Clinical and Genetic Spectrum of RYR1-Related Disease

Abstract

Introduction

In this study, we examined the genotype-phenotype characteristics of the cases with pathogenic/possibly pathogenic variants in the RYR1 gene that we follow in our clinic.

Methods

Data of patients who applied to our clinic and had pathogenic/possibly pathogenic/variant of unknown significance variants in the RYR1 gene were evaluated retrospectively. Patients were examined in terms of demographic, clinical, and individual genetic data, age of symptom-onset, sex, clinical features, clinical types, variants, cardiac involvement, muscle biopsy results, serum creatinine kinase (CK) levels, family history, and consanguinity.

Results

The variants were detected in 19 patients from 18 different families. The most common (n = 5, 38.4%) variant was the c.7880T>G (p.Val2627Gly) heterozygous change. 63.1% of our patients were male (n = 12) and 37.9% were female. Admission complaints included a floppy baby, developmental delay, or hyperCKemia. The most common clinical spectrum was malignant hyperthermia (MH) sensitivity (n = 8, 44.4%). We also identified four novel variants in our cohort.

Conclusion

RYR1 is known to be the gene most associated with MH. It is very important to manage and take precautions against possible comorbidities and anesthesia complications. For this reason, we think that RYR1 analyses should be given priority in the diagnostic algorithm.

Introduction

The RYR1 gene is composed of 106 exons and encodes 5,038 amino acids. This makes it one of the largest genes in the human genome. It is a skeletal muscle calcium release channel associated with excitation-contraction coupling [1]. The RYR1 gene encodes the ryanodine receptor which releases calcium from the sarcoplasmic reticulum [2]. Pathogenic variants of the RYR1 gene are the most common cause of congenital myopathies [3]. This variant is associated with a clinical spectrum including central core disease (CCD), multi-minicore disease (MmD), and malignant hyperthermia (MH) [4, 5]. Both dominant and recessive mutations have been reported in the RYR1 gene [2]. Recessive mutations predominate in patients with MmD, centronuclear myopathy (CNM), and congenital fiber-type disproportion [6–8]. Dominant mutations have usually been associated with CCD and/or MH susceptibility, which is a rare pharmacogenetic syndrome characterized by muscle rigidity and an increase in body temperature after exposure to anesthetics via inhalation [9, 10]. MH is a potentially fatal reaction that occurs in genetically susceptible individuals exposed to volatile anesthetics or succinylcholine. The RYR1 gene was the first gene linked to MHS and is involved in 34–86% of cases reported [11–19]. In addition to the RYR1 gene, genes such as CACNA1S and STATC3 are also associated with malignant hyperthermia [12]. It is a large gene and many variants have been associated with MHS, although only a minority of these have been demonstrated to be pathogenic.

Thus, the present study aimed to provide a comprehensive analysis of genotype-phenotype relationships of the RYR1 variants. We analyzed whether associations exist between variant type and location, clinical diagnosis, and severity of clinical features.

Methods

Patient Selection

Between January 2019 and December 2023, patients with a pre-diagnosis of neuromuscular diseases and/or hyperCKemia were enrolled. The diagnosis of RYR1 gene-related disease was established by clinical and genetic analysis. Demographic, clinical, and genetic data of the patients were reviewed retrospectively. The age of symptom-onset, sex, clinical features, clinical types of disease, cardiac signs, muscle biopsy results, family history, and consanguinity were noted. Serum creatine kinase (CK) levels, nerve conduction studies, electromyographic examinations, and genetic analysis results were also evaluated. While scrutinizing the demographic data, a solitary individual was extracted from every family cohort. This designated patient was ascertained to be the initial applicant to our clinic. The ethical approval was obtained from the Ethics Committee of the University of Health Sciences Izmir Tepecik Education and Research Hospital (Date: March 4, 2024, Decision number: 2024/02-24).

