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A GENETIC TEST 
FOR 
MALIGNANT HYPERTHERMIA 
A dissertation presented to Massey University in partial fulfilment of the 
requirements for the degree of Doctor of Philosophy in biochemistry. 
Rosemary L. Brown 
2000 
ACKNOWLEDGEMENTS 
I would like to extend my sincere gratitude to my principal supervisor, Dr. Kathryn Stowell for 
her enduring encouragement, dedication and reassurance, throughout the (prolonged) term of my 
PhD studies. Thanks also to Prof, John Tweedie for guidance, and enthusiastic assistance with 
computer-related tasks. 
This work would not have been possible without the dedication ofNeil Pollock, (specialist 
anaesthetist, Palmerston North hospital), who organised the collection ofblood and tissue 
samples and provided vital clinical expertise in the field ofMH. I would also like to thank other 
members of the Palmerston North hospital department of Anaesthesia and intensive care, for the 
provision of IVCT data and helpful discussion, particularly Ken Couchman and Mike Hodges. 
Many members of the former Department ofBiochernistry and the current IMBS have provided 
assistance, encouragement and enjoyable diversion along the way. In particular, I am indebted to 
Danielle James for her persistently cheerful dedication of to the task of endless DNA extractions, 
and for keeping tabs on the pedigree data. Much of this work also relied on the efficiency of 
Lorraine Berry, who was in charge the automatic sequencing. I would also like to thank friends 
and colleagues in the Twilight Zone, for friendship and technical advice, especially, Catherine, 
Robyn, Jakki, Bee and Carole. Special thanks to Stan Moore for his dedicated provision of comic 
relief, and likewise, the members of the IMBS touch team, numerous flatmates, and friends from 
the Massey alpine club. I am particularly indebted to Lee Davies for helping to salvage my data, 
and for reformatting and replacing my hard drive (sadly, on more than one occasion). 
I am sincerely grateful to my parents for their support and acceptance throughout the duration of 
my studies, and to Andrew, for his love and continued encouragement during my PhD and for 
abiding the protracted separation associated with the preparation of this thesis. 
TABLE OF CONTENTS 
ABSTRACT ......................................................................................................................................................................................................... i 
ABBREVIATIONS ............................................................................................................................................................................................. ii 
LIST OF FIGURES ............................................................................................................................................................................................ iii 
CHAP'fER ONE: INTRODUCTION ................................................................................................................ 1 
1.1 OVERVIEW OF THE GENETICS AND BIOCHEMISTRY OF MALIGNANT HYPERTHERMIA ........................................................ I 
1.2 CLINICAL FEATURES OF MALIGNANT HYPERTHERMIA ................................................................................................................... 2 
1.3 THE PORCINE MODEL OF MALIGNANT HYPERTHERMIA .................................................................................................................. 3 
1.4 DIAGNOSIS OF MALIGNANT HYPERTHERMIA ...................................................................................................................................... 4 
1.5 MOLECULAR COMPONENTS OF SKELETAL MUSCLE CONTRACTION ........................................................................................... 6 
1.6 THE PATHOPHYSIOLOGY OF MH ............................................................................................................................................................ 10 
1.7 MUSCLE DISORDERS ASSOCIATED WITH MH ........................................ ............................................................................................. l2 
1.8 THE GENETIC BASIS OF MALIGNANT HYPERTHERMIA ................................................................................................................... l3 
1.9 THE RY ANODINE RECEPTOR: STRUCTURE AND FUNCTION .......................................................................................................... 16 
1.10 EXCITATION-CONTRACTION COUPLING .............. ....................................... ............................................................................... ... 21 
1.11 MOLECULAR PREDICTIONS FOR MHS MUTATIONS .................................................................................................................... 23 
1.12 BIOCHEMICAL CHARACTERISATION OFMHS MUTATIONS ..................................................................................................... 24 
1.13 RESEARCH GOALS ................................................................................................................................................................................. 26 
CHAP'fER 1WO: MATERIALS AND METHODS ...................................................................................... 28 
2.1 PURITICATION OF GENOMIC DNA. .......................................................................................................................................................... 28 
2.2 PURITICATION OF HUMAN SKELETAL MUSCLE RNA ................................................................................... ..................................... 29 
2.3 STANDARD PCR PROCEDURES ................................................................................................................................................................. 32 
2.4 PROCEDURES FOR OPTIMISIMG PCR .................................................................................................................................................. , ... 34 
2.5 METHODS OF MUTATION DETECTION .................................................................................................................................................. 35 
2.6 ANALYSIS OF CHROMOSOME 19Q MARKERS ............................................................. ......................................................................... 40 
2.7 GENETIC LINKAGE ANAL YSIS .................................................................................................................................................................. 44 
2.8 SCREENING FOR NOVEL RYRI MUTATIONS BY RT-PCR .................................................................................................................. 46 
