Severe Neonatal RYR1 Myopathy With Pathological Features of Congenital Muscular Dystrophy

Abstract

The phenotypes associated with pathogenic variants in the ryanodine receptor 1 gene (RYR1, OMIM# 180901) have greatly expanded over the last few decades as genetic testing for RYR1 variants has become more common. Initially described in association with malignant hyperthermia, pathogenic variants in RYR1 are typically associated with core pathology in muscle biopsies (central core disease or multiminicore disease) and symptomatic myopathies with symptoms ranging from mild weakness to perinatal lethality. We describe a 2-week-old male patient with multiple congenital dysmorphisms, severe perinatal weakness, and subsequent demise, whose histopathology on autopsy was consistent with congenital muscular dystrophy. Immunohistochemical analysis of dystrophy-associated proteins was normal. Rapid exome sequencing revealed a novel heterozygous nonsense variant (p.Tyr661Ter) in RYR1, as well as a previously described RYR1 pathogenic variant associated with congenital myopathy (p.Phe4976Leu). This highlights the potential for RYR1 pathogenic variants to produce pathological findings most consistent with congenital muscular dystrophy.

INTRODUCTION

Pathogenic variants in the RYR1 gene, a 106 exon gene that encodes the skeletal muscle ryanodine receptor, cause dominant and recessive skeletal muscle disease. The ryanodine receptor is a critical component in excitation-contraction coupling, which allows stimulation of myofibers to be translated into myofibrillar contraction through the intracellular release of calcium (1, 2). Historically, mutations in RYR1 have been associated with 2 characteristic disease phenotypes, malignant hyperthermia (MH), and RYR1 myopathy (Table 1) (3). Patients with susceptibility to MH typically show no clinical symptoms unless there is exposure to succinylcholine or inhaled anesthetics, which can then induce life-threatening reactions including rhabdomyolysis, acidosis, muscle rigor, tachycardia, and bleeding abnormalities (4). RYR1 myopathies typically present with early onset muscle weakness and hypotonia of varying severity, possibly accompanied by macrocephaly, ophthalmoplegia, elevated creatine kinase, and type 1 fiber predominance (5–7). Depending on a patient’s RYR1 mutation, they may be predisposed for MH, RYR1 myopathy, or both (3). RYR1 myopathy also displays considerable pathological heterogeneity on skeletal muscle biopsy, which typically involves central core pathology but may also include pathology consistent with centronuclear myopathy and multiminicore myopathy (1, 8–10).

TABLE 1.

Clinical Syndromes Associated With RYR1 Pathogenic Variants

Phenotype	Clinical Features	Inheritance
RYR1 myopathy (including core and minicore myopathies and congenital fiber type disproportion)	Congenital myopathy characterized by motor developmental delay and mild proximal weakness most pronounced in the hip girdle musculature. Orthopedic complications include congenital hip dislocation and scoliosis. Risk for malignant hyperthermia. Significant phenotypic variability.	AD, AR
Minicore myopathy with external ophthalmoplegia	Shows overlapping features with central core disease but tends to be more clinically severe.	AR
Malignant hyperthermia susceptibility1/King Denborough syndrome	Triggering agents (volatile anesthetic agents or depolarizing muscle relaxants) can cause hyperthermia, skeletal muscle rigidity, tachycardia, or arrhythmia, respiratory and metabolic acidosis, and rhabdomyolysis. Facial dysmorphisms can include downslanting palpebral fissures and malar flattening.	AD

Inheritance patterns include autosomal dominant (AD) and autosomal recessive (AR).

Here, we report a case of severe RYR1-associated muscle disease with profound weakness and death at 17 days of life associated with compound heterozygous pathogenic variants, p.Tyr661Ter and p.Phe4976Leu. Genetic testing of the patient’s parents identified the p.Tyr661Ter variant as paternally inherited and the p.Phe4976Leu variant as maternally inherited. Autopsy evaluation of a quadriceps muscle revealed an unusual pathological phenotype, most consistent with congenital muscular dystrophy. Our findings further broaden the list of known pathogenic variants in RYR1 myopathy, while also providing an example of a striking but atypical pattern of skeletal muscle pathology in this disease.

