GENOMICS
12,335~3%
(19%)
Linkage Analysis of Spinal Muscular Atrophy R. J. DANIELS,* N. H. THOMAS,t R. N. MACKINNON * T. LEHNER,$ J OTT,* T. J. FLINT,* V. DUBOWlTZ,t J. IGNATIUS,~ M. DONNER,~~ K. ZERRES,~ M. RIETSCHEL,~ W. 0. C. COOKSON,~ L. M. BRZUSTOWICZ,# T. C. GILLIAM,# AND K. E. DAVIES* *Mo/ecu/ar Genetics Group, institute of Molecular Medicine, lohn Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom; tRoyal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, WI2 OHS, United Kingdom; *Department of Psychiatry, Columbia University, 722 West 168th Street, New York, New York 10032; SDepartment of Medical Genetics, Vaestoftitto, Kalevankatu 16, SF-00700 Helsinki, Fin/and; “Gyldenintie 8 8 21, SF-00200 Helsinki, Fin/and; %siitut fur Humangenetik der Universitat Bonn, Wilhelmstrasse 37, 8300 Bonn 1, Federal Republic of Germany;lINufie/d Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom; and #College of Physicians and Surgeons, 722 West 168th Street, Columbia University, New York, New York 10032 Received
June 12, 1991;
revised
Linkage data between four markers on chromosome 5 confirm and extend our previous studies that localized the mutation in spinal muscular atrophy to 5q11.2-q13.3. Localization of D5S6 by in situ hybridization refines the mapping of the defective gene to the region 5q12.2-q13. We also report the use of a highly informative PCR-based polymorphism with five alleles. This RFLP will be particularly useful for prenatal diagnosis where only old tissue samples from af-
fected individuals are available. The high heterozygosity of this locus should also assistin identifying recombinants that will Academic
refine
the genetic
mapping
of the mutation.
o 1992
Press. Inc.
INTRODUCTION The childhood spinai muscular atrophies (SMA) are a group of disorders inherited through an autosomal recessive mechanism and characterized by degeneration of the anterior horn cells and symmetrical weakness and wasting of voluntary muscles. Three broad groups can be defined on the basis of their clinical severity but these merge with and overlap each other (Dubowitz, 1978, 1989). Severe SMA (type I; acute SMA; Werdnig-Hoffmann disease) has an onset before birth or in early infancy, the affected child is severely paralyzed and never achieves the ability to sit or stand unaided and normally dies within the first few years of life. Individuals affected by the intermediate form (type II) achieve the ability to sit but not to walk unaided. Their prognosis depends on the degree of respiratory involvement but they may survive into adolescence and adulthood. Mild SMA (type III; Kugelberg-Welander disease; chronic SMA) has a later onset, patients achieve the ability to walk and usually have a good long-term prognosis, dependent on the degree of muscle weakness and of associated respiratory involvement,
October
1, 1991
We have demonstrated that all three types of SMA map to the same region of chromosome 5 at 5q11.2-q13.3 (Brzustowicz et al., 1990; Gilliam et aZ., 1990). A similar localization has been found by other investigators (Melki et al., 1990a,b). Here, we present an extension of these analyses in 31 more families and the fine mapping of one of the closely linked loci, and we report the use of a highly informative polymorphism (D5S204) that should be very helpful in the prenatal diagnosis of this disorder. MATERIALS
AND METHODS
Clinical details. The families were studied by clinical teams in the United Kingdom, Germany, Finland, and the United States. The diagnosis was established on the basis of clinical examination showing symmetrical proximal muscle weakness with absence of upper cranial nerve involvement. Hype- or areflexia was present. Patients with unusual distribution of weakness were excluded as were patients with other central nervous system abnormalities. The affected individuals were classified into severe, intermediate, and mild on the basis of maximal motor performance as outlined above. The diagnosis was confirmed by electromyography and/or muscle biopsy in at least one affected member of the family. Thirty-one families containing 69 affected individuals were studied. One family had 3 children affected by severe SMA. Thirteen families were affected by intermediate SMA and 6 by mild SMA, and 11 families had affected children of both intermediate and mild severity within the same sibship. The ages of onset and motor milestones are summarized in Table 1. The clinical details of the 16 families (40 affected children) used in our previous studies are described elsewhere (Munsat et ai, 1990). In situ hybridization. pM4 DNA was labeled with L3H]dCTP by nick-translation (Rigby et al., 1977), using a kit (Amersham), to a specific activity of 5 X 10’ cpm/gg. A slight human repetitive element in the insert was competed out with sonicated human DNA (Sealey et al., 1985) as described in Callen et al. (1988). Competed probe was hybridized in situ to normal male prometaphase chromosomes, at 0.2 ng/).d, as described in Simmers et al. (1986), except that denatured chromosomes were first quenched in ice-cold 2X SSC, pH 7.0, before dehydration in an ice-cold graded ethanol series. Slides were dipped in Ilford L4 photographic emulsion diluted 1:l with water and exposed for 8 days. Chromosome spreads were banded after in situ hybridization by the method of Zabel et al. (1983).
