GENOMICS
11,543-547
(1991)
Evidence for Genetic Heterogeneity in Malignant Hyperthermia Susceptibility R. C. LEVITT,* N. NOURI,* A. E. JEDLICKA,* V. A. McKusicK,t A. R. MARKS,* J. G. SHUTACK,!~ J. E. FLETCHER,~ H. ROSENBERG,~ AND D. A. MEmst *Department of Anesthesiology and Critical Care Medicine and t Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; # Department of Molecular Biology, Mt. Sinai Medical Center, New York, New York 10029; and 3 Department of Anesthesiology, Hahnemann University, Philadelphia, Pennsylvania 19102 Received
February26,
1991;revised
June 14, 1991
tochrome P450 gene CYPBA (19q13.1). Studying 21 families, MacLennan et al. (1990) found that MHS is tightly linked (lod score 4.20 at a linkage distance of 0 CM) to the skeletal muscle ryanodine receptor gene (RYDR), the calcium release channel of the sarcoplasmic reticulum localized near CYPBA on 19q13.1. On the basis of these linkage data, MacLennan et al. (1990) suggested that MHS is caused by a mutation in RYDR. Individuals with central core disease (CCD) are also at risk for MHS and the recent mapping of CCD to the MHS region of chromosome 19q suggests that these disorders may be caused by a genetic defect within the same gene (Haan et al., 1990; Kausch et al., 1990). These data further support the notion that one of the genetic loci that produce the MHS phenotype resides on chromosome 19q13.1. However, other data do not support the existence of a single genetic locus for MHS. For example, the linkage data reported do not explain an association between MHS and myotonic dystrophy, which maps to 19q13.2 (Johnson et al., 1988). Furthermore, malignant hyperthermia has been reported in patients with myotonia congenita (Gordon et al., 1986; Moulds and Denborough, 1974; King et al., 1972) and these patients are thought to react abnormally to in vitro contracture testing for MHS (Heiman-Patterson et al., 1988). However, the gene that causes myotonia congenita has been excluded from the region in which MHS has been mapped on chromosome 19 (Bender et aZ., 1990a; Koch et al., 1989). To test the hypothesis that MHS is produced by multiple genetic etiologies, we carried out further linkage analyses in families affected with this disorder. Here we report extended haplotype analyses in three unrelated families that indicate that markers in the 19q12-q13.3 linkage groups do not cosegregate with MHS. These data clearly demonstrate the first evidence of molecular genetic heterogeneity in MHS.
Malignant hyperthermia susceptibility (MHS) is a clinically heterogeneous pharmacogenetic disorder characterized by accelerated metabolism, hyperthermia, and frequently muscle rigidity. MHS is elicited by all commonly used potent inhalation anesthetics and depolarizing neuromuscular blockers and remains an important cause of death due to anesthesia. Recent linkage studies suggest a single genetic locus for this disorder on chromosome 19q13.1. The results of our linkage analyses exclude several loci on 19q13.1 as a site for the gene(s) that produces the Ml-W phenotype in three unrelated families and clearly establish genetic heterogeneity in this disorder. These results are consistent with the hypothesis that the genetic defect that alters thermoregulation may vary in MHS and that clinical variability in the expression of MEW may be o 1991 Academic pre~e, IUC. explained by genetic heterogeneity.
INTRODUCTION
Genetic heterogeneity has been suspected in malignant hyperthermia susceptibility (MHS) because the clinical expression is highly variable (Gronert, 1980; McPherson and Taylor, 1982). Genetic heterogeneity is further suggested by an association between MHS and other neuromuscular disorders, including myotonia congenita (Heiman-Patterson et al., 1988a, b), central core disease (Denborough et al., 1973), KingDenborough syndrome (King and Denborough, 1973), and myotonic dystrophy (Saidman et al., 1964). Although alternative genetic etiologies have been proposed (McPherson and Taylor, 1982), recent linkage studies have identified only one genetic locus in MHS. In three extended Irish pedigrees McCarthy et al. (1990) localized this disorder to 19q12-13.2 and suggested, using multipoint linkage analyses, that a single genetic locus for MHS resides near the cy543
Copyright 0 1991 All rights of reproduction
o&3&7543/91 $3.00 by Academic Press, Inc. in any form reserved.
LEVITT
544 METHODS
An established protocol was used as a standardized test to determine the MHS phenotype (Fletcher and Rosenberg, 1985; Allen et al., 1990) and is based on skeletal muscle contractures in vitro to halothane or caffeine. Surgical biopsies were conducted with nerve blocks and sedation. Muscle strips of the Vastus lateralis were contracture tested as described previously in detail (Fletcher and Rosenberg, 1985; Allen et al., 1990). The sensitivity and specificity of the MHS contracture test as determined in our laboratory have also been described elsewhere (Fletcher and Rosenberg, 1985; Allen et al., 1990). Based on these established protocols the following diagnoses can be made: MH-susceptible (MHS), MH-negative (MHN), and MH-equivocal (MHE) . We examined four unrelated families in detail. These families were identified by a proband who suffered a clinical episode of MHS and recovered. We selected only those families that demonstrate autosoma1 dominant inheritance for MHS. Extended haplotypes were constructed by hand using the described markers (including those with up to 11 recognized alleles), and these techniques demonstrate that in each case the biologic parents have been included with high probability. Nuclei from whole blood samples were isolated and lysed, and then high-molecular-weight DNA was purified in a cesium chloride density gradient using centrifugation. The DNA was digested with a restriction enzyme or amplified by polymerase chain reaction (Saiki et al., 1988). Duplicate DNA samples were analyzed for informative markers on each individual. Restriction fragment length polymorphisms were demonstrated using 0.6-1.0% agarose gel electrophoresis, Southern blotting (Southern, 19X5), and hybridization to random primed labeled (Feinberg and Vogelstein, 1983) DNA probes. Repetitive sequence polymorphisms (CA),, were demonstrated as previously described (Weber and May, 1989). The probes utilized included D19S49 (Weber et al., 1990); D19S7 (~4.1 ATCC (Shaw et al., 1986) ); D19S75 (Weber, 1990); D19S9 (pIJ2 ATCC (Brook et al., 1984) ); RYDR (rabbit cDNA (Marks et al., 1989; MacLennan et al., 1990) ). D19S47 (Weber and May, 1989); CYP2A (cDNA ATCC (Wainwright et al., 1985) ); ATPlA3 (cDNA ATCC (Harley et al., 1988) ); D19S16 (pJSB11 ATCC (Schepens et al., 1987) ); D19S19 (LDR152 (Bartlett et aZ., 1987) ); APOCB (Weber and May, 1989); D19S22 (pEFD4.2 ATCC (Nakamura et al., 1988) ); and PRKCG (lbPKCgl9 cDNA ATCC (Johnson et al., 1988) ). (Anonymous DNA segments detecting polymorphisms are referred to by “D” numbers (Shows and McAlpine, 1982).)