Genetic Analysis

For the molecular genetic evaluation, two Custom Target Capture NGS Panels (Custom Limb Girdle Muscular Dystrophy NGS Panel covering 31 genes and Custom Neuromuscular Disorders NGS Panel” covering 291 genes) (Celemics, Inc., Seoul, Korea) were used. Both NGS panels were covering ANO5, CAPN3, CAV3, DAG1, DES, DNAJB6, DYSF, FKTN, FLNC, FRKP, GAA, GMPPB, HNRNPDL, ISPD, LIMS2, LMNA, MYOT, PLEC, POMGNT1, POMK, POMT1, POMT2, SGCA, SGCB, SGCD, SGCG, TCAP, TNPO3, TRAPPC11, TRIM32, and TTN genes. After genomic DNA samples were extracted, libraries were prepared. Target capture NGS was performed on an Illumina MiSeq NGS System (Illumina, Inc., San Diego, CA, USA). FASTQ sequencing files were collected and transferred to “SEQ” variant analysis software (Genomize, Istanbul, Turkey).

Identification of variants

The data were analyzed using “SEQ” variant analysis software (Genomize, Istanbul, Turkey) according to the reference genome of GRCh37(h19). Variants with all ClinVar submissions that were benign or likely benign were excluded. The variant interpretation was done according to the standards and guidelines released by the American College of Medical Genetics (ACMG) [20].

Statistical Analysis

The data obtained in the study were entered into a database created in the Statistical Package for the Social Sciences (IBM SPSS Statistic Version 22, IBM Inc., Chicago, IL, USA) program, and statistical analyses were performed using the same program. The mean, standard deviation, median, minimum, and maximum values of continuous variables were calculated.

Results

Patient Characteristics

Genetic Characteristics

In our study, we detected 13 different variants (77.7%) in 19 individuals spanning across 18 familial units. Genetic analysis could not be performed on all members of the families included in the study, owing to an array of factors including financial constraints and limitations related to health insurance coverage. We were able to perform a genetic analysis on certain family members of only 8 patients.

The heterozygous variant was detected in 17 patients, homozygous variant was detected in 1 patient, and the compound heterozygous state was found in the remaining one and confirmed by parental analysis. The c.7880T>G (p.Val2627Gly) change in the RYR1 gene (n = 5, 38.4%) was the most frequent single detected variant (Table 1). We detected four novel heterozygous variants in our patients (Table 1). According to ACMG guidelines, they are predicted as likely pathogenic variants [20].

Table 1.

