Identification of Heterozygous and Homozygous Individuals with the Novel RYR1 Mutation Cys35Arg in a Large Kindred

The proband, individual IV:6, who is of Sicilian origin, suffered a suspected MH episode in 1979 when he was 5 yr old while undergoing a general anesthetic for a diagnostic bronchoscopy. After induction of general anesthesia with halothane, succinylcholine was administered for muscle relaxation. The patient immediately experienced severe masseter spasm, rendering tracheal intubation impossible. Tachycardia (150 beats/min) rapidly developed, along with tachypnea and generalized rigidity. The volatile anesthetic was discontinued, and the lungs were hyperventilated with 100% oxygen. Although dantrolene was not readily available at that time, he survived the episode and made an uneventful recovery. Postoperative creatine kinase (CK) was increased (3,253 IU/L). At the age of 16 yr old, he was referred to the Lille MH diagnostic center to evaluate MH susceptibility by CHCT. No indication of muscle disease was found on clinical examination, and his resting CK level was normal. There was no history of muscular disease or unexpected anesthetic reactions in the family. No patient complained of muscle faintness or muscle cramps. Eighteen patients from this pedigree were investigated by physical examination, CHCT (the diagnosis of MH susceptibility was based on skeletal muscle contractures by the CHCT performed according to the European malignant hyperpyrexia group protocol), [15]histo-enzymologic study, and DNA analysis.

Genomic DNA was isolated from blood preserved in edetic acid with informed consent from patients. Linkage analysis was performed using the Linkage 5.2 program. [16]Members of the pedigree were genotyped using chromosome 19 microsatellite markers flanking the RYR1 locus (D19S191, D19S220, D19S190, and D19S223).

Following RNA extraction and cDNA synthesis from a muscle sample obtained from the proband, the entire RYR1 cDNA was amplified by polymerase chain reaction in short overlapping segments for single stranded conformational polymorphism analysis, [17]but we were unsuccessful in identifying a RYR1 mutation in the proband.

We then investigated the proband's RYR1 cDNA, sequencing cloned RYR1 polymerase chain reaction fragments. Initially, we chose to clone and sequence the first 2,000 base pairs (bp) of the RYR1 cDNA, because the majority of previously described mutations are located in this region. [12]Total RNA was extracted from a 100-mg muscle biopsy sample obtained from individual IV:6 by guanidinium thiocyanate extraction. [18]First-strand cDNA synthesis was carried out using 200 ng random hexamers, 1 ug total RNA, and 15 units of avian myeloblastosis virus reverse transcriptase in a final volume of 20 ul. Polymerase chain reaction primers were designed using the published human RYR1 cDNA sequence. [19]Overlapping 500 bp products were amplified by polymerase chain reaction using the first strand cDNA as a template. Ligation into the TA cloning vector pCRII (In Vitrogen Corporations, San Diego, CA) was carried out according to the manufacturer's instructions. [20]Recombinant plasmids were sequenced using the T7 sequencing kit (Pharmacia Biotech, Uppsala, Sweden).

To amplify the region bearing the mutation from genomic DNA, the cDNA sequence of the first 500 bp was determined by standard dideoxy sequencing methods and revealed the presence of a substitution of C for T at nucleotide 103 in 4 independent clones (Figure 1). This transition mutation results in the replacement of a cysteine with an arginine at codon position 35. We amplified the smallest region possible to avoid inclusion of unknown intronic DNA. Polymerase chain reaction primers 78F (5'-CGTGCTCAAGGAGCAGCTCAAGCT-3') and 105R (5'-CCGAAGCCCTCGGCGACCAGG-3') were designed to amplify the mutation site and a flanking base on either side. Polymerase chain reaction amplification of genomic DNA and cDNA yielded the expected 48 bp product, demonstrating that no intron existed in the region amplified using the primers selected.

