RYR1-Related Myopathies: Clinical,Histopathologic and Genetic Heterogeneity Among 17 Patients from a Portuguese Tertiary Centre

Abstract

Background: Pathogenic variants in ryanodine receptor type 1 (RYR1) gene are an important cause of congenital myopathy. The clinical, histopathologic and genetic spectrum is wide.

Objective: Review a group of the patients diagnosed with ryanodinopathy in a tertiary centre from North Portugal, as an attempt to define some phenotypical patterns that may help guiding future diagnosis.

Methods: Patients were identified from the database of the reference centre for Neuromuscular Disorders in North Portugal. Their data (clinical, histological and genetic) was retrospectively accessed.

Results: Seventeen RYR1-related patients (including 4 familial cases) were identified. They were divided in groups according to three distinctive clinical characteristics: extraocular muscle (EOM) weakness (N = 6), disproportionate axial muscle weakness (N = 2) and joint laxity (N = 5). The fourth phenotype includes patients with mild tetraparesis and no distinctive clinical features (N = 4). Four different histopathological patterns were found: centronuclear (N = 5), central core (N = 4), type 1 fibres predominance (N = 4) and congenital fibre type disproportion (N = 1) myopathies. Each index case, except two patients, had a different RYR1 variant. Four new genetic variants were identified. All centronuclear myopathies were associated with autosomal recessive inheritance and EOM weakness. All central core myopathies were caused by pathogenic variants in hotspot 3 with autosomal dominant inheritance. Three genetic variants were reported to be associated to malignant hyperthermia susceptibility.

Conclusions: Distinctive clinical features were recognized as diagnostically relevant: extraocular muscle weakness (and centronuclear pattern on muscle biopsy), severe axial weakness disproportionate to the ambulatory state and mild tetraparesis associated with (proximal) joint laxity. There was a striking genetic heterogeneity, including four new RYR1 variants.

INTRODUCTION

Pathogenic variants in the ryanodine receptor type 1 (RYR1) gene have been identified as the most common cause of congenital myopathies, in several large cohorts [1, 2]. This gene codifies the ryanodine receptor, a calcium release channel of the skeletal muscle sarcoplasmic reticulum with a crucial role in excitation–contraction coupling. RYR1 is a large gene (106 exons) but it has three mutational hotspot regions: region 1 (exons 2–17, amino acid residues 35–614), region 2 (exons 39–46, amino acid residues 2,163–2,458) and region 3 (exons 85–104, amino acid residues 3,916–4,942) [3].

The best characterized entity associated to RYR1 is the dominantly inherited central core disease with malignant hyperthermia susceptibility, a potentially life-threatening pharmacogenetic reaction to volatile anesthetics and/or depolarising muscle relaxants [4]. However, in recent years, an increasing range of additional histopathological and clinical variants have been associated with RYR1 variants, including with recessive inheritance [5, 6].

We aimed to review the clinical, histopathologic and genetic characteristics of the patients diagnosed with ryanodinopathy followed in our tertiary centre. In face of the great variability described in literature, we attempted to define some key features of ryanodinopathies that could help future diagnosis.

MATERIALS AND METHODS

Centro Hospitalar do Porto – Hospital de Santo António is the reference centre for Neuromuscular Disorders in North Portugal.

Adult and paediatric patients with the genetic diagnosis of ryanodinopathy were identified from the Centre database. Their clinical records and muscle biopsies were retrospectively reviewed. As muscle magnetic resonance imaging is still not available in our centre in the routine clinical practice, we do not have such data.

This retrospective series of cases is in accord with the ethical standards of the Committee on Human Experimentation of our institution and in accord with the Helsinki Declaration of 1975.

RESULTS

Seventeen patients with ryanodine myopathy, including four families (FAM), were identified and included. Table 1 summarises the clinical, histopathological and genetic data of the patients (P1 to P17).

Clinical manifestations

At the time when data were retrospectively collected, the patients’ mean age was 22.3 years old (range 5 months-51 yo). Twelve patients (70.6% ) were female.

The disease onset was reported by most patients or their parents as having occurred congenitally or during the first year of life; only two patients noticed the first symptoms between 2yo and school age.