Genetic characteristics of our patients

Patients	Age, gender	Clinical type	Variant	Predicted protein change	Zygosity	Classification	Transcript	Novel	Inheritance
P-1	1, F	KDS	c.3389G>A	p.Arg1130His	Het	Variant of unknown significance (PM2, PP3)	ENST00000359596.3		AD
P-2	10, M	MHS	c.1756A>T	p.ıle586Phe	Het	LP (PM1, PM2, PP3)	ENST00000359596.3 (NM_000540.3)	Yes	AD
P-3	2, F	CM1A	c.2870C>T	p.thr957met	Het	Variant of unknown significance (PM2, PP3)	ENST00000359596.3 (NM_000540.3)		AD
P-4	7, F	MHS	c.14731G>A	p.Glu4911Lys	Het	Lp (PM1, PM2, PP3, PP5)	ENST00000359596.3 (NM_000540.3)		AD
P-5	2, M	CM1A	c.3901C>T	p.Arg1301Cys	Het	Variant of unknown significance (PM2, PP2)	ENST00000359596.3 (NM_000540.3)		AD
P-6	11, F	MHS	c.3901C>T	p.Arg1301Cys	Het	Variant of unknown significance (PM2, PP2)	ENST00000359596.3 (NM_000540.3)		AD
P-7	3, F	MHS	c.7007G>A	p.Arg2336His	Het	P (PM1, PM2, PP3, PP5)	ENST00000359596.3 (NM_000540.3)		AD
P-8	4, M	MHS	c.7007G>A	p.Arg2336His	Het	P (PM1, PM2, PP3, PP5)	ENST00000359596.3 (NM_000540.3)		AD
P-9	10, M	CM1A	c.11550C>A	p.Asn3850Lys	Het	Variant of unknown significance (PM2, PP3)	ENST00000359596.3 (NM_000540.3)		AD
P-10	1, F	CM1B	c.1931G>T --- c.2354G>A	Arg644Leu --- p.Gly785Asp	Comp. Het	Lp (PM1, PM2, PP3)	ENST00000359596.3 (NM_000540.3)	Yes	AR
P-11	2, M	CM1A	c.7880T>G	p.Val2627Gly	Het	Variant of unknown significance (PM2, PP2)	ENST00000359596.3 (NM_000540.3)		AD
P-12	7, M	MHS	c.7880T>G	p.Val2627Gly	Het	Lp (PM2, PP2, PP3, PM5)	ENST00000359596.3 (NM_000540.3)		AD
P-13	18, M	MHS	c.7880T>G	p.Val2627Gly	Het	Lp (PM2, PP2, PP3, PM5)	ENST00000359596.3 (NM_000540.3)		AD
P-14	17, M	KDS	c.7261G>A	p.Ala2421Thr	Het	Lp (PM2, PP2, PP3, PM5)	ENST00000359596.3 (NM_000540.3)		AD
P-15	15, M	KDS	c.7880T>G	p.Val2627Gly	Het	Lp (PM1, PM2, PP2, PP3, PM5)	ENST00000359596.3 (NM_000540.3)		AD
P-16	5, M	CM1A	c.6548+1G>C		Het	Lp (PVS1, PM2)	ENST00000359596.3 (NM_000540.3)	Yes	AD
P-17	6.5, M	CM1A	c.488G>A	p.Arg163His	Hom	Lp (PVS1, PM2)	ENST00000359596.3 (NM_000540.3)		AD
P-18	3, M	CM1A	c.13768G>T	p.Glu4590Ter	Het	Lp (PVS1, PM2)	ENST00000359596.3 (NM_000540.3)	Yes	AD
P-19	3, F	MHS	c.7880T>G	p.Val2627Gly	Het	Lp (PM2, PP2, PP3, PM5)	ENST00000359596.3 (NM_000540.3)		AD

F, female; MHS, malignant hyperthermia susceptibility; KDS, King-Denborough syndrome; CM1A, congenital myopathy 1A; CM1B, congenital myopathy 1B; AD, autosomal dominance; AR, autosomal recessive; M, male.

Clinical and Demographic Characteristics

In total, seven (36.8%) patients were female and twelve (65.7%) were male. The median age was 6.63 (range, 1–18) years, and the median time from onset of symptoms to diagnosis (for symptomatic patients) was 3.7 (range: 0.5–11) years. Clinical and demographic signs are summarized in Table 1. The rate of consanguineous marriage was 38% (n = 7). Our patient collective was drawn up out of patients with the following variants: eight MHS-1 (44.4%), seven congenital myopathy 1A (38.8%), one congenital myopathy 1B (5.5%), and two King-Denborough syndrome (11.1%) (Table 1). Hypotonia, neurodevelopmental delay, and hyperCKemia in routine laboratory tests were common findings. Fourteen (82.3%) of all patients had high CK. Muscle biopsy was not performed in all patients. For this reason, we could not perform a histopathological classification. Electromyography was performed on eight (42.1%) patients. While normal nerve conduction velocities and myopathic motor unit potentials were observed in 4 patients (50%), electromyographic evaluation was normal in the remaining 4 patients (50%). Cardiological assessments including electrocardiography and echocardiography were performed in twelve (70%) patients and revealed normal.

Clinical Presentation

We describe our patients according to the RYR1-related disease types in the OMIM (Online Mendelian Inheritance in Man).