2.9 REAGENTS AND CHEMICALS .................................................................................................................................................................... 49 
CHAP'fER THREE: SCREENING FOR PUBLISHED RYRl MUTATIONS ............................................. 50 
3.1 APPROACH TO THE SEARCH FOR REPORTED MH MUT ATIONS ..................................................................................................... 50 
3.2 MUTATION SCREENING BY PCR-RFLP ANALYSIS .............................................................................................................................. 52 
3.3 SCREENING FOR PUBLISHED RYRI MUTATIONS BY DIRECT SEQUENCE ANAL YSIS ............................................................. 69 
CHAP'fER FOUR: GENETIC LINKAGE ANALYSIS OF A LARGE MHS PEDIGREE .......................... 72 
4.1 CH PEDIGREE STRUCTURE ........................................................................................................................................................................ 72 
4.2 PRINCIPLES OF GENETIC LINKAGE ANALYSIS ................................................................................................................................... 73 
4.3 DATA COLLECTION ..................................................................................................................................................................................... 83 
4.4 SEGREGATION OF MHS WITH CHROMOSOME 19Q MARKERS IN THE CH FAMILY ................................................................. 93 
4.5 TWO-POINT LINKAGE ANALYSIS .......................................................................................................................................................... 105 
4.6 MUL TIPOINT LINKAGE ANALYSIS ........................................................................................................................................................ 112 
4.7 DISCUSSION OF RESULTS ........................................................................................................................................................................ 116 
CHAPTER FIVE: 
IDENTIFICATION OF MUTATIONS BY SEQUENCE ANALYSIS OF RYRl cDNA .............................. 122 
5.1 SEARCH FOR A NOVEL RYRI MUTATIONIN THE CH FAMILY ..................................................................................................... l22 
5.2 RT?PCR STRATEGY ..................................................................................................................................................................................... l25 
5.3 RT-PCR METHODOLOGY .......................................................................................................................................................................... 130 
5.4 RESULTS: RT-PCR AND RYRI SEQUENCE ANALYSIS ...................................................................................................................... 132 
5.5 IDENTFICATION OF RYRI MUTATIONS IN OTHER MHS PROBANDS .......................................................................................... !40 
5.6 IDENTIFICATION OF NOVEL RYRI MUTATIONS IN MHS PROBANDS BY SSCP ANALYSIS ................................................. 148 
5.7 OVERVIEW OF RYRI MUTATION ANALYSIS ...................................................................................................................................... l53 
CHAPTER SIX: GENOTYPE I PHENOTYPE RELATIONSHIPS FOR RYRl MUTATIONS ............... 158 
6.1 INVESTIGATING LINKAGE BETWEEN MHS AND T48261... .............................................................................................................. 158 
6.2 THE RELATIONSHIP BETWEEN IVCT THRESHOLDS AND EVIDENCE FOR LINKAGE ............................................................ 160 
6.3 STATISTICAL ANALYSIS OF THE IVCT DATA .................................................................................................................................... l63 
6.4 IDENTIFICATION AND DISCUSSION OF DATA INCONSISTENCIES .............................................................................................. l69 
6.5 FACTORS CONTRIBUTING TO GENOTYPE/PHENOTYPE DISCREPANCIES IN THE CH FAMILY .......................................... 172 
6.6 COMPARISON OF PHENOTYPES ASSOCIATED WITH DIFFERENT RYRI MUTATIONS ........................................................... l76 
6.7 MHS IN ASSOCIATION WITH SUDDEN INFANT DEATH IN THE LARGE MAORI FAMILY ...................................................... l80 
6.8 SUMMARY AND IMPLICATIONS FOR DIAGNOSIS ............................................................................................................................. l82 
6.9 IMPLICATIONS FOR THE STRUCTURE AND FUNCTION OF THE CALCIUM RELEASE CHANNEL ....................................... 189 
6.10 FUilJRE MOLECULAR CHARACTERISATION OF THE NOVEL THR4826ILE MUTATION ................................................. 198 
REFERENCES ................................................................................................................................................. 200 
APPENDICES .................................................................................................................................................. 224 
A I  PRIMERS FOR PCR AND SEQUENCING ................................................................................................................................................... AI 
A2 RYRI CDNA SEQUENCE, PRIMERS AND POL YMORPHISMS ............................................................................................................ A5 
A3 ONLINE RESOURCES: GENETIC MAPS AND MARKERS .................................................................................................................. Al4 
A4 CHROMOSOME !9 Q MAP ......................................................................................................................................................................... Al5 
A5 NZ MHS FAMILIES: SUMMARY OF IVCT AND GENETIC INVESTIGATION ................................................................................ Al7 
?6 CLINICAL CASE REPORTS FOR PATIENTS WITH RYR! MUTATIONS ......................................................................................... Al9 
?7 PATIENT CONSENT FORMS, GENETIC TESTING ............................................................................................................................. A23 
AS STNnSTICAL METHODS AND FORMULAE ........................................................................................................................................ A29 
-?? THE SEGREGATION OF THE T48261 WITH MHS IN THE COMBINED CH FAMILY PEDIGREE ...................................... INSERT 
, . 