All patients had a myopathic syndrome with thin appearance of muscle bulk, along with other specific features:

Extraocular muscle (EOM) weakness (Fig. 1, Panel A): This was present in six patients (including family FAM.I) and associated with different severities of tetraparesis. Patients 1 and 2 (two siblings aged 28 and 34yo) constitute our most disabled patients; they have never gained the ability to walk; they also have facial, bulbar, respiratory and spinal muscles weakness and were submitted to surgery in the second decade of life due to a rapid evolving scoliosis. Patient 3 (aged 24yo) has an intermediate presentation in terms of severity, as she has a slight tetraparesis but with a significant weakness of the axial muscles. She also had early scoliosis surgery and needs non-invasive ventilation. Patient 4 (aged 3yo) presented with a mild motor delay as she only acquired the ability to walk at the age of 24 months. Patients 5 (aged 37yo) and 6 (aged 33yo) have proximal tetraparesis with no other associated features apart from facial muscle weakness, also shared by the other patients.

Severe axial muscle weakness (Fig. 1, panel D): Patients 7 (FAM.II) and 8 (unrelated, aged 17 and 20yo) had rapidly progressive scoliosis that led to surgery at the age of 16 and 14yo, respectively. The second one has also been using non-invasive ventilation since that time. They also have mild facial muscle weakness. Both patients maintain unassisted walking and none has bulbar muscle involvement.

Joint laxity (Fig. 1, panel F): In two families, FAM.III and FAM.IV, the coexistence of weakness and laxity was the main key to the diagnosis. In the first kindred, the index case was patient 9, a 4yo girl with normal development and a mild to moderate proximal tetraparesis, mainly affecting the lower limbs, associated with an evident laxity in axial and limbs joints (shoulders and elbows) noticed at the age of two. Patient 10, her mother (aged 20yo), has recurrent patellar dislocations and the same pattern of laxity but with less weakness. This worsened transiently during her second pregnancy. She gave birth to our youngest patient, patient 11, who was born with tetraparesis (mainly upper limbs), laxity, facial and transitory bulbar involvement, needing to be fed by a nasogastric tube in the neonatal period. She progressively improved in months and at present, aged 12 months, she achieved to sit unsupported. In FAM.IV, the daughter (P12, aged 9yo) was also the index case, with moderate tetraparesis, elbow laxity and past history of congenital hip dislocation. Her mother (P13, aged 38 yo) was found to have also past history of congenital hip dislocation and easy fatigue. On examination, a mild tetraparesis was revealed. So, apart from patient 11 in her first month of life, no patient in this group had facial, bulbar or axial muscle involvement.

The fourth phenotype includes four patients (P14 to P17) with a mild tetraparesis and no distinctive clinical feature. The two patients with later reported age of onset belong to this group (P14 and P15). They also have facial muscles weakness but no other additional feature. Patient 14 belongs to FAM.II and is a maternal uncle of patient 7 (different phenotype). Patient 17 presented with neonatal arthrogryposis that required several surgical procedures. He was only able to walk at the age of 6 and became fully functional.

None of the patients has cardiac involvement.

Eight patients (47.1% ) are not aware of any family history suggestive of myopathy.

Histopathology

Fourteen patients (82.4% ) had a muscle biopsy, at a mean age of 10.9 (1–29) yo. The examined muscle (deltoid) was abnormal in every patient, with four histopathologic myopathic patterns found (Fig. 1, panels B to G): centronuclear (N = 5), centralcore (N = 4), type 1 fibres predominance (T1FP; N = 4) and congenital fibre type disproportion (CFTD; N = 1). All patients with EOM weakness had the centronuclear pattern, except P6 who showed CFTD. From the four centralcore patterns identified, three belong to patients with isolated mild tetraparesis with or without facial weakness. The remaining belongs to patient 12, with the laxity component. The group of patients with T1FP was more heterogeneous in terms of clinical presentation but both patients with severe axial muscles involvement having this histopathologic pattern. From the other two T1FP patients, one was the youngest patient with the laxity (P11) and the other was one of the less severely affected patients (P15).

In the extraocular muscles weakness phenotype, the majority of cases showed severe myopathic changes. In the other phenotypes, the numbers are too small to drawing conclusions (Table 2).