Amplification of the region flanking the T103C mutation was achieved using primers 78F and 105R and resulted in a 48 bp amplicon. To abolish a common Acil site, eight bases 3' to the mutation Acil site created by the T103C exchange, a guanine at position 16 of the 105R primer was replaced with an adenosine (underlined in primer sequence). Amplification was carried out using a hot start of 95 degrees Celsius for 5 min, followed by 32 cycles of 94 degrees Celsius for 1 min, 56 degrees Celsius for 1 min, and 72 degrees Celsius for 1 min. Cycling was followed by a final extension of 72 degrees Celsius for 10 min.

The presence of the T103C transition creates a new Acil site in the amplified product, which results after digestion, in two fragments, of 25 bp and 23 bp. Amplified DNA products in which the mutation is absent are not cleaved by Acil. Products amplified from patients heterozygous for the mutation should yield fragments of 48 bp, 25 bp and 23 bp, whereas products from patients homozygous for the mutation should yield fragments of 25 bp and 23 bp (Figure 2). Digestion products were electrophoresed through 18% polyacrylamide gels, stained with ethidium bromide, and visualized by ultraviolet irradiation. This restriction enzyme assay was used to test for segregation of the mutation in the MHS family, and to test for the presence of the mutation in unrelated affected and healthy individuals.

The CHCT and serum CK values of the family members are shown in Table 1. The MHS pedigree is shown in Figure 3and is unusual in that the parents, III:7 and III:8, both identified as MHS, are first-degree cousins. No specific myopathic signs were found on histo-enzymologic examination. The CHCT values are presented under four headings; the tension generated in each muscle strip at 2% halothane and 2 mM caffeine, respectively, and the concentration of halothane and caffeine required to generate a threshold tension of 0.2 g. The biopsies from the two homozygote members were the most sensitive of all samples to caffeine. Both had the lowest threshold concentration of caffeine (0.5 mM) and had the strongest contractures at a 2 mM concentration. The two homozygotes and three heterozygotes reached the threshold tension at the lowest amount of halothane used (0.5% v/v). Homozygote IV:4 achieved the highest muscle tension at 2% halothane volume. However the next highest tension of 2.3 g was achieved by the heterozygote IV:11. Homozygote IV:6 had the third highest contracture at 2% halothane. Therefore, the two homozygotes could only be differentiated from other susceptible heterozygous family members by caffeine sensitivity.

Linkage analysis generated a maximal pairwise lod score of 3.7 and 3.26 in favor of linkage to MHS for the D19S220 and D19S223 markers, respectively, at a recombination fraction of 0.00. All affected individuals share a common haplotype; 13–8-2–9 for the D19S191, D19S220, D19S190, and D19S223 markers, respectively (Figure 3). This result indicates that the RYR1 gene is linked to MH in the MHS individuals in this pedigree. Haplotype analysis indicates that the proband, IV:6, and his sibling, IV:4, were homozygous for the mutant allele, having inherited the affected paternal and maternal haplotypes (Figure 3).

A search for the eight known MH mutations was negative. DNA sequencing identified a T to C transition at nucleotide position 103, resulting in the substitution of an arginine for cysteine 35, representing the most N-terminal MHS mutation reported to date in the RYR1 gene (Figure 1).

Mutation analysis in the pedigree shows that the mutant allele is present in the 13–8-2–9 haplotype and segregates fully with the MHS phenotype, generating a lod score of 4.65 in favor of linkage. The amplified products are completely cleaved to 25 bp and 23 bp fragments in the proband IV:6 and his sibling IV:4, confirming that these two individuals are homozygous for the Cys35Arg mutation. The consanguineous parents III:7 and III:8 are heterozygous for the mutation (Figure 2). The mutation was not detected in 200 normal chromosomes, or in 65 unrelated MHS samples.

Linkage analysis has identified two members of a MHS pedigree who have inherited identical affected RYR1 haplotypes, indicating that these individuals are homozygous for a RYR1 mutation. RYR1 cDNA of a known homozygote was cloned and partially sequenced, resulting in the detection of a novel T103C transition, resulting in the substitution of cysteine 35 for an arginine. This is the most N terminal mutation detected, to date. The mutation was not present in 200 normal chromosomes, indicating that the change is not a polymorphism. The exchange was not detected in 65 MH-susceptible individuals and appears to be unique to this particular family. The cysteine 35 residue is otherwise conserved across 8 RYR receptors so far sequenced from 5 species, including human and Drosophila (Figure 4).