MATERIALS AND METHODS

Case History

This male patient was born at an outside institution at 35 weeks 3 days, weighing 2489 g with a prenatal history of polyhydramnios and multiple congenital anomalies. He was intubated shortly after birth and transferred to our institution at 2 weeks of life. Upon arrival, he was extubated but required ongoing airway support with nasal cannula. His physical examination at the time of transfer demonstrated axial and appendicular hypotonia, reduced motor activity, and a high-pitched cry. He was macrocephalic with a large elongated anterior fontanelle. Dysmorphic craniofacial features included hypertelorism, downward slanting palpebral fissures, a broad nasal bridge, and midface hypoplasia. Oral exam revealed retromicrognathia, a high arched palate, and poor suck reflex with the accumulation of copious secretions. Palmar and plantar grasp reflexes were absent. His hands showed a single palmar crease, thenar hypoplasia, cortical thumbs, ulnar deviation, and flexion contractures of the fingers. Elbows and knees also had flexion contractures. Nerve conduction studies demonstrated absent sensory responses and lower extremity motor responses but normal upper extremity responses. Concentric needle electrode examination demonstrated diffuse fibrillation potentials, mildly reduced amplitudes of minimal complexity and increased recruitment. This was felt to be indicative of a diffuse myopathic disease consistent with a severe congenital myopathy or dystrophic process. Given the medical complexity, lack of improvement with appropriate medical intervention and predicted poor outcome, the parents opted to proceed with palliative care and he died shortly after, at 17 days of life.

Family History

The patient’s mother was G7P1423. Pregnancy history was significant for 2 first-trimester pregnancy losses, an intrauterine fetal death at 36 weeks, and 3 live births (1 delivered at 33 weeks with polyhydramnios, clubbed feet, and macrocephaly that died shortly after birth due to absent respiratory effort; another born at 33 weeks with polyhydramnios and macrocephaly; and a third full-term birth with unremarkable phenotype), in addition to the current patient.

Laboratory Studies

Laboratory studies included a basic metabolic profile, carbohydrate deficient transferrin, urine organic acids, very long chain fatty acids, TSH, Free T4, creatine kinase, chest X-ray, echocardiogram, and renal ultrasound, STAT FISH for aneuploidy, and DNA deletion/duplication array. These assays did not provide diagnostic information that was suggestive of a specific diagnosis.

Pathological Studies

Isopentane-frozen and glutaraldehyde-fixed muscle tissue was further processed for light and electron microscopy, respectively, using standard clinical practices (11). Histochemical stains evaluated included hematoxylin and eosin, Gomori trichrome, NADH, SDH, COX, ATPase (at pH 4.3, 4.6, and 9.4), PAS, and Oil Red O. Immunohistochemical evaluation for spectrin, utrophin, dystrophin (using DYS1, DYS2, DYS3, and BMD hotspot antibodies), β-dystroglycan, α-sarcoglycan, γ-sarcoglycan, dysferlin, and merosin was performed to assess the expression of proteins related to muscular dystrophy, and CD3 and CD68 expression provided assessment for lymphocyte and macrophage infiltration in the tissue. In addition, Pax7 and developmental myosin heavy chain were evaluated by immunofluorescence to assess for markers of muscle regeneration. Antibodies used in these studies are shown in Table 2. Electron microscopy was performed using standard techniques at the Medical College of Wisconsin Electron Microscopy Core Facility.

TABLE 2.

List of Antibodies Used in the Pathological Characterization of This Case

Antigen	Vendor	Antibody Name	Dilution Used
Dystrophin rod domain	Novocastra	DYS1	1:50
Dystrophin C-terminus	Novocastra	DYS2	1:50
Dystrophin N-terminus	Novocastra	DYS3	1:20
Dystrophin, Becker muscular dystrophy hotspot	DSMB	Clone 7G1	1:20
Utrophin	Novocastra	NCL-DRP2	1:10
Spectrin	Novocastra	NCL-SPEC1	1:100
Alpha dystroglycan	Upstate	IIH6	1:50
Alpha dystroglycan	Upstate	VIA4-1	1:30
Beta dystroglycan	Novocastra	NCL-b-DG	1:200
Alpha sarcoglycan	Novocastra	NCL-a-SARC	1:100
Gamma sarcoglycan	Novocastra	NCL-g-SARC	1:100
Dysferlin	Novocastra	Hamlet-1	1:20
Emerin	Novocastra	NCL-EMERIN	1:50
Merosin (laminin alpha 2)	Vector labs	VP-M648	1:300
CD3	Novocastra	LN10	Undiluted
CD68	Dako	EBM11	Undiluted
Pax7	DSMB	PAX7	1:25
Myosin heavy chain, developmental	Leica	NCL-MHCd	1:25