Malignant Hyperthermia Susceptibility 1: MHS-1

One of the two most common clinical spectrum was MHS-1 (n = 8, 42.1%). The median age was 8.5 (range: 3–18) years, and an autosomal dominant inheritance was demonstrated in all patients. All of the patients had heterozygous variants. The median CK level was 1,900 (range: 58–5,500) U/L (Table 1). While 4 patients presented with asymptomatic hyperCKemia, the remaining 4 patients had varying degrees of muscle weakness. None of the patients had dysmorphic findings, neuromotor delay, or hypotonia. We segregated the same variant in the parents (mother or father) of 5 patients. We detected moderate proximal muscle weakness and hyperCKemia in the parents of some of the patients in this particular group (n = 3). Individuals in this group were also diagnosed with MHS along with their children. The remaining two parents were asymptomatic. We performed genetic analysis on these patients for other neuromuscular and metabolic diseases. The only variant detected was in the RYR1 gene. Therefore, we accepted these patients as both manifesting carriers and MHS-1. We informed the patients and their parents about malignant hyperthermia and continued the follow-up. Genetic analysis could not be performed on the parents of the remaining 2 patients for the aforementioned reasons. There were two siblings in this group, and their father was also diagnosed with MHS-1 (patients 7 and 8) (Fig. 1). We initiated a male sibling with low-dose dantrolene because he had frequent attacks of rhabdomyolysis. Dantrolene 0.25 mg/kg was started. The dantrolene dose was increased by 0.25 mg/day/week up to three doses of 0.75 mg/kg/day. He is monitored without rhabdomyolysis attacks.

Fig. 1.

Pedigree of patient 7 and 8’s family (c.7007G>A p.Arg2336His).

Congenital Myopathy 1A, Autosomal Dominant, with Susceptibility to Malignant Hyperthermia: CM1A (Phenotype MIM Number 117000)

Congenital myopathy-1A with susceptibility to malignant hyperthermia is a disorder of skeletal muscle characterized by muscle weakness primarily affecting the proximal muscles of the lower limbs beginning in infancy or early childhood. There were seven (41.1%) patients in this group. The median age was 3.7 (range: 1–10) years, and an autosomal dominant inheritance was demonstrated in all patients. The age at first presentation median was age 0.7 (range, neonate: 1) years. The mean CK level was 341 (range: 86–990) U/L. Their neurological evaluation was abnormal (axial hypotonicity, dominance proximal muscle weakness, neurodevelopmental delay, and absent DTR). Since none of our patients had a muscle biopsy, histopathological classification (CCD, multimini-core disease, etc.) was not performed.

Congenital Myopathy 1B, Autosomal Recessive: CM1B (Phenotype MIM Number 255320)

Congenital myopathy 1B is a disorder of skeletal muscle characterized by severe hypotonia and generalized muscle weakness apparent soon after birth or in early childhood with neuromotor delay, generalized muscle weakness, and difficulty walking or running. A 12-year-old male patient presented with neurodevelopmental delay and difficulty walking and hyperCKemia. Family histories were unremarkable, but he had consanguineous parents (first cousins). His neurological examination was abnormal (dominance proximal muscle weakness, inability to walk, absent DTR). We detected the c.488G>A (p.Arg163His) variant (likely pathogenic, homozygous) in the RYR1 gene. In addition, the same variant was detected in his parents as heterozygous. Clinical follow-up for CM1B continues (Fig. 2).

Fig. 2.

Pedigree of patient 17’s family (c.488G>A (p.Arg163His).

King-Denborough Syndrome

Two male and 1 female patients with King-Denborough syndrome (KDS) had a similar disease course. All of the patients were accompanied by endocrinologic (micropenis, short stature, etc.) and orthopedic (scoliosis) abnormalities. Another distinguishing feature was the infantile-onset. We detected three different variants in 3 patients (Table 1). The parents were healthy. Genetic analysis of patient 14’s parents could not be performed. The parents of the other patient (patient 15) variant were not detected. We think that 3 patients have KDS because no other variant was detected in the genetic analysis and their clinically distinctive features.

Characteristics of Our Novel Variants Patients

Here we describe 3 patients who had novel mutations. We detected four novel heterozygous variants in our patients (Table 1). The c.1756A>T variant detected at patient 2, c.1931G>T variant detected at patient 10, the c.6548 + 1G>C variant detected at patient 16, and the c.13768G>T variant detected at patient 18 had extremely low allele frequencies in population databases (PM2), in silico algorithms showed pathogenic predictions (PP3) and with additional criteria such as loss of function of the gene (PVS1), according to ACMG guidelines, they are predicted as likely pathogenic variants (20). There were no variants listed in the databases and literature. We interpreted these variants as novel pathogenic variants (Table 1). Of whom, 3 had CM1A (patients 10, 16, and 18), and the remaining one had MHS (patient 2).