ABSTRACT 
Malignant Hyperthermia (MH) is an inherited disorder of skeletal muscle in which an abnormality 
in the regulation of calcium release from internal stores can result in a fatal hypermetabolic 
reaction on exposure to general anesthetics. Mutations in the gene encoding the skeletal muscle 
ryanodine receptor/ calcium release channel (R YR 1) have been linked to MHS in 50% of overseas 
families examined, and at least five additional MH susceptibility loci have since been proposed. 
Current diagnosis of MH in New Zealand relies on the in vitro contracture testing ( IVCT) of 
excised muscle bundles with caffeine and halothane. The genetic basis ofMH in NZ families was 
investigated, with the goal of developing genetic tests to replace the muscle biopsy test. 
A search for previously published RYR1 mutations in susceptible members of 33 NZ MH families 
revealed three RYR1 mutations; Arg163Cys, Gly34 1Arg, and Gly2434Arg, which eo-segregated 
completely with susceptibility to MH (MHS). None of the 1 7  published RYRI mutations were 
detected in a local MHS Maori family in which several anaesthetic deaths have occurred. This is 
the largest characterised MH family in the world. An examination of the segregation of a panel of 
chromosome 1 9q markers with MHS in over 200 members of this family revealed that MHS was 
linked to the RYR1 -flanking markers. This implicated the involvement of a novel RYRI defect . 
The entire 1 5 . 3  kb R YR 1 coding region was combed for mutations by R T-PCR and automatic 
sequence analysis. A novel point mutation was detected that changed threonine 4826 to isoleucine 
in the C-terminal region of the RyR l protein. This mutation was not found in 220 chromosomes 
from the normal population, or in 94 members of the family who had been diagnosed MHN 
(normal) .  A screen for the mutation in 2 1 0  key family members revealed a direct correlation 
between inheritance of the mutation and highly abnormal muscle contracture results in 36 
individuals. 22 MHS individuals lacked the mutation; consequently the false positive rate of the 
IVCT and the possible segregation of at least one additional MHS gene complicated genetic 
linkage analysis. These problems were addressed by investigating increasingly stringent models for 
MH diagnosis .  
Four additional novel R YR 1 mutations were detected in other MHS families investigated by 
sequence analysis of cDNA and genomic DNA, Arg40 1 Cys, Arg2452Trp, Arg2454His and 
His4833Tyr. The detection of the Thr4826IIe and His 4833Tyr mutations established the channel 
domain of the ryanodine receptor as a new MHS domain. Genetic testing for MHS can now be 
applied with caution to predict MH susceptibility in approximately 40 % of at-risk individual in 
NZ, thus reducing the number of patients requiring an expensive and invasive surgical procedure. 
A 
ACRS 
ARMS 
ASO 
bp 
BRL 
cDNA 
Caf. 
CaM 
CFLP 
CHCT 
CK 
CS 
ddNTP 
dH20 
DEPC 
DHPR 
DMD 
DMSO 
dNTP 
ds DNA 
DTT 
ECC 
EDTA 
EEO 
EMHG 
EtBr 
Ha I. 