DSMB, Developmental Studies Hybridoma Bank.

Molecular Studies

Rapid exome sequencing was conducted in the Advanced Genomics Sequencing Laboratory at the Medical College of Wisconsin utilizing HiSeq 2500 (Illumina, San Diego, CA) sequencer. Exome capture was carried out using SureSelect XT Human All Exon V4 baits (Agilent, Santa Clara, CA) to minimum exome wide average coverage exceeding 85×. Variants were annotated and prioritized using previously published methods (12). Variants of interest were confirmed using site-specific PCR and sequenced on the ABI 3730xl DNA Genetic Analyzer (Applied Biosystems, Foster City, CA).

RESULTS

Skeletal Muscle Pathology

Pathological analysis of the frozen quadriceps muscle specimen displayed findings that were most characteristic of a congenital muscular dystrophy phenotype (Fig. 1). Hematoxylin and eosin staining revealed highly variable myofiber size (including small and large myofibers), with moderate to marked endomysial fibrosis. There was marked diffuse mononuclear cell infiltration in the muscle, which corresponded to a mixture of CD3-positive lymphocytes, CD68-positive macrophages, and Pax7-positive satellite cells. Basophilic fibers indicative of active regeneration were rare, but immunostaining with developmental myosin heavy chain identified a significant subpopulation of positive fibers, which is overall consistent with a moderate degree of active/recent regeneration. Evidence of active muscle fiber degeneration included myofibers displaying myophagocytosis and myonecrosis. Rare internally nucleated fibers were present, but not the extent that is typical for a centronuclear myopathy. Peripheral nerve twigs present in the specimen were unremarkable. Given the presence of active and chronic degenerative changes, immunohistochemical panels for dystrophinopathy, congenital muscular dystrophy, and limb-girdle muscular dystrophy immunostain panel were performed. All immunostaining results were appropriate in comparison to control, and not suggestive of a specific muscular dystrophy. ATPase stains revealed moderate type 1 fiber predominance (∼60%–70% of fibers), and a subpopulation of type 1 fibers accounted for the largest fibers in the specimen. Other standard histochemical stains did not detect structures suggestive of a specific muscle disorder, and specifically no evidence of cores or minicores. Electron microscopy revealed significant myofibrillar disarray in many fibers and small areas with osmiophilic staining (although not sufficient for characterization as nemaline rods or bodies). There was no evidence of triad abnormalities, cores, minicores, or mitochondrial pathology on ultrastructural examination.

FIGURE 1.

Histopathological findings consistent with congenital muscular dystrophy. (A, B) Hematoxylin and eosin-stained sections of muscle reveal variation in fiber size, mononuclear cell infiltrate, degenerating fibers, and regenerating fibers. (C) An NADH stain shows differences in organelle concentration, but no definitive cores, minicores, necklace fibers, or spoked-wheel architecture. Immunostaining to characterize the mononuclear infiltrate revealed a mixture of (D) CD3-positive lymphocytes, (E) CD68-positive macrophages, and (F) Pax7-positive satellite cells. Scale bar: 40 μm.

Molecular Studies

Exome sequencing trio analysis and a pathological evaluation by muscle biopsy were pursued in parallel. Sequencing revealed a novel heterozygous nonsense change, NM_000540, c.1982G>A (p.Tyr661Ter) as well as a previously described pathogenic variant in trans configuration, c.14928C>G (p.Phe4976Leu) (Fig. 2) associated with congenital myopathy (13). The p.Phe4976Leu missense change lies within the transmembrane/luminal and pore forming region of the channel on the C-terminus of the protein known to harbor central core disease causing pathogenic variants. Exome results identified that the c.1982G>A (p.Tyr661Ter) variant was paternally inherited and that the c.14928C>G (p.Phe4976Leu) variant was maternally inherited.

