A genome wide search for susceptibility loci in three European malignant hyperthermia pedigrees+
A genome wide search for susceptibility loci in three European malignant hyperthermia pedigrees + Rachel L. Robinson1,*, Nicole Monnier2, Werner Wolz3, Martin Jung4, André Reis4, Gudrun Nuernberg4, Julie L. Curran5,6, Koen Monsieurs7,8, Paul Stieglitz9, Heytens8, Ruth Fricker10, Christine van Broeckhoven7, Thomas Deufel11, Phil M. Hopkins5, Joel Lunardi2 and Clemens R. Mueller3
1Department of Biology, University of Leeds, Leeds LS2 9JT, UK, 2BECP-EA 2019, Departement de Biologie Moleculaire et Structurale, CEA Grenoble et UJF, F-38054 Grenoble cedex 09, France, 3Institut für Humangenetik der Universität Würzburg, Biozentrum, D-97074 Würzburg, Germany, 4Microsatelliten-Labor, Max-Delbrück-Centrum, D-13122 Berlin, Germany, 5Academic Unit of Anaesthesia, St James University Hospital, Leeds LS9 7TF, UK, 6Molecular Medicine Unit, University of Leeds, St James University Hospital, Leeds LS9 7TF, UK, 7Laboratory of Neurogenetics, Born-Bunge Foundation, University of Antwerp, Department of Biochemistry, Antwerpen, Belgium, 8Department of Intensive Care, University Hospital Antwerp, Antwerp, Belgium, 9Departement Anesthésie- Reanimation 1, CHU Grenoble, 217X, F-38043 Grenoble cedex, France, 10Abteilung Allg. Anaesthesie und Intensivmedizin B, AKH, A-1090 Wien, Austria and 11Institut für Klinische Chemie und Laboratoriumsdiagnostik, Klinikum der Universität Jena, D-07740 Jena, Germany
Received January 23, 1997;Revised and Accepted March 12, 1997
Malignant hyperthermia (MH) is an autosomal dominant disorder which is potentially lethal in susceptible individuals on exposure to commonly used inhalational anaesthetics and depolarising muscle relaxants. Crises reflect the consequences of disturbed skeletal muscle calcium homeostasis. Susceptibility was first localised to chromosome 19q13.1 and the skeletal muscle ryanodine receptor, RYR1 (the calcium release channel of the sarcoplasmic reticulum). Defects in this gene have been identified which cosegregate with the MHS phenotype and evidence as to their potential causal roles has accumulated. MH has, however, been shown to be genetically heterogeneous, additional loci on chromosomes 3q, 17q and 7q being proposed. Pedigrees remain in Europe where linkage status is still unclear. In a collaborative search of the human genome conducted with three pedigrees whose disease status was classified according to the European IVCT protocol we have evidence to suggest that at least two further loci exist for MH susceptibility. One of these locates to chromosome 1q, the site of a candidate gene, CACNL1A3, encoding the [alpha]-subunit of the dihydropyridine receptor. The second region resides on chromosome 5p to where no known candidate has been mapped to date. The third family exhibited inconclusive results which suggests the existence of at least one other locus. This study adds to the evidence for considerable genetic heterogeneity in MH and will provide a route to further our understanding of the molecular pathology of the condition.
Malignant hyperthermia (MH) is a life threatening disorder triggered in susceptible individuals on exposure to commonly used inhalational anaesthetics, e.g. halothane and the depolarising muscle relaxant suxamethonium (succinyl choline). From clinical data, susceptibility to MH in humans is inherited as an autosomal dominant condition (1 ), the frequency in the population being estimated at 1:10 000 (2 ).
Patients suffering an MH crisis develop masseter or generalised skeletal muscle rigidity, rising body temperature, acidosis, hypoxia and rhabdomyolysis. At present the only reliable means to determine patient susceptibility is by an invasive in vitro contracture test (IVCT), whereupon the contracture responses of viable muscle tissue to in vitro exposure to halothane and caffeine, respectively, are assessed (3 ).