Genetics

High genetic heterogeneity was found in this cohort of patients. Except for patients 4 and 5, each index case had different pathogenic variants in heterozygosity, the majority being of the missense type. Five pathogenic variants had not previously published in the literature (P4, P5, P6 and P12). As detailed in Table 1, variants found in compound heterozygosity were more dispersed along the RYR1 gene. Cases with autosomal recessive (AR) inheritance patterns were more frequent in the group of patients with EOM involvement (P1 to P6). In addition, all the centronuclear histopathologic presentations were also found in this group. In the remaining patients, there was a predominance of pathogenic variants in hotspot 3 (exons 93 to 106) along with autosomal dominant (AD) inheritance patterns. All the central core histopathologic patterns were found in this last group. The variant c.7843 C>T (p.Arg2615Cys) identified in patient 8 has a paternal origin; considering that the patient’s father is asymptomatic, its clinical significance remains unclear.

Malignant hyperthermia events were not reported in any of the patients or in their family members. However, three variants were previously associated with malignant hyperthermia susceptibility, by in vitro or functional contracture tests. They were identified in the first family with laxity (FAM.III), in one of the least disabled patients with EOM involvement (P15) and in one of the four patients with mild tetraparesis and facial weakness only (P16).

DISCUSSION

We report a rather heterogeneous group of 17 patients (including four families) with RYR1-related myopathies.

Clinically, all patients have muscle weakness and thin appearance of muscle bulk. We divided them in major groups according to three distinctive features: EOM weakness (N = 6), severe axial muscle weakness (N = 2) and marked joint laxity (N = 5). The fourth phenotype had mild tetraparesis with or without facial weakness and no clinical distinctive feature (N = 4).

There was no significant association between EOM weakness and clinical severity, as the patients who presented with this feature were quite heterogeneous in this respect (considering ambulation, respiratory and bulbar involvement). This has also been previously reported [16].

There is clear disproportion between axial weakness and the ambulatory state in patients 3, 7 and 8 who were submitted to scoliosis surgery early in life while maintaining ambulation so far. This has been described by Colombo et al. in a large published series of congenital myopathies, 44.4% of whom with the genetic diagnosis of RYR1 related myopathy [2]. They also report that in RYR1-related myopathies, feeding difficulties can be transitory, as occurred with patient 11. Of interest, four out of six patients with EOM weakness also have axial weakness, with three of them having been submitted to early surgery. Therefore, the combination of these two patterns of muscle weakness may thus point towards the diagnosis of ryanodinopathy. Concerning the third phenotype that we describe, the association of RYR1 myopathy and laxity has been previously reported [7]. In our patients, laxity was mostly observed in proximal joints like shoulder, elbow, hip and knee, which may also help in the distinction from other myopathies with prominent joint hypermobility (e.g. collagen VI-related dystrophies). In this regard, muscle biopsy can also guide genetic testing.

In terms of histopathology, among the 14 patients who had muscle biopsy, four different myopathic patterns were found, previously described as being associated with RYR1 myopathies: centronuclear (N = 5), central core (N = 4), T1FP (N = 4) and CFTD (N = 1).

In our cohort, all centronuclear myopathies were associated with EOM weakness. Wilmshurst and collegues identified RYR1 mutations in 17 patients from a cohort of 24 patients with unresolved centronuclear myopathy. All patients except one had EOM weakness [8]. This association has also been described by other authors [9, 10].

Three of the patients with the T1FP pattern were already published by our group, emphasising the absence of central cores. The authors suggest that T1FP and involvement of axial muscles may be an important element to consider RYR1 as a candidate gene [11]. In Maggi and colleagues’ series of 66 patients with a congenital myopathy, the identification of RYR1 mutations in 4 patients with isolated type 1 uniformity or predominance suggested that RYR1 mutations are a relatively common cause of these histological findings [12]. Sato et al. published ten unrelated Japanese patients diagnosed with congenital neuromuscular disease with uniform type 1, four of whom (40% ) had RYR1 mutations. These patients had milder clinical features compared with those without RYR1 mutations [13]. Our findings do not support such a clear association between T1FP and milder clinical involvement in RyR1 patients because half of them had severe axial involvement (P7 and P8).

Clarke et al. identified RYR1 mutations in four out of seven families with typical CFTD in whom other known genetic causes had been excluded. The authors suggested that RYR1 is a relatively common cause of this clinico-pathological pattern. They also noted an association between the presence of RYR1 mutations and ophthalmoplegia, proposing that it may be the most specific clinical indicator of RYR1 mutations in the setting of CFTD as this is not reported in the other known causes [14]. In agreement, our only CFTD case had EOM weakness.