ET AL. RESULTS
AND
DISCUSSION
The segregation data for Family 1 are illustrated in Fig. 1A. The diagnosis of MHS was established in the proband (11-2) from this family by her clinical response to anesthesia. Anesthesia was induced with halothane and nitrous oxide. Muscle rigidity and an irregular heart rhythm were observed following the administration of succinylcholine. Postoperative muscle soreness was noted and CPK levels rose to 3132 on the evening of surgery. The grandparents (I1, I-2) were not tested for MHS; however, both of their affected children (11-2, 11-4) inherited the same chromosome from their father (I-l) but different chromosomes 19 from their mother (I-2). In the second generation, the proband (11-2) transmitted this same chromosome 19 haplotype to her affected son (III-l). However, individual III-3 was found to be MHS by contracture testing, and inherited only a portion of this maternal haplotype demonstrating recombination between MHS and D19S49, D19S7, RYDR, D19S47, CYP2A, and D19S19. Individuals II3 and III-Z also demonstrate recombination on this same chromosome between D19S19 and D19S22. These represent the minimum number of crossovers that have occurred and suggest that in this family the gene responsible for MHS is located distal to D19S19, or elsewhere in the genome. The segregation data from Family 2 are illustrated in Fig. 1B. The proband in this family experienced an increase in exhaled CO,, an elevated temperature, and a decreased serum pH during anesthesia. His CPK rose to over 87,000 postoperatively, and he noted significant muscle weakness and soreness for several weeks. The maximum contracture response was 2.2 g to 3% halothane and 0.2 g to 2 mM caffeine. Affected individuals II-1 and III-l share a chromosome 19 haplotype inherited from individual I-l. The proband (11-3) and his affected sons (111-3,111-4) inherited in common a different chromosome 19 haplotype from individual I-l. However, an unaffected son (111-5)also inherited this same chromosome 19 haplotype, thus excluding it as carrying the defective gene producing MHS. Therefore, recombination between MHS and D19S49, D19S9, RYDR, D19S47, CYPBA, ATPlA3, APOC2, and PRKCG has occurred; the most likely explanation for these data is that the markers in the 19q12-13.3 linkage groups do not cosegregate with MHS in this family. The segregation data from Family 3 are illustrated in Fig. 1C. The proband in this family underwent anesthesia for the surgical correction of strabismus. He developed fever (38°C at end of surgical procedure, 38.2”C maximum) and increased heart rate and was treated promptly with dantrolene. His CPK rose postoperative to 9800. The proband’s maternal aunt (indi-
GENETIC
HETEROGENEITY
A
IN
MHS
545
B
I
I
\-
III D19S49 D19S.7 RYDR DWS47
t-II-7 D B -
A B A D A m
IFI I A m
uu
n 0 H-IS
N
Q
0
0
MHN
q
8
not tested
1 --+
MHE
(MHRXN)
]
rl
n 0 MHS N @ MHE Cl 0 MHN k!
8
---,
FIG. 1. Segregation data for selected informative polymorphisms groups. Genes and markers are placed in the order that the literature not yet been accurately mapped were placed in the order suggested only two-point analyses of MHS with each marker were performed.
vidual 11-4) apparently died from malignant hyperthermia. Individuals I-l and II-1 are MHN, which excludes these chromosome 19 haplotypes from harboring a defective gene producing MHS. Individual II-3 is unrelated to II-2 and is presumed normal. Affected individuals II-Z and III-1 (the proband) inherit in common a normal chromosome 19 haplotype from I-l, suggesting that the gene causing MHS in this family is located elsewhere in the genome. In this smaller family, there is recombination between MHS and D19S49, D19S75, RYDR, D19S47, and APOCB. The chromosomal localization of a genetic locus causing one form of MHS (MHS-1) to 19q13.1 has allowed us to test the hypothesis that MHS is a geneti-
not tested
(MH RXN)
within genes and anonymous DNA markers in the 19q12-q13.3 linkage suggests they occur (6,16,23,24,29,33,34,39-41). Markers that have by these three families. This is purely for purposes of illustration since (A) Family 1; (B) Family 2; (C) Family 3.
cally heterogeneous group of disorders. Lod scores of -2.00 were obtained from D19S49 at 8 = 0.09, RYDR at 0 = 0.06, D19S47 at 0 = 0.05, CYPBA at f3 = 0.02, and APOCB at 0 = 0.07 (Table 1). Multipoint analyses were not performed because an accurate map of these multiple loci in this region is not yet available. However, based on the exclusion results obtained from the three families described, RYDR can certainly be excluded as a candidate locus for MHS in any of these families. In a fourth small family, recombination could not be demonstrated between MHS and D19S49, D19S47, D19S16, BCL3, CKMM, and PRKCG. The data presented suggest at least one additional genetic locus (MHS-2) distinct from that
546
LEVITT
TABLE Lod
Scores
1
between MHS on Chromosome Recombination
0.01
0.05
0.10
ET
AL. is supported by MDA, Foundation for Anesthesia Education and Research, National Institutes of Health (GM47145), and the Malignant Hyperthermia Association of the United States. Special thanks to Dr. M.C. Rogers for his continued support of this work.
and Markers 19 fraction 0.20
REFERENCES 0.30
0.40 1.