According to the responses to the standardised IVCT protocol of the European MH group, patients are assigned the status of MH susceptible (MHS), MH normal (MHN) or MH equivocal (MHE). The last category encompasses patients who react abnormally to one but not both of the triggering agents used, MHE(h) positive response to halothane and MHE(c) positive response to caffeine. Both the MHS and MHE groups are regarded as clinically at risk for MH, although no definite diagnosis can be made for MHE individuals. It is accepted that the MHE group incorporates some false positive test results. This category is consequently regarded as `phenotype unknown' in linkage analyses in order to permit maximum specificity for differentiation of MHS/MHN phenotypes. False positive assignments would confound such studies.
Using a LINKAGE based package, MSIM (23 ,24 ) computed the average and maximum two-point lod scores for each family over 500 replicates using an anonymous marker with five alleles, heterozygosity of 0.8 and at varying recombination fractions. The following max lods were to be expected at a recombination fraction of [theta] = 0.05: pedigree VER: 4.46, pedigree AN2: 2.94, pedigree WZ99: 4.98. The program also generated data concerning the number of expected maximum lod scores greater than a given constant (1, 1.5, 2, 2.5, 3.0) which gave an indication of the frequency of possible spurious associations (data not shown).
Segregation of chromosome 1 markers with MHS phenotype. The pattern of inheritance of alleles for PCR based microsatellites on the long arm of chromosome 1 are shown in Figure 1 . One haplotype, 4-2-3-4, segregates with the MHS phenotype throughout the entire pedigree. This interval is bound by proximal and distal recombination events at markers D1S422 and D1S419, which delimit the candidate region to 30 cM. Within this region, 1q32, the MH candidate CACNL1A3 encoding the [alpha]-subunit of DHPR has been previously mapped (25 ). The marker CACNL1A3 (15 ) is intragenic to this candidate and segregates exclusively with the MHS phenotype (Fig. 1 ). Linkage analysis. Two-point lod scores calculated between the MHS trait and genetic markers from 1q are shown in Table 1 . Several markers fail to show maximum lod scores at [theta] = 0, indicating reduced informativity and/or recombination events. The intragenic marker to CACNL1A3, being highly informative generated a maximum lod score at [theta] = 0 of +4.38. This was corroborated by map specific multipoint linkage calculations (Fig. 3 ). In light of the positive lod scores obtained using a marker intragenic to an actual MH candidate gene and the clear segregation of haplotypes, remaining chromosomes were not fully investigated for linkage. Although this means exclusion data is incomplete for other regions, the lod of +4.38 compared well to the theoretical maximum of +4.46 obtained by MSIM calculations.
The complete genomic search was conducted with this family. The structure of this pedigree limited the maximum estimated lod score attainable with a marker of five alleles at a recombination fraction of 0.05 to +2.94 (MSIM calculation). In the event of markers/individuals showing reduced informativity, intermediate lod scores were obtained. As a direct consequence, it was difficult to achieve convincing exclusion data (lod of -2) for many regions of the genome as well as, of course, significantly positive lod scores (>3). Despite this limitation a positive region was localised to chromosome 5p.Segregation of chromosome 5 markers with MHS phenotype. Figure 2 shows the segregation of alleles for markers specific to the short arm of chromosome 5. One haplotype 1-4-3-1-3-5, spanning a region of 29 cM, cosegregates with the MHS phenotype throughout the pedigree with the exception of MHS individual 105 which is fully recombinant for the entire `high risk' haplotype. On review of the clinical data for this individual, although positive contractures were obtained (>0.2 g at threshold levels of halothane and caffeine [2%/2 mM]), only one out of three caffeine tests and one out of three halothane tests conducted yielded such results (Table 2 ). The fact that only two out of six tests were positive with borderline values is a rare occurrence in clinical studies from this laboratory and could indicate the possibility of a false positive IVCT result. There was no significant evidence of segregation with markers from alternative regions (data not shown).
Linkage analysis. Two-point lod scores between the MHS trait and genetic markers are presented in Table 3 with multipoint analyses presented in Figure 4 . None of the two-point lod scores exceeds the significance level of +3, due to the presence of a single recombinant individual. If, however, the phenotype of individual 105 were to be reassigned as MHE for linkage analysis, which serves to limit the risks associated with incorporation of a borderline classification, a lod score close to that predicted by MSIM calculations would be achieved. After considering the limited power of the pedigree structure, the consistency of lod scores obtained for this region, and the exclusion data of most other markers tested, the mapping of a novel MH locus to chromosome 5 seems well supported despite the single recombinant individual.