HE PES 
IP3R 
IVCT 
kb 
KR 
MH 
MBE 
MHN 
MHS 
MPC 
m RNA 
MOPS 
NCE 
PAGE 
PCR 
PMCA 
PSS 
RE 
ABBREVIATIONS 
absorbance 
amplification-created restriction site 
amplification refractory mutation system 
allele-specific oligonucleotide 
base pair 
Bethseda research laboratories 
complementary DNA 
Caffeine 
calmodulin 
cleavase fragment length polymorphism 
caffeine/halothane contracture test (North American protocol) 
Creatine phosphokinase 
chromosome 
2' ,3 '-dideoxyribonucleotide 5 '-triphosphate 
distilled water 
diethylpyrocarbonate 
dihydropyridine receptor 
Duchenne muscular dystrophy 
dimethyl sulphoxide 
deoxyribonucleoside 5 '-triphosphate 
double-stranded DNA 
dithiothreitol 
Excitation-contraction coupling 
ethylene diamine tetra-acetate 
electroendosmosis 
European Malignant Hyperthermia Group 
ethidium Bromide 
halothane 
N-[2-hydroxyethyl]piperazine-N'-[ 4-butanesulphonic acid] 
inositol 1 ,  4 ,5-trisphosphate receptor 
in vitro contracture test (European protocol) 
kilo base 
Krebs-Ringer 
malignant hyperthermia 
malignant hyperthermia equivocal diagnosis 
malignant hyperthermia negative diagnosis 
malignant hyperthermia susceptible diagnosis 
magnetic particle concentrator 
Messenger RNA 
3-[N-morpholino ]propanesulphonic acid 
Na+1Ca+2 exchanger 
polyacylamide gel electrophoresis 
polymerase chain reaction 
plasma membrane calcium ATPase 
porcine stress syndrome 
restriction endonuclease 
11 
RFLP 
RNase 
rRNA 
RT 
RT-PCR 
RYR 
RyR 
SDS 
SERCA 
ss DNA 
SSCP 
SR 
TAE 
Taq 
TBE 
TC 
TE 
TEMED 
Tm 
restriction fragment length polymorphism 
ribonuclease 
ribosomal RNA 
reverse transcriptase 
reverse transcription-polymerase chain reaction 
ryanodine receptor (gene) 
ryanodine receptor (protein) 
sodium dodecyl sulphate 
Sarco( en do )plasmic reticulum A TPase 
single-stranded DNA 
single-stranded conformation polymorphism 
sarcoplasmic reticulum 
Tris-acetate-EDTA buffer 
Thermus aquaticus 
Tris-borate-EDT A buffer 
Terminal cisternae 
Tris-EDTA buffer 
N,N,N' ,N' -tetramethylenediamine 
melting temperature 
lll 
LIST OF FIGURES 
FIGURE 1-1 ION CHANNELS INVOLVED IN SKELETAL MUSCLE ECC COUPLING ....................... ..................... 9 
FIGURE 1-2 A PROPOSED :MECHANISM FOR THE INDUCTION OF HUMAN AND PORCINE MH .................... 11 
FIGURE 1-3 TIIREE-DI:MENSIONAL STRUCTURAL RECONSTRUCTION OF RYR . ......... ................................ 18 
FIGURE 1-4 PROPOSED INTERACTIONS BETWEEN TilE DHPR a1 SUBUNIT AND RYR1 ................................ 22 
FIGURE 1-5 LOCATION OF RYR1 MHS AND CCDMUTATIONS ...................................... ................................... 24 
FIGURE 3-1 EFFECTS OF [MG+2] ON PCR AMPLIFICATION YIELD AND SPECIFICITY .. ................. . . ... 53 
FIGURE 3-2 SCREENING FOR PUBLISHED RYR1 MUTATIONS BY PCR-RFLP.................................. .. 54 
FIGURE 3-3 DETECTION OF THE C487T (ARG163CYS) MUTATION BY PCR-RFLP ........... . ..... 55 
FIGURE 3-4 MANUAL SEQUENCE ANALYSIS OF TilE C487T (ARG163CYS) MUTATION ............................... . 56 
FIGURE 3-5 AUTOMATED SEQUENCE ANALYSIS OF THE C487T (ARGI63CYS) MUTATION ........ ................ 56 
FIGURE 3-6 SEGREGATION TilE ARG163CYS (C487T) MUTATION IN FAMILY 35 ..................... ................... 58 