FIGURE 2.

Genetic testing findings indicative of a pathogenic variant in RYR1. Electropherogram plots of dideoxy Sanger traces confirm next-generation sequencing results. The heterozygous c.1982G>A is predicted to cause ap.Tyr661Ter change (top) as compared to reference. While the heterozygous c.14928C>G is predicted to cause a p.Phe4976Leu change (bottom) compared to reference sequence.

DISCUSSION

Historically, the large size of RYR1 limited genetic testing to clinical or pathological situations that were considered “classic” (14). Recent advances in genomic sequencing technologies have allowed the evaluation of RYR1 pathogenic variants in situations outside of these classic symptomatic or pathological scenarios (14), and have expanded the range of disorders that are associated with RYR1 pathogenic variants. Aside from being a large protein, RYR1 has multiple isoforms and genetic variants in the gene are known to show reduced penetrance. These factors compound the ability to predict genotype-phenotype correlations accurately.

The possibility of MH remains one of the greatest clinical concerns in patients with pathogenic variants in RYR1. While in vitro studies are available to assess risk for MH, this is complicated by reduced penetrance and variable expressivity (Reviewed in [3, 15]). Early studies seemed to suggest that variants in the conserved C-terminus were more likely to present with myopathy while central and N-terminal variation could present with either phenotype. The variants discussed in this case involve a C-terminal region stop codon in one of the SPRY structural domains but given the premature termination of the transcript, it is predicted to undergo nonsense-mediated decay. Familial studies have identified a subset of RYR1 variants that result in an MH-selective phenotype and others with a mixed phenotype, however, clear correlations with specific genetic regions are lacking.

The murine Ryr1 knock out model is neonatal lethal, which has hampered additional investigation and options for treatment development in RYR-related myopathies (16). Studies of muscle from Ryr1-null mice have indicated that MH-only variants increase the basal sarcoplasmic reticulum calcium channels to a point insufficient to alter net calcium content, while myopathic or mixed phenotype variants result in calcium depletion within the sarcoplasmic reticulum (17). This calcium release is utilized in the in vitro contracture screen for the human MH phenotype.

This patient case report further illustrates the known genetic and pathological diversity characteristic in RYR1 myopathies. The presence of myofiber degeneration, myofiber regeneration, fibrosis, and a mononuclear cell infiltrate including inflammatory and satellite cells is most consistent with a dystrophic process, as it indicates a combination of degenerative and regenerative muscle processes that are of varying ages. While the pathological findings of a congenital muscular dystrophy are not typically associated with RYR1 muscle disease, the clinical phenotype observed in this patient is consistent with the natural clinical course seen in severe RYR1 myopathy. In addition, a recent report described a patient with a similarly severe neonatal course due to RYR1 pathogenic variant, with reported pathology including myofiber atrophy, adipose tissue infiltration, and endomysial fibrosis (5). While classic features of muscular dystrophy including active degeneration and recent regeneration were not described, the previously reported paper likely represents another example of a dystrophic phenotype in the context of a RYR1 pathogenic variant. These cases highlight the value for considering RYR1 pathogenic variants in the evaluation of patients with severe neonatal dystrophic disease, as the identification of these variants may significantly impact clinical decision-making. The clinical and electrodiagnostic features of the patient reported here are also consistent with a patient with a severe congenital muscular dystrophy, including the macrocephaly reported elsewhere in congenital RYR1 mutations (5–7).

Dr Lawlor is a compensated member of Scientific Advisory Boards for Audentes Therapeutics, Solid Biosciences, and Ichorion Therapeutics and he also receives research support from these companies. He is also a consultant for Wave Life Sciences, Dynacure, and Valerion Therapeutics. Dr Harmelink is a compensated member of the advisory boards for Sarepta Therapeutics, Biogen, Inc., Avexis, and PTC, Inc., a consultant for Biogen, Inc. and Connected Research and Consulting, and receives grant research support from CureSMA and the Muscular Dystrophy Association.