The results described from a genome wide search in three large European MH pedigrees with status classified according to the European IVCT protocol, provide evidence for the existence of a novel MH susceptibility locus on chromosome 1q31-q32 and suggestive evidence for a further locus on chromosome 5p. These findings further substantiate the occurrence of genetic heterogeneity within this disorder adding to the current loci identified in European pedigrees on chromosomes 19q, 3q and 7q (EMHG unpublished results; 18 ,21 ).
Studies on the Grenoble family, VER, suggest a novel candidate locus for MH. An intragenic marker to the gene encoding the [alpha] subunit of DHPR (CACNL1A3) shows significantly positive results which complement lod scores obtained with additional markers typed in this region. Although the positive region spans a 30 cM interval, the lod score obtained with the intragenic marker strongly suggests a role of CACNL1A3 in the pathology of MH in this family.
In skeletal muscle excitation-contraction (E-C) coupling, the progression of T-tubule depolarisation leading to SR calcium release is presumed to be mediated by the DHPR (L-type voltage sensor at the T-tubule) and the ryanodine receptor (at the SR). A mechanical E-C coupling model proposes that a T-tubule depolarisation-induced conformational change in the DHPR is sensed by RYR via a direct physical interaction between the two receptors resulting in the opening of RYR, release of calcium from the SR and muscle contraction (26 ,27 ). The five protein subunits of DHPR, encoded by five separate genes, have therefore been considered as plausible candidates for genetic defects in MH (21 ). So far, a single MH pedigree has been linked to the gene for the [alpha]2/[delta]-subunit on chromosome 7q (18 ). Work is in progress to identify potential causative mutations in the 5.6 kb transcript of the [alpha] subunit of the VER family.
The Belgian family, AN2, gave positive lod scores for an extended haplotype on chromosome 5p with exclusion of almost all remaining chromosomal regions (data not shown). Results were, however, confounded by the presence of a `fully recombinant' MHS individual, 105. This, in theory, could be due to errors in marker genotyping, errors in phenotyping (the IVCT) or because the genetic model of MH as a single gene disorder is wrong. Errors in marker typing appear highly unlikely since an extended contiguous haplotype of chromosome 5p shows positive lod scores. However, the IVCT results of the single recombinant individual differ significantly from those of the other MHS members of the family. As can be seen from the IVCT details in Table 2 , out of six individual muscle bundles tested, only one each was weakly positive for caffeine and halothane, respectively. We therefore favour a phenotyping error as the most likely explanation. To account for the possibility that more than one gene might contribute to the MH phenotype in this family, we have varied the disease allele frequency, thus allowing for independent MHS alleles to be contributed by the unrelated spouses. Even at a frequency as high as 1 in 100, the lods did not change significantly (data not shown). The fact that the entire genome search failed to pinpoint any other region with comparable results further implicates the 29 cM region identified in the aetiology of MH in this family. This leaves the task of identification of a candidate gene a formidable one. Positional cloning requires high resolution mapping (within 1 cM) to reduce the search to a tractable region, an unrealistic possibility in this project. Future work will consequently be dependent upon the systematic evaluation of known genes within this region and the investigation of mapped sequence tagged sites. This in itself poses a challenging but more manageable prospect (28 ).
This study has highlighted the problems faced in the genetic analysis of MH pedigrees. Although recognised as a heterogeneous disorder, a preponderance (~50%) of pedigrees appear to be linked to RYR1 on 19q (EMHG, unpublished results). The contribution of other loci, to date, has been limited to single pedigrees, e.g. linkages demonstrated to 3q in one German family (21 ) and 7q in another German pedigree (18 ). In response to the heterogeneity observed in MH, analysis has been limited to single large pedigrees, the pooling of family data in this case being inappropriate and invalid. This restricts the ability to utilise recombination events observed in different pedigrees to refine the boundaries of a linked interval to a realistic size (29 ).