This is the first report of a genotyped homozygous proband arising from consanguineous parents. An MHS patient homozygous for a known mutation (Arg614Cys) was reported recently. [7]However, in this pedigree, there are many discrepancies between the CHCT results and haplotypes of markers for the MHS1/RYR1 region, including the C1840T transition:(1) all MHS patients do not have the mutation, casting doubt on the causality of this mutation in this family;(2) the haplotype of the MHS patient reported to be homozygous for the mutation is not compatible with the inheritance of an identical set of alleles from her parents;(3) one false-negative CHCT result must have occurred in this pedigree;(4) the haplotype of the father of the homozygote is reconstructed because the father is deceased. In our pedigree, the haplotypes of the two potential MHS homozygotes are compatible with such an inheritance, especially because their parents are directly related. There are no discrepancies between the CHCT results and the haplotypes of markers for the MH locus. A lod score greater or equal to +3 has been found in this pedigree, strongly suggesting linkage to the RYR1 gene.

The presence of the two MHS RYR1 alleles in the homozygotes clearly influenced the caffeine CHCT results, because it was possible to distinguish heterozygotes from homozygotes by comparing the level of contracture achieved at any caffeine concentration (Table 1). However, no clear differentiation could be achieved using the halothane CHCT results. This outcome may be explained by the fact that halothane and caffeine are known to have different modes of action on calcium release in skeletal muscle cells. Whereas the caffeine results differentiated the homozygotes from the rest of the family, this test alone was not sufficient to detect susceptibility in all heterozygous family members, because one MHE(h) individual was recorded.

Depending on the amino acid substitution and location within the protein primary structure, a mutation in the RYR1 gene can give rise to a range of phenotypes. Some human mutations appear to have no effect on phenotype in the absence of triggering agents. In other cases, the mutation is manifested even in the absence of triggering agents. Certain susceptible patients complain of persistent muscle cramps, with associated elevated blood CK values indicating excessive muscle contractures and damage. [21,22]The presence of the Arg615Cys mutation in swine is associated with muscle hypertrophy. [6]In this case, the mutated calcium channel appears to open for longer than normal periods of time, releasing extra amounts of calcium during the contracture process, which could lead to hypertrophy. [6,23]A more deleterious change to the channel structure might allow the release of even greater amounts of calcium into the myoplasm, which could give rise to many of the characteristic features observed in central core disease cases: loss of mitochondria, Z disk streaming, and muscle atrophy. [11]It has been suggested that the inheritance of homozygous dominant alleles is associated with a more severe phenotype. [24]One MHS patient with a chronic myopathy was reported in a pedigree in which both parents were typed MHS by the CHCT, suggesting that the patient was either a homozygote or a compound heterozygote. [25]Subsequently, homozygosity in this patient was ruled out, and it is likely she has inherited two independent, and not necessarily identical, MHS mutations. The current mutation does not appear to have any affect on phenotype in the absence of halothane or caffeine, because both homozygous patients had no signs of muscle abnormality, and had normal serum CK values. This indicates that the Cys35Arg substitution alters myoplasmic calcium regulation in a distinct manner from other mutations that may be detected in the absence of triggering agents. Further biochemical and electrophysiologic characterization will be necessary to elucidate the mechanistic nature of these mutations on RYR1 channel function and correlation with the observed phenotype in affected MHS individuals.

Individual IV-19 was tested according to the European malignant hyperpyrexia group protocol by Dr. L. Heytens (Antwerp MH diagnostic unit, Belgium), and individual III-12 was tested by Dr. Cozzolino (Naples MH diagnostic unit, Italy.) We are grateful to both of them for supplying the CHCT results.