DlSS49 Family Family Family
1 2 3
Total RYDR Family Family Family
1 2 3
Total DlSS47 Family Family Family
1 2 3
Total CYPZA Family Family Family
1 2 3
Total APOC2 Family Family Total
2 3
-1.70 -4.32 -2.79
-0.45 -1.17 -1.28
-0.22 -0.65 -0.91
-0.06 -0.23 -0.50
-0.01 -0.06 -0.26
-0.00 -0.01 -0.11
-8.81
-2.90
-1.78
-0.79
-0.33
-0.12
-2.22 -1.97 -2.10
-0.93 -0.60 -0.74
-0.60 -0.34 -0.48
-0.28 -0.13 -0.26
-0.11 -0.04 -0.16
-0.03 -0.01 -0.08
-6.29
-2.27
-1.42
-0.67
-0.31
-0.12
-1.92 -2.10 -2.10
-0.71 -0.46 -0.74
-0.44 -0.23 -0.48
-0.19 -0.05 -0.26
-0.08 -0.00 -0.16
-0.02 -0.00 -0.08
-6.12
-1.91
-1.15
-0.50
-0.24
-0.10
-1.92 -2.00 0.30
-0.71 -0.56 0.26
-0.44 -0.31 0.22
-0.19 -0.10 0.13
-0.08 -0.03 0.06
-0.02 -0.00 0.02
-3.62
-1.01
-0.53
-0.16
-0.05
-0.00
-4.22 -2.79
-1.17 -1.28
-0.65 -0.91
-0.23 -0.50
-0.06 -0.26
-0.01 -0.11
-7.01
-2.45
-1.56
-0.73
-0.32
-0.12
Note. Lod scores were calculated autosomal dominant disease; MHE not used in the analyses.
using LIPED and untested
(22) assuming an individuals were
proposed by others and which is not situated on 19q13.1 and does not represent the RYDR locus. Additional linkage analyses will be necessary to identify the chromosomal location of MHS-2 and evaluate whether other genetic loci (MHS-3, etc.) may also exist. These data are consistent with the hypothesis that clinical variability in the expression of MHS is associated with molecular genetic heterogeneity. Furthermore, additional studies may disclose whether a particular clinical phenotype is associated with a specific genetic locus. Ultimately, the evaluation of genetic heterogeneity in MHS will be critical to the development of noninvasive, presymptomatic, diagnostic tests, using molecular genetics.
2.
3.
4.
5.
6.
7.
8.
9.
10. 11. 12.
13.
14.
15.
ACKNOWLEDGMENTS We thank Dr. J. L. Weber for unpublished data regarding his microsatellite markers, Ms. Kelly Battaglia for her secretarial assistance, and Ms. Joanna Strayer for her creative artwork. R.C.L.
16.
ALLEN, G. C., FLETCHER, J. E., HUGGINS, F. J., CONTI, P. A., AND ROSENBERG, H. (1990). Caffeine and halothane contracture testing in swine using the recommendations of the North American Malignant Hyperthermia Group. Anesthesiology 72: 71-76. BARTLETT, R. J., PERICAK-VANCE, M. A., YAMAOKA, L., GILBERT, J., HERBSTFZEITH, M., HUNG, W. Y., LEE, J. E., MoHANDAS, T., BRUNDS, G., LABERGE, C., THIBAULT, M. C., Ross, D., AND ROSES, A. D. (1987). A new probe for the diagnosis of myotonic muscular dystrophy. Science 235: 16481650. BENDER, K., SENFF, H., STEIERT, A., LAGODNY, H., WIENKER, T. F., AND KOCH, M. (1990a). Linkage study of myotonia congenita and paramyotonia congenita. Clin. Genet. 36: 92-99. BENDER, K., SENFF, H., WIENKJZR, T. F., SPIESS-KIEFER, C., AND LEHMANN-HORN, F. (199Ob). A linkage study of malignant hyperthermia (MH). Clin. Genet. 37: 221-225. BROOK, J. D., SHAW, D. J., MEREDITH, L., BRUNS, G. A. P., ANLI HARPER, P. S. (1984). Localization of genetic markers and orientation of the linkage group on chromosome 19. Hum. Genet. 68: 282-285. BRUNNER, H. G., SMEETS, H., LANBERMON, M. M., COERWINKEL-DRIESSEN, M., VAN OOST B. A., WIERINGA, B., AND ROPERS, H. H. (1989). A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19. Genomics 5: 589-595. DENBOROUGH, M. A., DENNE?T, X., AND ANDERSON, R. M. (1973). Centeral core disease and malignant hyperpyrexia. Br. Med. J. 1: 272-273. FEINBERG, A., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. FLETCHER, J. E., AND ROSENBERG, H. (1985). In vitro interaction between halothane and succinylcholine in human skeletal muscle: Implications for malignant hyperthermia and masseter muscle rigidity. Anesthesiology 63: 190-194. GORDON, A. S., BRITT, B. A., AND PRITZKER, K. P. H. (1986). Myotonia and malignant hyperpyrexia. Muscle Nerve 9: 221. GRONERT, G. (1980). Malignant hyperthermia. Anesthesiology 53: 395-423. HAAN, E. R., FREEMANTLE, C. J., MCCURE, J. A., FRIEND, K. L., AND MULLEY, J. C. (1990). Assignment of the gene for central core disease to chromosome 19. Hum. Genet. 2: 187190. HARLEY, H. G., BROOK, J. D., JACKSON, C. L., GLASER, T., WALSH, K. V., SARFARAZAI, M., KENT, R., LAGER, M., KOCH, M., HARPER, P. S., LEVENSON, R., HOUSMAN, D. E., AND SHAW, D. J. (1988). Localization of a human Na+, K+-ATPase (Y subunit gene to chromosome lSq12+q13.2 and linkage to the myotonic dystrophy locus. Genomics 3: 380-384. HEIMAN-PA?TERSON, T., MARTINO, C., ROSENBERG, H., FLETCHER, J. E., AND TAHMOUSH, A. J. (1988b). Malignant hyperthermia in myotonia congenita. Neurology 38: 810-812. HEIMAN-PATTERSON, T., ROSENBERG, H., FLETCHER, J. E., AND TAHMOUSH, A. J. (1988a). Halothane-caffeine contracture testing in neuromuscular diseases. Muscle Nerve 11: 453-457. HOLM, C., KIRCHGESSNER, T. G., SVENSON, K. L., FREDRIKSON, G., NILSSON, S., MILLER, C. G., SHNELY, J. E., HEINZ-
GENETIC
17.