Linkage studies in MH are also complicated by the problem of ascertaining the MHS phenotype. As a diagnostic test for clinical purposes and for accurate genetic analysis, it is important to distinguish between normal and susceptible populations as precisely as possible. Work is ongoing within the EMHG to refine this procedure and achieve the maximum standardisation of protocols, laboratory methods and interpretation of results between centres (EMHG, unpublished work). The MHE classification recognises an inevitable overlap of IVCT test results between normal and abnormal populations and directly provides a means to standardise results between laboratories. From a clinical perspective such individuals are treated as at risk. From a geneticist's viewpoint their exclusion from genetic analysis although reducing the statistical power of pedigrees, greatly improves the validity of the MHN/MHS assigned genotypes by maximising IVCT specificity. In fact, previous linkage studies with MH pedigrees have shown that ~50% of MHE cases inherit high risk haplotypes (EMHG, unpublished results). Consequently, the exclusion of these individuals from linkage studies is paramount as the risk of reaching false positive linkage would be considerable, e.g. MHE individuals 24 and 21 from VER inherit high risk haplotypes. In both cases the classification of these patients as MHS would have considerable effects on linkage results.
This study has mapped an additional MH susceptibility locus, to a candidate gene, CACLN1A3 on 1q and suggests the involvement of another locus on chromosomes 5p. This adds to the already substantial degree of heterogeneity reported in MH pedigrees. An additional pedigree included in the genome wide search failed to show linkage to chromosomes 19q, 17q, 7q, 3q, 1q and 5p which infers the existence of at least one further MH susceptibility locus. The continuously unfolding pattern of heterogeneity in European MH pedigrees implicates the involvement of several genes in the aetiology of this phenotypically homogeneous disorder. This implies that a multitude of mutations in different genes disrupt, at the molecular level, the normal processes of muscle calcium homeostasis and E-C coupling in susceptible individuals on application of a suitable trigger. Future MH research should therefore provide a route to understand this fundamental process.
Pedigrees were selected in a collaborative initiative supported by the EMHG, the two families described in detail originating from laboratories in Grenoble and Antwerp. Pedigrees are shown in Figures 1 and 2 . Tables 4 and 2 summarise IVCT results from individual family members which were performed according to the European standard protocol (3 ,30 ). All MHS individuals of the AN2 pedigree gave an unusually weak response to the trigger challenge (Table 2 ). This may reflect a peculiar feature of the underlying, yet unidentified genetic defect. In the initial stages a third pedigree from Vienna was also included. This is not discussed in detail here for reasons apparent. Prior to their inclusion in the project candidate loci on 19q, 7q and 3q were excluded in the parent laboratories on the basis of at least two recombination events. `Power' calculations for each family were conducted using a LINKAGE based approach.
Results of the in vitro contracture tests in pedigree VER
Family VER
Contractures observed (g)
Assigned
individual
2 mmol/l caffeine
2% halothane
phenotype
08
-
-
ND
09
0
0
MHN
10
-
-
ND
II.1
-
-
ND
11
0.2
0.5
MHS
II.2
-
-
ND
06
0.4
0.7
MHS
01
1.6
2.7
MHS
02
0.1
0.2
MHE(h)
12
-
-
ND
II.3
-
-
ND
07
0
0
MHN
47
0.2
1.0
MHS
46
0.3
0.3
MHS
19
0.2
0.4
MHS
20
0
0
MHN
21
0.05
0.4
MHE(h)
22
0.2
0.3
MHS
23
0
0
MHN
24
0
0.25
MHE(h)
04
0.3
1.4
MHS
03
0
0
MHN
III:11a
-
-
Clinical reaction
05
0.2
0.4
MHS
29
0.5
1.9
MHS
aClinical history of proband III:11. Aged 30 years, was anaesthetised for internal fixation of fractured femur. Tachycardia, extrasystole and hypercapnia developed 30 min after injection of suxamethonium and administration of isoflurane. Severe hyperthermia (40oC) was observed after 90 min when a diagnosis of malignant hyperthermia was made. Administration of isoflurane was stopped and dantrolene administered. However hypoxemia, acidosis and hyperkaliemia caused two cardiac arrests, the second leading to death of the patient.