FIGURE 3-7 PCR-RFLPDETECTION OF TilE GL Y2434ARG MUTATION IN FAMILY 5 ....................................... 59 
FIGURE 3-8 CONFIRMATION OF TilE GLY2434ARG MUTATION BY AUTOMATIC DNA SEQUENCE ANALYSIS 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  00 
FIGURE 3-9 SEGREGATION OF THE ARG3434CYS MUTATION IN FAMILY 24 .................................................. 62 
FIGURE 3-10 PCR-RFLP SCREEN FOR ARG2458IDS/CYS AND ARG2163CYS MUTATIONS ........... .................. . 64 
FIGURE 3-11 CORRECTION OF SEQUENCE ERRORS IN INTRON 46 ................................................ ................ ... 65 
FIGURE 3-12 AMPLIFICATION CREATED RESTRICTION SITES TO SCREEN FOR TYR522SER . ...................... 67 
FIGURE 3-13 DEMONSTRATION OF ACRS SCREEN FOR TilE TYR522SER MUTATION.............. .. ................ 68 
FIGURE 3-14 DIRECT MANUAL SEQUENCE ANALYSIS OF PUBLISHED RYRl MUTATION SITES ................... 70 
FIGURE 4-1 TilE EFFECT OF THIRD GENERATION DATA ON PHASE DETERMINATION ............................... 71 
FIGURE 4-2 SEGREGATION OF RYRl RFLP MARKERS IN NZ :MEMBERS OF FAMILY 94 .................. ... ..... 84 
FIGURE 4-3A RFLP ANALYSIS OF TilE SER2862 POL YMORPIDSM WTIB CFO L . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . .  85 
FIGURE 4-3B PCR-RFLP ANALYSIS OF RYRl POL YMORPIDSMS WTIB TAQ I AND FOK I . . . .. . . . . . . . .. . . .. .. . . . ... . . .. . .  86 
FIGURE 4-4 LOCATION OF RYRl MARKERS ............................................................. .......................................... 87 
FIGURE 4-5 ANALYSIS OF THE R YR-CA DINUCLEOTIDE REPEAT MARKER IN THE CHEA PEDIGREE .. .... 90 
FIGURE 4-6 ANALYSIS OF TilE Dl9S220 DINUCLEOTIDE REPEAT MARKER IN THE CHES PEDIGREE .... ... 90 
FIGURE 4-7 ANALYSIS OF TilE Dl9S47 DINUCLEOTIDE REPEAT MARKER IN THE CHEH PEDIGREE ..... .... 91 
FIGURE 4-8 ANALYSIS OF TilE Dl9S47 DINUCLEOTIDE REPEAT MARKER IN THE CHES PEDIGREE ....... .. 91 
FIGURE 4-9 ANALYSIS OF THE Dl9S 190 TRINUCLEOTIDE REPEAT MARKER IN THE CHEA PEDIGREE ...... 92 
FIGURE 4-10 SEGREGATION OF CHROMOSOME 19Q MARKERS WTIB MHS IN THE CHEHPEDIGREE ........ .. 94 
FIGURE 4-11 SEGREGATION OF CHROMOSOME 19Q MARKERS WTIB MHS IN THE CHEH PEDIGREE .......... 96 
FIGURE 4-12 SEGREGATION OF CHROMOSOME 19Q MARKERS WTIB MHS IN THE CH2 PEDIGREE ............ 98 
FIGURE 4-13 SEGREGATION OF CHROMOSOME 19Q MARKERS WTIB MHS IN THE CH3 PEDIGREE ........... 101 
FIGURE 4-13 ANALYSIS OF THE TAQ I MARKER IN TilE FAMILY OF A SIDS CHILD ....... ........................... .. 103 
FIGURE 4-14 ASSOCIATION OF THE MHS-LINKED HAPLOTYPE WTIB SUDDEN UNEXPLAINED DEATH . . . 104 
FIGURE 4-15 INFLUENCE OF THE PHENOCOPY PARA:METER ON SUPPORT FOR LINKAGE TO RYR1 ......... 107 
FIGURE 4-17 MULTIPOINT LINKAGE ANALYSIS OF TilE CHI PEDIGREE ......................................................... 114 
FIGURE 4-18 MULTIPOINT LINKAGE ANALYSIS OF TilE CH2 PEDIGREE ......................................................... 115 
IV 
FIGURE 4-19 MULTIPOINT LINKAGE ANALYSES OF DIE CH3 PEDIGREE ........................................................ 115 
FIGURE 5-1 SUBJECTS CHOSEN FOR Ml.ITATION SCREEN ............................................................................... 124 
FIGURE 5-2 RNA GEL ELECTROPHORESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  125 