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
HETEROGENEITY
MANN, C., SPARKES, R. S., MOHANDAS, T., LUSIS, A. J., BELFRAGE, P., AND SCHOTZ, M. C. (1988). Hormone-sensitive lipase: Sequence, expression, and chromosomal localization to 19 cent-13.3. Science 241: 1503-1506. JOHNSON, K., JONES, P. J., SPURR, N., NIMMO, E., et al. (1988). Linkage relationships of the protein kinase C gamma gene which exclude it as a candidate for myotonic dystropy. Cytogenet. Cell Genet. 48: 13-15. KAUSCH, K., GRIMM, T., JANKA, M., LEHMANN-HORN, F., WIERINGA, B., AND MILLER, C. R. (1990). Evidence for linkage of the central core disease locus to chromosome 19q. J. Neurol. Sci. D&k 549. KING, J. O., AND DENBOROUGH, M. A. (1973). Anesthesia induced malignant hyperpyrexia in children. J. Pediutr. 83: 3740. KING, J. D., DENBOROUGH, M. A., AND ZAPF, P. W. (1972). Inheritance of malignant hyperpyrexia. hncet 1: 365-370. KOCH, M., HARLEY, H., SARFARZAI, M., BENDER, K., WIENKER, T., ZOLL, B., AND HARPER, P. S. (1989). Myotonia congenita (Thomsen’s disease) excluded from the region of the myotonic dystrophy locus on chromosome 19. Hum. Genet. 82: 163-166. LATHROP, G. M., LALOUEL, J. M., JULIER, C., AND OTT, J. (1984). Strategies for multilocus linkage analysis in humans. Proc. Natl. Acad. Sci. USA 81: 3443-3446. LEBEAU, M. M., RYAN, D., JR., AND -RICK-VANCE, M. A. (1989). Report of the committee on the genetic constitution of chromosomes 18 and 19. Cytogenet. Cell Genet. 61: 338357. MACLENNAN, D. H., DUFF, C., ZOFUATO, F., F~JJII, J., et al. (1990). Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature 343: 559-561. MARKS, A. R., TEMPST, P., HWANG, K. S., TAIJBMAN, M. B., INUI, M., CHADWICK, C., FLEISCHER, S., AND NADAL-GINARD, B. (1989). Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 86: 8683-8687. MCCARTHY, T. V., SANDRA HEALY, J. M., HEFFRON, J. J. A., LEHANE, M., DEUFEL, T., LEHMANN-HORN, F., FARRALL, M., AND JOHNSON, K. (1990). Localization of malignant hyperthermia susceptibility locus to human chromosome 19q1213.2. Nature 343: 562-564. MCPHERSON, E., AND TAYLOR, C. A. (1982). The genetics of malignant hyperthermia: Evidence for heterogeneity. Am. J. Med. Genet. 11: 273-285. MOULDS, R. F. W., AND DENBOROUGH, M. A. (1974). Identification of susceptibility to malignant hyperpyrexia. Br. Med. J. 2: 245-247.
IN
29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
MHS
547
NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., LALOUEL, J. M., AND WHITE, R. (1988). A primary map of ten DNA markers and two serological markers for human chromosome 19. Genomics 3: 67-71. SAIDMAN, L. J., HAVAFQ E. S., AND EGER, E. I. (1964). Hyperthermia during anesthesia. JAMA 190: 1029-1032. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. SCHEPENS, J., SMEETS, H., HULSEBOS, T., BRUNNER, H., AND WIERINGA B. (1987). Isolation of a polymorphic DNA sequence pJSBl1 (D19S16) from the human chromosome 19cen-q13.2 region linked to the myotonic dystrophy (DM) gene. Nucleic Acids Res. 16: 3192. SCHONK, D., COERWINKEL-DRIESSEN, M., VAN DALEN, I., OERLJZMANS, F., SMEETS, B., SCHJZPENS, J., HULSEBOS, T., COCKBURN, D., BOYD, Y., DAVID, M., RETTIG, W., SHAW, D., ROSES, A., ROPERS, H., AND WIEFUNGA, B. (1989b). Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q. Genomics 4: 384-396. SCHONK, D., VAN DIJK, P. E., RIEGMAN, P., TRAPMAN, J., HOLM, C., CRAIG, I. W., SILLEKENS, P., VAN VENROOY, W., ROPERS, H. H., AND WIEXUNGA, B. (1989a). Subregional assignment of six genes on chromosome 19q. Cytogenet. Cell Genet. 51: 1075. SHAW, D. J., MEREDITH, A. L., SARFARAZI, M., HARLEY, H. G., HUSON, S. M., BROOK, J. D., BIJF~ON, L., Lrrr, M., MOHANDAS, T., AND HARPER, P. S. (1986). Regional localizations and linkage relationships of seven RFLPs end myotonic dystrophy on chromosome 19. Hum. Genet. 74: 262-266. SHOWS, T. B., AND MCALPINE, P. J. (1982). The 1981 catalogue of assigned human genetic markers and report of the nomenclature committee. Cytogenet. Cell Genet. 32: 221-245. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. WAINWRIGHT, B. J., WATSON, E. K., SHEPHARD, E. A., AND PHILLIPS, I. R. (1985). RFLP for a human cytochrome P-450 gene at 19ql3.1-qter (HGMS provisional designation CYPI). Nucleic Acids Res. 13: 4610. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396. WEBER, J. L., MAY, P. E., AND KAPPEL, C. (1990). Dinucleotide repeat polymorphism at the D19S49 locus. Nucleic Acids Res. 18: 1927. WEBER, J. L. (1990). Dinucleotide repeat polymorphism at the D19S75 locus. Nucleic Acids Res. 18: 4639.