Genomic DNA was prepared from EDTA whole blood according to standard methods in the parent laboratory (31 ,32 ). Working DNA stocks of 10 ng/[mu]l were prepared in the microsatellite laboratory at the Max-Delbrück-Centrum, Berlin. Sample DNA (40 ng) was amplified in 10 [mu]l PCR reactions performed in 96-well microtitre plates. One primer of each pair was fluorescently labelled with one of HEX, TET or FAM dyes (Applied Biosystems).
In total 28 chromosome specific marker sets (M. Jung et al., in preparation) comprising 333 microsatellite markers were investigated which generated a map with average spacing of ~11 cM and heterozygosity of 80%. Of these, 285 markers were reliably amplified over 28 cycles each of 94oC/5 min; 94oC/30 s; 30 s at primer specific annealing temperature followed by 72oC/30 s and a final extension of 72oC/10 min. Each reaction was carried out using a final concentration of 1.5 mM MgCl2, 100 [mu]M dNTPs and 0.4 U Taq polymerase (Perkin Elmer Amplitaq). Marker loci and primer sequences have all been previously reported (33 ,34 ,35 ) although >90% of primer sequences have been redesigned (M. Jung et al., in preparation). Chromosomal panels were arranged in sets of up to 15 markers: five HEX-labelled, five FAM-labelled and five TET-labelled, enabling fragment sizes to be differentiated with ease on 6% acrylamide/7 M urea gels.
Prior to analysis on an ABI 373 DNA sequencer, PCR product quality was assessed on 3% agarose gels. Markers were pooled in a ratio of 3 [mu]l FAM:6 [mu]l HEX:2 [mu]l TET with 10% of the final pooled volume taken and mixed with 3 [mu]l of combined loading buffer and standard (2.7 [mu]l formamide/blue dextran loading buffer: 0.3 [mu]l TAMRA labeled standard: pUC18 DNA, fragment sizes: 71; 132; 187; 218; 253; 317; 347; 393). Gels were run for 6-8 h at a constant voltage of 700 V.
Analysis was performed using the GENESCAN 672 software (Applied Biosystems) as described in the manufacturer's manual. GENOTYPER V1.1 was then used as described in the manual to `call alleles', alleles being defined as the highest two peaks within the expected size range. All sizing data were tabulated and checked for inconsistencies prior to linkage analysis.
Marker files generated by the automated sequencer were exported from GENOTYPER and analysed using LINKRUN software (T. F. Wienker, unpublished). Using LINKAGE V5.21 (36 ) two-point lod scores were calculated between each marker locus and MH under the standard genetic model defined by the EMHG genetics section and reported elsewhere (37 ,38 ): the disease allele frequency was 1/10 000; penetrance of the index case was taken as 1.0; penetrance of the MHS heterozygote was set at 0.98; the phenocopy rate was set at 0.02 and MHE individuals were assigned unknown status and did not contribute to the lod scores of the pedigrees. Evidence is accumulating that the frequency of positive IVCT results may be higher than that of clinical MH episodes. Linkage calculations were therefore repeated for a representative subset of markers setting the disease allele frequency at 1/1000 and 1/100, respectively. The resulting changes to the lods were minimal, in no case exceeding the first decimal. Data are, therefore, not presented in detail.
Lod score summary tables were compiled and data graphically represented in LODVIEW EXCEL V5.0 (39 ). Where gaps occurred in the genetic map or where `positive' regions arose, additional intercalating markers from the Research Genetics PanelTM were analysed. Where results were consistently positive, haplotypes were drawn by hand and by CRIMAP V2.41 (23 ) in an attempt to refine the position of the putative locus.
This work was supported by grants from the Association Francaise contre les Myopathies (AFM) to J.L., and from the Deutsche Forschungsgemeinschaft to C.R.M. We would like to thank the staff at the MDC, Berlin: Barbara Beyert and Thomas Wienker, Marie-Anne Shaw from Leeds, and Ann Lofgren from Antwerp for their help during this project. R.L.R. is a BBSRC PhD student.
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*To whom correspondence should be addressed. Tel: +44 113 233 3087; Fax: +44 113 244 1175; Email: rrobinso@hgmp.mrc.ac.uk
+We wish to dedicate this paper to the memory of Alistair D. Stewart who inspired and guided us in the initial stages of this work until his untimely death in November 1995.
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