillsome difficulties to overcome specially when dealing withsequence variants with unknown clinical significance. Thedevelopment of new approaches based on next generationsequencing should increase the diagnostic yield in this fieldand may bring some additional clues to the apparent geno-type and/or phenotype discrepancies reported in somepatients.We suggest that the predominance of type 1 fibers andinvolvement of axial muscles may be an important elementto consider the RYR1 gene as candidate.No funding was provided to conduct this study.References1. Quane KA, Healy JM, Keating KE, et al. Mutations in the ryanodinereceptor gene in central core disease and malignant hyperthermia.Nat Genet. 1993;5:51-55.2. Jungbluth H, Sewry CA, Muntoni F. Core myopathies. Semin PediatrNeurol. 2011;18:239-249.3. Klein A, Suzanne L, Munteanu L, et al. Clinical and genetic findings ina large cohort of patients with ryanodine receptor 1 gene-associatedmyopathies. Human Mutation;00:1-8.4. Amburgey K, Bailey A, Hwang JH, et al. Genotype-phenotype cor-relations in recessive RYR1-related myopathies. Orphanet J Rare Dis.2013;8:117.5. Dlamini N, Voermans NC, Lillis S, et al. Mutations in RYR1 are acommon cause of exertional myalgia and rhabdomyolysis. Neuro-muscular Disorders. 2013;23:540-548.6. Løseth S, Voermans NC, Torbergsen T, et al. A novel late-onsetaxial myopathy associated with mutations in the skeletal mus-cle ryanodine receptor (RYR1) gene. J Neurol. 2013;260:1504-1510.7. Sewry CA, Muller C, Davis M, et al. The spectrum of pathology incentral core disease. Neuromuscul Disord. 2002;12:930-938.8. Zhou H, Jungbluth H, Sewry CA, et al. Molecular mechanisms andphenotypic variation in RYR1-related congenital myopathies. Brain.2007;130:2024-2036.9. Gambelli S, Malandrini A, Berti G, et al. Inheritance of a novel RYR1mutation in a family with myotonic dystrophy type 1. Clin Genet.2007;71:93-94.10. Sato I, Wu S, Ibarra CA, et al. Congenital neuromuscular disease withuniform type 1 fiber and RYR1 mutation. Neurology. 2008;70:114-122.11. Jungbluth H, Zhou H, Sewry CA, et al. Centronuclear myopathydue to a de novo dominant mutation in the skeletal muscleryanodine receptor (RYR1) gene. Neuromuscul Disord. 2007;17:338-345.12. Wilmshurst JM, Lillis S, Zhou H, et al. RYR1 mutations are a commoncause of congenital myopathies with central nuclei. Ann Neurol.2010;68:717-726. http://dx.doi.org/10.1002/ana.22119.13. Kossugue PM, Paim JF, Navarro MM, et al. Central core disease dueto recessive mutations in RYR1 gene: is it more common thandescribed? Muscle Nerve. 2007;35:670-674.14. Zhou H, Brockington M, Jungbluth H, et al. Epigenetic allelesilencing unveils recessive RYR1 mutations in core myopathies. Am JHum Genet. 2006;79:859-868.

Ryanodine myopathies without central cores-clinical, histopathologic, and genetic description of three cases.

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Ryanodine myopathies without central cores-clinical, histopathologic, and genetic description of three cases.

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