• Patient 10: a seventeen-month-old female (patient 10) presented with hypotonia and neurodevelopmental retardation. She was born to consanguineous parents (second cousins) at 38 weeks of gestation with a 3,000 g of birth weight. Neurological examination revealed axial hypotonia, myopathic face, and absent DTR. She has poor head control and assisted sitting was present. Serum CK level was normal. Developmental stages were retarded, and there was no regression. In molecular genetic investigation, heterozygous c.1931G>T, c.2354G>A (p.Arg644Leu), and c.2354G>A (p.Gly785Asp) variants were detected. This result was considered to be consistent with compound heterozygosity since each parent carried one mutation. All genotypes were verified by Sanger sequencing and segregation analysis. The patient was diagnosed with CM1B based on the genetic results.

• Patient 16: a 6-year-old male presented with unsteady walking and neurodevelopmental retardation. He was born to unrelated parents at 40 weeks of gestation with a 3,400 g of birth weight. Neurological examination revealed axial hypotonia and Gower’s sign. Serum CK level was normal. Developmental stages were retarded without any regression. Genetic analysis identified a heterozygous novel variant in the RYR1 gene (c.6548 + 1G>C). In addition, genetic analysis could not be performed on our patient’s parents. A 3 years old male patient (patient 18) presented as floppy baby with neurodevelopmental retardation and hyperCKemia. Personal and family histories were unremarkable. Neurological examination revealed axial hypotonia and absent DTR. Serum CK level was high (990 U/L). Developmental stages were retarded and there was no regression. He was diagnosed with CM1A by genetic results c.13768G>T (p.Glu4590Ter) novel heterozygous variant of the RYR1 gene. Genetic analysis could not be performed on our patient’s parents. No other variants were detected in the genetic analysis of our three patients (patients 10, 16, 18) and metabolic tests were normal. In addition, brain MRI and cardiological examination of all patients were unremarkable.

• Patient 2: a 9-year-old male patient presented with difficulty getting up from sitting and climbing stairs. Personal and family histories were unremarkable except for consanguineous parents (first cousins). Neurological examination revealed only moderate proximal muscle weakness. Serum CK level was high (1,764–2,391 U/L). Genetic analysis identified a novel heterozygous c.1756A>T (p.Ile586Phe) variant in the RYR1 gene. Clinical follow-up for MHS continues. While no variant was detected in the mother, the same variant was found in his father. Moderate proximal muscle weakness and hyperCKemia were also detected in his father. No other variants were detected in the genetic analysis of our patient and metabolic tests were normal. Our patient’s father was also diagnosed. However, this individual may also be a manifest carrier. Therefore, information was given regarding malignant hyperthermia.

Discussion

The RYR1 gene is one of the largest human genes, including 106 exons. So far, hundreds of RYR1 gene mutations have been identified, many of which are associated with different types of diseases [4]. The RYR1 gene mutations are associated with a variety of diseases, including CCD, MmD, and MH [4, 5]. Moreover, the association of variants in this gene with an anesthetic emergency such as malignant hyperthermia has been known for years [21]. For these reasons, we thought it would be important to report the clinical manifestations of variants in the RYR1 gene.