The classic description of a RYR1 congenital myopathy was a core myopathy but this paradigm has changed. Most of the previously cited papers that describe non-core histologic presentations propose that cores may evolve overtime, in a dynamic model. None of our patients have repeated the muscle biopsy and the mean age at muscle biopsy was in fact lower in central core (7.7yo) than in non-central core presentations (11.1 yo), as opposed to what would be expected based on the possible development of cores with age increasing. The small number of patients may contribute to this finding.

Concerning the genetic profiles found in our patient cohort there is a high genetic heterogeneity and variability. The majority of cases had different RYR1 variants including four described for the first time in this work.

It is not infrequent to find variant of uncertain significance (VUS) during these studies. Particularly those of missense type pose additional interpretation difficulties and as consequence the clinical utility of these studies is sometimes limited. According to the most recent guidelines for sequence variants interpretation from the American College of Medical Genetics and Genomics, several lines of evidences should be considered when performing variant pathogenicity assessment [15].

As an example, the 2348 C>T (p.Ser783Leu) variant if interpreted individually would be classified as VUS (class 3), in spite of the bioinformatics analysis suggesting its deleterious effect and absence from population variant databases. However, when interpreted together with the c.1342A>T (p.Ile448Phe) variant (detected in another patient of our laboratory cohort and considered likely pathogenic) would be in favour of being causative for RYR1-related myopathy. Nonetheless, it would be important to further characterize the functional properties of these variants as previously demonstrated in the literature [16, 17].

We attempted to correlate the sequence variants distribution found in this cohort and their impact on the structure and/or activity of ryanodine receptor type 1 using the information available in previous reports.

It is well known that the majority of patients with autosomal dominant central core disease have RYR1 pathogenic variants that are clustered in the hydrophobic COOH-terminal region (hotspot domain 3) of this receptor. Zhou et al. published a series of 28 patients in which the dominant RYR1 variants were mainly found in the C-terminal and only in the central domain of RYR1. These authors also stated that most patients with dominant C-terminal variants had the classical central core phenotype, characterized by variable degrees of proximal weakness and absence of significant bulbar, respiratory or extraocular involvement [18]. We have also found these milder phenotypes in our patients with AD inheritance, having variants located in hotspot 3 and central cores in their muscle biopsies. It is interesting to note that the majority of pathogenic variants found in our AD patients are within a small stretch of 38 residues (4861–4898) of the protein. Since the high (near-atomic) resolution structure of RyR1 has been recently described, it is now evident that these residues correspond to critical segments of the ion-conducting pathway: the luminal loop, the pore helix and the selectivity filter [19]. In particular, the p.Arg4893Trp variant (found in P7 and P14) may affect one of the potential residues that constitute the inner cation-binding site. These structures were proposed to be a hotspot for disease-related mutations [9, 19]. Some variants in this region have been further characterized, namely the p.Ile4898Thr variant found in family FAM.III, which was found to create a dominant-negative suppression of RYR1 channel calcium ion permeation [20].

The genetic variants associated with autosomal recessive inheritance and other histopathological patterns (e.g. centronuclear) are distributed along the entire coding sequence [5, 21]. Our series is concordant with this description. Among 26 patients with RYR1 pathogenic variants, Maggi and colleagues (2013) identified compound heterozygous variants consistent with recessive inheritance distributed throughout the RYR1 coding sequence in 15 patients (57.7% ) [12]. Amburgey and collaborators attempted to establish genotype-phenotype correlations in a large cohort of recessive RYR1-related cases [9]. Our data is in accordance with the main observations of this work. Hypomorphic mutations (nonsense and frame-shifting, expected to reduce RyR1 expression) were identified in AR non-central core myopathies (P1 to P5) and in association with more severe clinical phenotypes. In our cohort, all patients with AR inheritance pattern and at least one hypomorphic allele had EOM weakness and centronuclear myopathy. This was previously described by other previous works [6, 22].

Regarding malignant hyperthermia, we identified three genetic variants (five patients) with proven susceptibility. In concordance with previous reports, this susceptibility may exist even in the context of a mild and/or non centralcore RYR1 myopathy [23].

In conclusion, there is a great variability in clinical presentation, genetics and muscle pathology in patients with ryanodinopathies but some clinical features may be diagnostically relevant: extraocular muscle weakness (with centronuclear pattern on muscle biopsy), severe axial weakness disproportionate to the ambulatory state and mild tetraparesis associated with (proximal) joint laxity.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.