11,543-547
(1991)
Evidence for Genetic Heterogeneity in Malignant Hyperthermia Susceptibility R. C. LEVITT,* N. NOURI,* A. E. JEDLICKA,* V. A. McKusicK,t A. R. MARKS,* J. G. SHUTACK,!~ J. E. FLETCHER,~ H. ROSENBERG,~ AND D. A. MEmst *Department of Anesthesiology and Critical Care Medicine and t Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; # Department of Molecular Biology, Mt. Sinai Medical Center, New York, New York 10029; and 3 Department of Anesthesiology, Hahnemann University, Philadelphia, Pennsylvania 19102 Received
February26,
1991;revised
June 14, 1991
tochrome P450 gene CYPBA (19q13.1). Studying 21 families, MacLennan et al. (1990) found that MHS is tightly linked (lod score 4.20 at a linkage distance of 0 CM) to the skeletal muscle ryanodine receptor gene (RYDR), the calcium release channel of the sarcoplasmic reticulum localized near CYPBA on 19q13.1. On the basis of these linkage data, MacLennan et al. (1990) suggested that MHS is caused by a mutation in RYDR. Individuals with central core disease (CCD) are also at risk for MHS and the recent mapping of CCD to the MHS region of chromosome 19q suggests that these disorders may be caused by a genetic defect within the same gene (Haan et al., 1990; Kausch et al., 1990). These data further support the notion that one of the genetic loci that produce the MHS phenotype resides on chromosome 19q13.1. However, other data do not support the existence of a single genetic locus for MHS. For example, the linkage data reported do not explain an association between MHS and myotonic dystrophy, which maps to 19q13.2 (Johnson et al., 1988). Furthermore, malignant hyperthermia has been reported in patients with myotonia congenita (Gordon et al., 1986; Moulds and Denborough, 1974; King et al., 1972) and these patients are thought to react abnormally to in vitro contracture testing for MHS (Heiman-Patterson et al., 1988). However, the gene that causes myotonia congenita has been excluded from the region in which MHS has been mapped on chromosome 19 (Bender et aZ., 1990a; Koch et al., 1989). To test the hypothesis that MHS is produced by multiple genetic etiologies, we carried out further linkage analyses in families affected with this disorder. Here we report extended haplotype analyses in three unrelated families that indicate that markers in the 19q12-q13.3 linkage groups do not cosegregate with MHS. These data clearly demonstrate the first evidence of molecular genetic heterogeneity in MHS.
Malignant hyperthermia susceptibility (MHS) is a clinically heterogeneous pharmacogenetic disorder characterized by accelerated metabolism, hyperthermia, and frequently muscle rigidity. MHS is elicited by all commonly used potent inhalation anesthetics and depolarizing neuromuscular blockers and remains an important cause of death due to anesthesia. Recent linkage studies suggest a single genetic locus for this disorder on chromosome 19q13.1. The results of our linkage analyses exclude several loci on 19q13.1 as a site for the gene(s) that produces the Ml-W phenotype in three unrelated families and clearly establish genetic heterogeneity in this disorder. These results are consistent with the hypothesis that the genetic defect that alters thermoregulation may vary in MHS and that clinical variability in the expression of MEW may be o 1991 Academic pre~e, IUC. explained by genetic heterogeneity.
INTRODUCTION
Genetic heterogeneity has been suspected in malignant hyperthermia susceptibility (MHS) because the clinical expression is highly variable (Gronert, 1980; McPherson and Taylor, 1982). Genetic heterogeneity is further suggested by an association between MHS and other neuromuscular disorders, including myotonia congenita (Heiman-Patterson et al., 1988a, b), central core disease (Denborough et al., 1973), KingDenborough syndrome (King and Denborough, 1973), and myotonic dystrophy (Saidman et al., 1964). Although alternative genetic etiologies have been proposed (McPherson and Taylor, 1982), recent linkage studies have identified only one genetic locus in MHS. In three extended Irish pedigrees McCarthy et al. (1990) localized this disorder to 19q12-13.2 and suggested, using multipoint linkage analyses, that a single genetic locus for MHS resides near the cy543
Copyright 0 1991 All rights of reproduction
o&3&7543/91 $3.00 by Academic Press, Inc. in any form reserved.
LEVITT
544 METHODS
An established protocol was used as a standardized test to determine the MHS phenotype (Fletcher and Rosenberg, 1985; Allen et al., 1990) and is based on skeletal muscle contractures in vitro to halothane or caffeine. Surgical biopsies were conducted with nerve blocks and sedation. Muscle strips of the Vastus lateralis were contracture tested as described previously in detail (Fletcher and Rosenberg, 1985; Allen et al., 1990). The sensitivity and specificity of the MHS contracture test as determined in our laboratory have also been described elsewhere (Fletcher and Rosenberg, 1985; Allen et al., 1990). Based on these established protocols the following diagnoses can be made: MH-susceptible (MHS), MH-negative (MHN), and MH-equivocal (MHE) . We examined four unrelated families in detail. These families were identified by a proband who suffered a clinical episode of MHS and recovered. We selected only those families that demonstrate autosoma1 dominant inheritance for MHS. Extended haplotypes were constructed by hand using the described markers (including those with up to 11 recognized alleles), and these techniques demonstrate that in each case the biologic parents have been included with high probability. Nuclei from whole blood samples were isolated and lysed, and then high-molecular-weight DNA was purified in a cesium chloride density gradient using centrifugation. The DNA was digested with a restriction enzyme or amplified by polymerase chain reaction (Saiki et al., 1988). Duplicate DNA samples were analyzed for informative markers on each individual. Restriction fragment length polymorphisms were demonstrated using 0.6-1.0% agarose gel electrophoresis, Southern blotting (Southern, 19X5), and hybridization to random primed labeled (Feinberg and Vogelstein, 1983) DNA probes. Repetitive sequence polymorphisms (CA),, were demonstrated as previously described (Weber and May, 1989). The probes utilized included D19S49 (Weber et al., 1990); D19S7 (~4.1 ATCC (Shaw et al., 1986) ); D19S75 (Weber, 1990); D19S9 (pIJ2 ATCC (Brook et al., 1984) ); RYDR (rabbit cDNA (Marks et al., 1989; MacLennan et al., 1990) ). D19S47 (Weber and May, 1989); CYP2A (cDNA ATCC (Wainwright et al., 1985) ); ATPlA3 (cDNA ATCC (Harley et al., 1988) ); D19S16 (pJSB11 ATCC (Schepens et al., 1987) ); D19S19 (LDR152 (Bartlett et aZ., 1987) ); APOCB (Weber and May, 1989); D19S22 (pEFD4.2 ATCC (Nakamura et al., 1988) ); and PRKCG (lbPKCgl9 cDNA ATCC (Johnson et al., 1988) ). (Anonymous DNA segments detecting polymorphisms are referred to by “D” numbers (Shows and McAlpine, 1982).)