FIGURE 5-3 STRATEGY FOR THE AMPLIFICATION OF RYR1 CDNA ............................................................... 127 
FIGURE 5-4 DETECTION OF A PROBABLE UV-INDUCED MUTATION AMPLIFIED lN SECONDARY PCR .... 133 
FIGURE 5-5 PCR-ASSOCIATED DELETIONS lN RYR1 5' UTR AMPLIFIED FROM CDNA ................................. 135 
FIGURE 5-6 DETECTION OF NOVEL RYR1 POL YMORPHISMS BY SEQUENCE ANALYSIS OF CDNA .......... 136 
FIGURE 5-7 SEQUENCE ANALYSIS OF THE T4826I MUTATION ........................................................................ 138 
FIGURE 5-8 SSCP DETECTION OF THE THR4826ILE MUTATION ...................................................................... 139 
FIGURE 5-9 PCR PRODUCTS ENCOMPASSlNG RYR1 MUTATION-RICH REGIONS ......................................... 140 
FIGURE 5-10 SSCP DETECTION OF THE ARG2452TRP MUTATION 1N FAMILY 36 ............................................ 141 
FIGURE 5-11 IDENTIFICATION OF THE ARG401CYS MUTATION ....................................................................... 143 
FIGURE 5-12 SEGREGATION OF THEARG401CYS MUTATION lN FAMILY 70 .................................................. 143 
FIGURE 5-13 SSCP ANALYSIS OF THE G1021A (GLY341ARG) MUTATION IN FAMILY 24 ............................... 145 
FIGURE 5-14 DETECTION OF THE G1021A (GLY341ARG) MUTATION .............................................................. 146 
FIGURE 5-15 SEGREGATION OF THE GL Y341ARG MUTATION WITH MH lN FAMILY 24 ................................ 147 
FIGURE 5-16 SSCP DETECTION OF THE HIS4833TYR MUTATION ...................................................................... 149 
FIGURE 5-17 CHARACTERISATION OF THE HIS4833TYR MUTATION ............................................................... 149 
FIGURE 5-18 PROXIMITY OF NOVEL C-TERMINAL RYR1 MUTATIONS ........................................................... 150 
FIGURE 5-19 SEGREGATION OF THE HIS4833TYR MUTATION lN FAMILY 1... ................................................. 150 
FIGURE 5-20 IDENTIFICATION OF THE ARG2454HIS AND ARG2452TRP MUTATIONS .................................... 152 
FIGURE 6-1 DISTRIBUTION OF IVCT DATA FOR SUBJECTS WITH AND WITHOUT THR4826ILE ............... 164 
FIGURE 6-2 DOSE RESPONSE CURVES FOR PATIENTS WITH AND WITHOUT THR4826ILE ...................... 166 
FIGURE 6-3 DISTRIBUTION OF IVCT DATA ........................................................................................................ 167 
FIGURE 6-4 CORRELATION BETWEEN IVCT RESPONSE AND THE THR4826ILE MUTATION ....................... l71 
FIGURE 6-5 DISCORDANCE BETWEEN THR4826ILE AND MHS IN THE CHES FAMILY ............................... 174 
FIGtJR? 6-6 COMPARISON OF IVCT DATA FOR DIFFERENT RYR1 MUTATIONS ........................................... 178 
FIGURE 6-7 CONSERVATION OF AMINO ACIDS SURROUNDING MHS MUTATIONS ...................................... 91 
FIGURE 6-8 GLUTAMlNE 3756 IN THE HUMAN RYR1 SEQUENCE IS NOT CONSERVED .............................. 192 
FIGURE 6-9 CONSERVATION OF THR4826 AND HIS4833 AMONG RYR ISOFORMS ........................................ 193 
FIGURE 6-10 RELATIVE POSffiONS OF RYR1 CHANNEL MUTATIONS ACCORDING TO MODELS FOR RYR1 
TRANSMEMBRANE TOPOLOGY ...................................................................................................... 196 
V