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Novel Variants Near the Central Domain of RYR1 in Two Malignant Hyperthermia-Susceptible Families from Taiwan

Chen, Po-Liang, MD*; Chang, Yao-Wen, MS; Chen, Chun-Yu, MD; Hsiang, Yung-Chih, MS; Day, Yuan-Ji, MD, PhD†‡§

doi: 10.1213/ane.0b013e3181ae94c7
General Article: Brief Report
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BACKGROUND: Our primary objective was to detect malignant hyperthermia (MH)-susceptible persons and thereby prevent MH episodes. We identified variants in the ryanodine receptor isoform 1 using molecular pedigree analysis.

METHODS: Nineteen exons covering major hotspots were chosen for the primary screening by polymerase chain reaction, denaturing high performance liquid chromatography, and confirmed by direct sequencing.

RESULTS: Three novel variants involving amino acid changes were identified in two unrelated families as Met2698Arg, Glu2724Lys in exon 51 and Leu2785Val in exon 53.

CONCLUSIONS: Three novel ryanodine receptor isoform 1 variants located either near or within the central domain might predispose carriers to MH.

From the *Department of Anesthesiology, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan, ROC; †Department of Medical Research, Transgenic & Molecular Immunogenetics Laboratory, Chang Gung Memorial Hospital; ‡Department of Anesthesiology, Linko Chang Gung Memorial Hospital; and §Department of Medicine, Graduate Institute of Clinical Medical Science, Chang Gung University, Taoyuan, Taiwan, ROC.

Accepted for publication April 10, 2009.

Supported by Chang Gung Memorial Hospital Research Grant CMRPG330034.

Address correspondence and reprint requests to Yuan-Ji Day, MD, PhD, Department of Anesthesiology, Transgenic & Molecular Immunogenetics Laboratory, Chang Gung Memorial Hospital, No. 5, Fushin St., 333 Gueishang Taoyuan, Taiwan, ROC. Address e-mail to yd3j.uva@msa.hinet.net.

The “gold standard” in vitro contracture test and caffeine and halothane contracture test for diagnosis of malignant hyperthermia (MH)1–3 is invasive, expensive, and inconvenient. However, the modified guidelines after 2000 make the diagnosis of MH more accessible for patients and families who lack access to the gold standard tests.4–6 These guidelines include genetic screening of ryanodine receptor isoform 1 (RYR1) as an alternative method for identifying probands as MH-susceptible (MHS) after clinical evidence of MH susceptibility. The accumulating data reveal that mutations of RYR1 found in MHS people are mostly single mutations located in three hotspots.7–9 However, there could be other undetected mutations outside these three regions, because screening the entire RYR1 is not routinely performed. A large scale analysis might find other mutations that could contribute to the aberrant physiological behavior of RYR1.

We initiated a molecular genetic screening program for MH probands in our hospital. A well-documented mutation, located at the central domain, was detected in one family (Family A), and three novel variants of RYR1 near the vicinity of the central domain were detected in two unrelated families (Families B and C).

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CLINICAL COURSE

The clinical course of the MH patients in Families B and C with novel variants of RYR1 are the following.

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MHS Patient in Family B

A 70-yr-old man weighing 61 kg underwent elective gastric surgery under general anesthesia. Anesthesia was induced with IV thiamylal, fentanyl, and cisatracurium, and maintained with sevoflurane and O2. The minute ventilation was set to 6.2 L/min. Generalized muscular rigidity developed about half an hour after tracheal intubation. This was accompanied by tachycardia (up to 120 bpm), increase of ETco2 (35–65 mm Hg), and elevated body temperature (36.5°C up to >39.0°C). Arterial blood gas at this time was pH 7.28, Paco2 66 mm Hg, Pao2 342 mm Hg, HCO3 24 meq/L, base excess −4.3 mmol/L, and SaO2 99%. The concurrent biochemistry data were Na+ 133 meq/L, K+ 5.5 meq/L, Cl 101 meq/L, Ca2+ 9.9 mg/dL, blood glucose 176 mg/dL, and lactate 4.2 mg/dL. We instituted our MH protocol, which included administration of IV dantrolene and discontinuation of the sevoflurane. The clinical signs subsided subsequently, except brownish urine that persisted for several days. The levels of serum creatinine kinase and serum myoglobin peaked at 11000 U/L and 1750 μg/L, respectively.

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MHS Patient in Family C

An 83-yr-old man age weighing 58 kg underwent elective vertebroplasty of his lumbar spine under general anesthesia. Anesthesia was induced with IV thiamylal, fentanyl, and cisatracurium, and maintained with desflurane and O2. The minute ventilation was set to 6 L/min. After patient positioning, his heart rate increased to 115 bpm, ETco2 increased to 60 mm Hg, and body temperature increased to 38.5°C. His lungs were manually hyperventilated, but airway resistance was normal and hyperventilation failed to reduce the ETco2. Arterial blood gas at that time was pH 7.2, Paco2 63 mm Hg, Pao2 311 mm Hg, HCO3 20.8 meq/L, base excess −8.3 mmol/L, and SaO2 98%. The biochemistry data were Na+ 130 meq/L, K+ 6.5 meq/L, Cl 98 meq/L, Ca2+ 9.3 mg/dL, blood glucose 192 mg/dL, and lactate 7.6 mg/dL. We instituted our MH protocol, which included administration of IV dantrolene and discontinuation of the desflurane. The clinical signs gradually subsided. The levels of serum creatinine kinase and serum myoglobin peaked at 8000 U/L and 1200 μg/L, respectively.