ET AL. RESULTS
AND
DISCUSSION
The segregation data for Family 1 are illustrated in Fig. 1A. The diagnosis of MHS was established in the proband (11-2) from this family by her clinical response to anesthesia. Anesthesia was induced with halothane and nitrous oxide. Muscle rigidity and an irregular heart rhythm were observed following the administration of succinylcholine. Postoperative muscle soreness was noted and CPK levels rose to 3132 on the evening of surgery. The grandparents (I1, I-2) were not tested for MHS; however, both of their affected children (11-2, 11-4) inherited the same chromosome from their father (I-l) but different chromosomes 19 from their mother (I-2). In the second generation, the proband (11-2) transmitted this same chromosome 19 haplotype to her affected son (III-l). However, individual III-3 was found to be MHS by contracture testing, and inherited only a portion of this maternal haplotype demonstrating recombination between MHS and D19S49, D19S7, RYDR, D19S47, CYP2A, and D19S19. Individuals II3 and III-Z also demonstrate recombination on this same chromosome between D19S19 and D19S22. These represent the minimum number of crossovers that have occurred and suggest that in this family the gene responsible for MHS is located distal to D19S19, or elsewhere in the genome. The segregation data from Family 2 are illustrated in Fig. 1B. The proband in this family experienced an increase in exhaled CO,, an elevated temperature, and a decreased serum pH during anesthesia. His CPK rose to over 87,000 postoperatively, and he noted significant muscle weakness and soreness for several weeks. The maximum contracture response was 2.2 g to 3% halothane and 0.2 g to 2 mM caffeine. Affected individuals II-1 and III-l share a chromosome 19 haplotype inherited from individual I-l. The proband (11-3) and his affected sons (111-3,111-4) inherited in common a different chromosome 19 haplotype from individual I-l. However, an unaffected son (111-5)also inherited this same chromosome 19 haplotype, thus excluding it as carrying the defective gene producing MHS. Therefore, recombination between MHS and D19S49, D19S9, RYDR, D19S47, CYPBA, ATPlA3, APOC2, and PRKCG has occurred; the most likely explanation for these data is that the markers in the 19q12-13.3 linkage groups do not cosegregate with MHS in this family. The segregation data from Family 3 are illustrated in Fig. 1C. The proband in this family underwent anesthesia for the surgical correction of strabismus. He developed fever (38°C at end of surgical procedure, 38.2”C maximum) and increased heart rate and was treated promptly with dantrolene. His CPK rose postoperative to 9800. The proband’s maternal aunt (indi-
GENETIC
HETEROGENEITY
A
IN
MHS
545
B
I
I
\-
III D19S49 D19S.7 RYDR DWS47
t-II-7 D B -
A B A D A m
IFI I A m
uu
n 0 H-IS
N
Q
0
0
MHN
q
8
not tested
1 --+
MHE
(MHRXN)
]
rl
n 0 MHS N @ MHE Cl 0 MHN k!
8
---,
FIG. 1. Segregation data for selected informative polymorphisms groups. Genes and markers are placed in the order that the literature not yet been accurately mapped were placed in the order suggested only two-point analyses of MHS with each marker were performed.
vidual 11-4) apparently died from malignant hyperthermia. Individuals I-l and II-1 are MHN, which excludes these chromosome 19 haplotypes from harboring a defective gene producing MHS. Individual II-3 is unrelated to II-2 and is presumed normal. Affected individuals II-Z and III-1 (the proband) inherit in common a normal chromosome 19 haplotype from I-l, suggesting that the gene causing MHS in this family is located elsewhere in the genome. In this smaller family, there is recombination between MHS and D19S49, D19S75, RYDR, D19S47, and APOCB. The chromosomal localization of a genetic locus causing one form of MHS (MHS-1) to 19q13.1 has allowed us to test the hypothesis that MHS is a geneti-
not tested
(MH RXN)
within genes and anonymous DNA markers in the 19q12-q13.3 linkage suggests they occur (6,16,23,24,29,33,34,39-41). Markers that have by these three families. This is purely for purposes of illustration since (A) Family 1; (B) Family 2; (C) Family 3.
cally heterogeneous group of disorders. Lod scores of -2.00 were obtained from D19S49 at 8 = 0.09, RYDR at 0 = 0.06, D19S47 at 0 = 0.05, CYPBA at f3 = 0.02, and APOCB at 0 = 0.07 (Table 1). Multipoint analyses were not performed because an accurate map of these multiple loci in this region is not yet available. However, based on the exclusion results obtained from the three families described, RYDR can certainly be excluded as a candidate locus for MHS in any of these families. In a fourth small family, recombination could not be demonstrated between MHS and D19S49, D19S47, D19S16, BCL3, CKMM, and PRKCG. The data presented suggest at least one additional genetic locus (MHS-2) distinct from that
546
LEVITT
TABLE Lod
Scores
1
between MHS on Chromosome Recombination
0.01
0.05
0.10
ET
AL. is supported by MDA, Foundation for Anesthesia Education and Research, National Institutes of Health (GM47145), and the Malignant Hyperthermia Association of the United States. Special thanks to Dr. M.C. Rogers for his continued support of this work.
and Markers 19 fraction 0.20
REFERENCES 0.30
0.40 1.