The most common clinical types in our study were MHS and CM. Malignant hyperthermia is an anesthetic emergency triggered by the use of volatile anesthetics (isoflurane, halothane, desflurane, etc.) and depolarizing relaxants (succinylcholine, etc.) [19]. Susceptibility to MH predominantly shows autosomal dominant inheritance, although not all reported cases are consistent with simple autosomal dominant inheritance [22–24]. It was reported that about three-fourths of MH-susceptible families have pathogenic variants in the RYR1 gene [24]. Therefore, anesthesia precautions should be taken in all individuals with the pathogenic variant within this particular gene. The genetic diagnosis of MHS can be challenging because of the large size of the RYR1 gene and often limited data available on its variants [25]. In our study, 41% of our patients were diagnosed with MHS. We also found the same variant in some parents. Sixty of these individuals had symptoms. These parents were diagnosed with MHS. The remaining 2 patients may also be manifest carriers with these findings. However, other tests for neuromuscular and metabolic diseases (genetic and metabolic) were normal. We accepted the remaining cases as manifest carriers. The manifesting carriers are an ongoing problem, especially AD genes. It has been reported in the literature that manifest carriers may have similar or milder symptoms of the disease [26]. The manifesting carrier in AD genes is complicated. Moreover, the situation is more complicated in a disease that causes isolated MHS clinics, such as RYR1. It is difficult to determine whether an individual with a pathogenic variant in the RYR1 gene, which is associated with a condition that can lead to malignant hyperthermia without clinical findings such as MHS, is a patient or a carrier. Additionally, clinical findings may appear in children in adolescence or adulthood. Therefore, while the examination and follow-up of these patients continue, it is necessary to make recommendations regarding malignant hyperthermia and rhabdomyolysis.

In the literature, the RYR1 gene has been reported mostly in association with congenital myopathies [4, 5]. In our study, congenital myopathy and MHS were the most common clinical presentations. The number of patients with MHS is significant, which can be attributed to the genetic-based nature of this study. No muscle biopsy was required for diagnosis in any of our patients. This result supports the view that the diagnosis of patients with asymptomatic or mild clinical symptoms will increase with the development of genetic analysis methods. Future developments in genetic analysis methods will make MH more obvious and interesting. We think that the incidence of diseases will change in the future with the development of genetic analysis methods and the increase in publication notifications regarding this. Additionally, newborn genetic sequencing, once implemented, will increase the need for diagnosis and management.

In addition, with the advent of next-generation gene sequencing and the increasing number of pathogenic variant genes, the disease spectrum of RYR1-related disease expanded. Another consequence of lacking a muscle biopsy in any of our patients was that we were unable to make histopathological classification. We think that the use of histopathological classification will decrease in the future as genetic diagnosis methods improve.

There are several limitations of this study, including its retrospective design and failure to perform genetic analysis on all parents. However, even with these limitations, our patients include four novel variants not previously described in the human gene database.

Conclusion

We detect variants very frequently in the RYR1 gene. Knowing the clinical spectrum of variants in this gene will help avoid diagnostic confusion. Finally, the RYR1 gene is known to be the gene most associated with malignant hyperthermia. It is important to know the risks such as management of possible comorbidities and anesthesia complications and to take precautions against them. Genetic analysis for hypotonic infants, patients with rhabdomyolysis as well as chronically elevated CK are important. In addition, since many with exertional heat illness or heat stroke harbor RYR1 DNA variants, those should be tested for RYR1 variants as should the family members of those with variants related to MH and to RYR1 myopathies. For this reason, we think that analysis of this gene should be prioritized in the diagnostic algorithm of cases evaluated due to hypotonic infants or asymptomatic CK elevation.

Statement of Ethics

This study protocol was reviewed and approved by the Ethics Committee of the University of Health Sciences Faculty of Medicine (Date: March 4, 2024, Decision number: 2024/02-24). Because it was a retrospective study, it was not necessary to obtain written informed consent from the participants. This retrospective review of patient data did not require ethical approval by local/national guidelines.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

There is no funding in this study.

Author Contributions

Gamze Sarıkaya Uzan: design, data analyses, evaluation of results, and writing of the manuscript. Berk Özyılmaz: design, data analyses, and writing of the manuscript. Gizem Doğan: design, data analyses, and evaluation of results. Figen Baydan, Yiğithan Güzin, and Pınar Gençpınar: data analyses and evaluation of results. Hande Gazeteci Tekin: design and evaluation of results. Nihal Olgaç Dündar: design, evaluation of results, and writing of manuscript.

Funding Statement

There is no funding in this study.

Data Availability Statement

Patient data were obtained from the patient files of our clinic. Further inquiries can be directed to the corresponding author.