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METHODS

The pedigree study was approved by our local ethics committee. Written informed consent was obtained from 31 people before the study, which included 3 MHS patients with a Larach score over 50,10 19 blood-related families, and 9 normal volunteers. The genomic DNA of each individual was extracted from 5 mL blood drawn by using “Gentra Puregene Blood Kit” (QIAGEN®). Primers for screening of 19 exons covering most reported RYR1 hotspots were designed. The exons were first amplified by polymerase chain reaction (PCR) programmed on a thermo-cycler (MJ Research-BioRad; Watertown, MA), gel electrophoresis, and the clean PCR products were subsequently screened by denaturing high performance liquid chromatography (DHPLC, Wave System 3500HT and Wave Maker software, Transgenomic®) for possible heteroduplex formation. Samples with positive findings were further confirmed by direct sequence. The ryanodine receptor-mediated calcium release in human B lymphocytes was detected by Hitachi F4500 fluorescence spectrophotometer under continuous stirring (excitation at 340/380 nm and emission at 510 nm, primers and protocols available on request).11–13

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RESULTS

The heteroduplex formations with early elution were observed at exon 14-15 and 46 in Family A (Fig. 1A) and at exon 51-53 in both Families B and C (Fig. 2A). The results of direct sequencing of PCR products with heteroduplex formation confirmed that variants/mutations at exon 46, 51, 53 correlated well with heteroduplex formation observed by DHPLC (Figs. 1B and 2B). The direct sequencing data also demonstrated that these three linked variants at exon 51, 53 were harvested in two unrelated families, which implies that variants detected at exon 51 and 53 may relate specifically to populations in Taiwan (Figs. 4B and C). No mutation was found in exon 14, 15, and 52 even though the heteroduplex formations were detected. Analysis of the detected variant/mutation sites demonstrated that all the variants/mutations were characterized with encoded amino acid switching, such as Arg2458His (exon 46), Met2698Arg and Glu2724Lys (exon 51), and Leu2785Val (exon 53). All four variants/mutations are either located within or near the vicinity of the central domain (exon 39-46) of RYR1. Moreover, the single mutation detected in Family A was located in the central domain (exon 46: Arg2458His). However, the three variants segregated together and detected in both Families B and C were located near the vicinity of the central domain (exon 51: Met2698Arg and Glu2724Lys, exon 53: Leu2785Val). No null or nonsense mutation was observed in these variant sites, suggesting that these variants/mutations may switch the amino acid and alter the protein structure, thus subsequently imposing aberrant physiological behavior on the ryanodine receptor. These variants/mutations rendered an increased sensitivity of B lymphocyte calcium release to RYR1 agonist 4-Cmc (Figs. 3A and B).

Figure 1.

Figure 1.

Figure 2.

Figure 2.

Figure 4.

Figure 4.

Figure 3.

Figure 3.

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DISCUSSION

This study has identified three novel inherited DNA variants within the RYR1 gene in two unrelated families. These variants caused encoded protein switching, which possibly associate with alteration of the ryanodine protein structure. Their central domain location might enable minor structural modification and an aberrant response of the ryanodine receptor, resulting in a leaky Ca2+ channel.14,15 Though the significance of variants in MH is uncertain because of lack of contracture studies, both the structural and positional effects of these three mutations may legitimately be considered possible candidates for MH susceptibility.

Some specific mutations of RYR1 have been demonstrated to have a geographic distribution, such as Gly341Arg and Gly2434Arg in the United Kingdom, Arg614Cys and Thr2206Met in Germany, and Val2168Met in Switzerland.7 These mutations are seldom observed in Asia, although Gly341Arg has been reported in Japan with a low incidence.16 Together with the previously reported novel mutation (Tyr522Cys on exon 14) in Taiwan,17 Chinese patients may have different RYR1 mutations associated with MH. Our finding of a common mutation at exon 46 (Arg2458His) in one family suggests that not all RYR1 have limited geographical distributions.8,16,18

Our results of variants in the vicinity of the central domain indicate a vulnerable region relevant to the regulation of calcium release and calmodulin sensitivity,14,15 thus implying that a single amino acid switch may alter function of the ryanodine receptor. However, we cannot exclude the possibility that there might be variants in other exons of the RYR1 that are critical to aberrant ryanodine receptor function. It is important to develop a simplified method to screen all 106 exons of RYR1 for each proband in the future investigation of MH.

In conclusion, in patients with clinical evidence of MH we identified unique variants of RYR1. This highlights the importance of examining all 106 exons of RYR1 when screening MH probands. The causative relationship between MH mutations of RYR1 and the location of their affected exon in different populations is open for further investigation.

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ACKNOWLEDGMENTS

The authors thank Lin, Chih-Chung, MD, PhD (Associate Professor, Department of Anesthesiology, Linko Chang Gung Memorial Hospital), Yang, Min-Wen, MD (Associate Professor, Department of Anesthesiology, Linko Chang Gung Memorial Hospital), Wong, Chung-Hang, MD (Associate Professor, Department of Anesthesiology, Chiayi Chang Gung Memorial Hospital) for invaluable assistance.

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