DlSS49 Family Family Family
1 2 3
Total RYDR Family Family Family
1 2 3
Total DlSS47 Family Family Family
1 2 3
Total CYPZA Family Family Family
1 2 3
Total APOC2 Family Family Total
2 3
-1.70 -4.32 -2.79
-0.45 -1.17 -1.28
-0.22 -0.65 -0.91
-0.06 -0.23 -0.50
-0.01 -0.06 -0.26
-0.00 -0.01 -0.11
-8.81
-2.90
-1.78
-0.79
-0.33
-0.12
-2.22 -1.97 -2.10
-0.93 -0.60 -0.74
-0.60 -0.34 -0.48
-0.28 -0.13 -0.26
-0.11 -0.04 -0.16
-0.03 -0.01 -0.08
-6.29
-2.27
-1.42
-0.67
-0.31
-0.12
-1.92 -2.10 -2.10
-0.71 -0.46 -0.74
-0.44 -0.23 -0.48
-0.19 -0.05 -0.26
-0.08 -0.00 -0.16
-0.02 -0.00 -0.08
-6.12
-1.91
-1.15
-0.50
-0.24
-0.10
-1.92 -2.00 0.30
-0.71 -0.56 0.26
-0.44 -0.31 0.22
-0.19 -0.10 0.13
-0.08 -0.03 0.06
-0.02 -0.00 0.02
-3.62
-1.01
-0.53
-0.16
-0.05
-0.00
-4.22 -2.79
-1.17 -1.28
-0.65 -0.91
-0.23 -0.50
-0.06 -0.26
-0.01 -0.11
-7.01
-2.45
-1.56
-0.73
-0.32
-0.12
Note. Lod scores were calculated autosomal dominant disease; MHE not used in the analyses.
using LIPED and untested
(22) assuming an individuals were
proposed by others and which is not situated on 19q13.1 and does not represent the RYDR locus. Additional linkage analyses will be necessary to identify the chromosomal location of MHS-2 and evaluate whether other genetic loci (MHS-3, etc.) may also exist. These data are consistent with the hypothesis that clinical variability in the expression of MHS is associated with molecular genetic heterogeneity. Furthermore, additional studies may disclose whether a particular clinical phenotype is associated with a specific genetic locus. Ultimately, the evaluation of genetic heterogeneity in MHS will be critical to the development of noninvasive, presymptomatic, diagnostic tests, using molecular genetics.
2.
3.
4.
5.
6.
7.
8.
9.
10. 11. 12.
13.
14.
15.
ACKNOWLEDGMENTS We thank Dr. J. L. Weber for unpublished data regarding his microsatellite markers, Ms. Kelly Battaglia for her secretarial assistance, and Ms. Joanna Strayer for her creative artwork. R.C.L.
16.
ALLEN, G. C., FLETCHER, J. E., HUGGINS, F. J., CONTI, P. A., AND ROSENBERG, H. (1990). Caffeine and halothane contracture testing in swine using the recommendations of the North American Malignant Hyperthermia Group. Anesthesiology 72: 71-76. BARTLETT, R. J., PERICAK-VANCE, M. A., YAMAOKA, L., GILBERT, J., HERBSTFZEITH, M., HUNG, W. Y., LEE, J. E., MoHANDAS, T., BRUNDS, G., LABERGE, C., THIBAULT, M. C., Ross, D., AND ROSES, A. D. (1987). A new probe for the diagnosis of myotonic muscular dystrophy. Science 235: 16481650. BENDER, K., SENFF, H., STEIERT, A., LAGODNY, H., WIENKER, T. F., AND KOCH, M. (1990a). Linkage study of myotonia congenita and paramyotonia congenita. Clin. Genet. 36: 92-99. BENDER, K., SENFF, H., WIENKJZR, T. F., SPIESS-KIEFER, C., AND LEHMANN-HORN, F. (199Ob). A linkage study of malignant hyperthermia (MH). Clin. Genet. 37: 221-225. BROOK, J. D., SHAW, D. J., MEREDITH, L., BRUNS, G. A. P., ANLI HARPER, P. S. (1984). Localization of genetic markers and orientation of the linkage group on chromosome 19. Hum. Genet. 68: 282-285. BRUNNER, H. G., SMEETS, H., LANBERMON, M. M., COERWINKEL-DRIESSEN, M., VAN OOST B. A., WIERINGA, B., AND ROPERS, H. H. (1989). A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19. Genomics 5: 589-595. DENBOROUGH, M. A., DENNE?T, X., AND ANDERSON, R. M. (1973). Centeral core disease and malignant hyperpyrexia. Br. Med. J. 1: 272-273. FEINBERG, A., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. FLETCHER, J. E., AND ROSENBERG, H. (1985). In vitro interaction between halothane and succinylcholine in human skeletal muscle: Implications for malignant hyperthermia and masseter muscle rigidity. Anesthesiology 63: 190-194. GORDON, A. S., BRITT, B. A., AND PRITZKER, K. P. H. (1986). Myotonia and malignant hyperpyrexia. Muscle Nerve 9: 221. GRONERT, G. (1980). Malignant hyperthermia. Anesthesiology 53: 395-423. HAAN, E. R., FREEMANTLE, C. J., MCCURE, J. A., FRIEND, K. L., AND MULLEY, J. C. (1990). Assignment of the gene for central core disease to chromosome 19. Hum. Genet. 2: 187190. HARLEY, H. G., BROOK, J. D., JACKSON, C. L., GLASER, T., WALSH, K. V., SARFARAZAI, M., KENT, R., LAGER, M., KOCH, M., HARPER, P. S., LEVENSON, R., HOUSMAN, D. E., AND SHAW, D. J. (1988). Localization of a human Na+, K+-ATPase (Y subunit gene to chromosome lSq12+q13.2 and linkage to the myotonic dystrophy locus. Genomics 3: 380-384. HEIMAN-PA?TERSON, T., MARTINO, C., ROSENBERG, H., FLETCHER, J. E., AND TAHMOUSH, A. J. (1988b). Malignant hyperthermia in myotonia congenita. Neurology 38: 810-812. HEIMAN-PATTERSON, T., ROSENBERG, H., FLETCHER, J. E., AND TAHMOUSH, A. J. (1988a). Halothane-caffeine contracture testing in neuromuscular diseases. Muscle Nerve 11: 453-457. HOLM, C., KIRCHGESSNER, T. G., SVENSON, K. L., FREDRIKSON, G., NILSSON, S., MILLER, C. G., SHNELY, J. E., HEINZ-
GENETIC
17.
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
HETEROGENEITY
MANN, C., SPARKES, R. S., MOHANDAS, T., LUSIS, A. J., BELFRAGE, P., AND SCHOTZ, M. C. (1988). Hormone-sensitive lipase: Sequence, expression, and chromosomal localization to 19 cent-13.3. Science 241: 1503-1506. JOHNSON, K., JONES, P. J., SPURR, N., NIMMO, E., et al. (1988). Linkage relationships of the protein kinase C gamma gene which exclude it as a candidate for myotonic dystropy. Cytogenet. Cell Genet. 48: 13-15. KAUSCH, K., GRIMM, T., JANKA, M., LEHMANN-HORN, F., WIERINGA, B., AND MILLER, C. R. (1990). Evidence for linkage of the central core disease locus to chromosome 19q. J. Neurol. Sci. D&k 549. KING, J. O., AND DENBOROUGH, M. A. (1973). Anesthesia induced malignant hyperpyrexia in children. J. Pediutr. 83: 3740. KING, J. D., DENBOROUGH, M. A., AND ZAPF, P. W. (1972). Inheritance of malignant hyperpyrexia. hncet 1: 365-370. KOCH, M., HARLEY, H., SARFARZAI, M., BENDER, K., WIENKER, T., ZOLL, B., AND HARPER, P. S. (1989). Myotonia congenita (Thomsen’s disease) excluded from the region of the myotonic dystrophy locus on chromosome 19. Hum. Genet. 82: 163-166. LATHROP, G. M., LALOUEL, J. M., JULIER, C., AND OTT, J. (1984). Strategies for multilocus linkage analysis in humans. Proc. Natl. Acad. Sci. USA 81: 3443-3446. LEBEAU, M. M., RYAN, D., JR., AND -RICK-VANCE, M. A. (1989). Report of the committee on the genetic constitution of chromosomes 18 and 19. Cytogenet. Cell Genet. 61: 338357. MACLENNAN, D. H., DUFF, C., ZOFUATO, F., F~JJII, J., et al. (1990). Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia. Nature 343: 559-561. MARKS, A. R., TEMPST, P., HWANG, K. S., TAIJBMAN, M. B., INUI, M., CHADWICK, C., FLEISCHER, S., AND NADAL-GINARD, B. (1989). Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 86: 8683-8687. MCCARTHY, T. V., SANDRA HEALY, J. M., HEFFRON, J. J. A., LEHANE, M., DEUFEL, T., LEHMANN-HORN, F., FARRALL, M., AND JOHNSON, K. (1990). Localization of malignant hyperthermia susceptibility locus to human chromosome 19q1213.2. Nature 343: 562-564. MCPHERSON, E., AND TAYLOR, C. A. (1982). The genetics of malignant hyperthermia: Evidence for heterogeneity. Am. J. Med. Genet. 11: 273-285. MOULDS, R. F. W., AND DENBOROUGH, M. A. (1974). Identification of susceptibility to malignant hyperpyrexia. Br. Med. J. 2: 245-247.
IN
29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
MHS
547
NAKAMURA, Y., LATHROP, M., O’CONNELL, P., LEPPERT, M., LALOUEL, J. M., AND WHITE, R. (1988). A primary map of ten DNA markers and two serological markers for human chromosome 19. Genomics 3: 67-71. SAIDMAN, L. J., HAVAFQ E. S., AND EGER, E. I. (1964). Hyperthermia during anesthesia. JAMA 190: 1029-1032. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S. J., HIGUCHI, R., HORN, G. T., MULLIS, K. B., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. SCHEPENS, J., SMEETS, H., HULSEBOS, T., BRUNNER, H., AND WIERINGA B. (1987). Isolation of a polymorphic DNA sequence pJSBl1 (D19S16) from the human chromosome 19cen-q13.2 region linked to the myotonic dystrophy (DM) gene. Nucleic Acids Res. 16: 3192. SCHONK, D., COERWINKEL-DRIESSEN, M., VAN DALEN, I., OERLJZMANS, F., SMEETS, B., SCHJZPENS, J., HULSEBOS, T., COCKBURN, D., BOYD, Y., DAVID, M., RETTIG, W., SHAW, D., ROSES, A., ROPERS, H., AND WIEFUNGA, B. (1989b). Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q. Genomics 4: 384-396. SCHONK, D., VAN DIJK, P. E., RIEGMAN, P., TRAPMAN, J., HOLM, C., CRAIG, I. W., SILLEKENS, P., VAN VENROOY, W., ROPERS, H. H., AND WIEXUNGA, B. (1989a). Subregional assignment of six genes on chromosome 19q. Cytogenet. Cell Genet. 51: 1075. SHAW, D. J., MEREDITH, A. L., SARFARAZI, M., HARLEY, H. G., HUSON, S. M., BROOK, J. D., BIJF~ON, L., Lrrr, M., MOHANDAS, T., AND HARPER, P. S. (1986). Regional localizations and linkage relationships of seven RFLPs end myotonic dystrophy on chromosome 19. Hum. Genet. 74: 262-266. SHOWS, T. B., AND MCALPINE, P. J. (1982). The 1981 catalogue of assigned human genetic markers and report of the nomenclature committee. Cytogenet. Cell Genet. 32: 221-245. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517. WAINWRIGHT, B. J., WATSON, E. K., SHEPHARD, E. A., AND PHILLIPS, I. R. (1985). RFLP for a human cytochrome P-450 gene at 19ql3.1-qter (HGMS provisional designation CYPI). Nucleic Acids Res. 13: 4610. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 388-396. WEBER, J. L., MAY, P. E., AND KAPPEL, C. (1990). Dinucleotide repeat polymorphism at the D19S49 locus. Nucleic Acids Res. 18: 1927. WEBER, J. L. (1990). Dinucleotide repeat polymorphism at the D19S75 locus. Nucleic Acids Res. 18: 4639.