335 All
OSSS-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
336
DANIELS
ET
AL.
DNA analysis. DNA was prepared from lymphocytes or EBVtransformed cell lines by methods described previously (Kunkel et al., 1977; Patterson et aZ., 1989). After digestion with the appropriate restriction enzyme, samples were electrophoresed in 0.8% agarose gels and Southern blotted onto Hybond N (Amersham International). Probes were randomly primed according to the method of Feinberg and Vogelstein (1983) and hybridized to the filters in 50% formamide, 4X SSC, 50 &sodium pyrophosphate, 8% dextran sulfate, 10X Denhardt’s at 42°C. Filters were washed in 1X SSC, 0.1% SDS at 65°C and autoradiographed for l-3 days. The probes used were pM4 (D5S6), JK53 (D5S112), p105-153Ra (D5S39), and p105-798Rb (D5S78) (Brzustowicz et aZ., 1990; Gilliam et al., 1990). PCR analysis. One 21-mer and one 22-mer primers flanking a region of 212-220 bp from the D5S204 locus were used in the amplification (Mankoo et al., 1990). The conditions were as follows: 50 ng DNA, 5 pmol of each primer, 1.5 mM MgCl,, 10 n&f Tris-HCl, pH 8.3, 50 mM KCl, 200 aM dNTPs, 1 unit Toq polymerase (Perkin-Elmer Cetus), 0.01% gelatin, and 1 ~1 of [?S]dATP at >lOOO Ci/mmol (Amersham) in a 25-~1 reaction. The template was amplified for 30 cycles with denaturation at 94’C, annealing at 6O”C, and extension at 72°C all for 1 min each, with one further cycle that included a 4-min 72°C extension. Half of 6-pl reaction volumes were run on 6% denaturing acrylamide gels at 50 W for 4 h and autoradiographed overnight or longer at room temperature. Linkage analysis. Linkage analysis including both two-point and multipoint analysis was carried out using the LINKAGE (Version 5.1) group of programs (Lathrop et al., 1984). For homogeneity testing the HOMOG program was used (Ott, 1985). The gene frequency estimate was used as previously published (Brzustowicz et al., 1990). RESULTS
Recent mapping in the SMA region of chromosome 5 has shown that there is a highly informative microsatellite polymorphism closely linked to D5S39 (Mankoo et al., 1990). This locus, D55204, maps in the same bacteriophage clone as D5S39. The products of the PCR amplification across this region were analyzed on a 6% acrylamide gel. A typical run is shown in Fig. 1. PCR products are only seen when the region of chromosome 5 containing this locus is present. The lane labeled chr5del contains the hybrid deleted for the region 5q11.2-q13.3 (Gilliam et aZ., 1989). Although there are a large number of background bands, the alleles can be clearly typed. In the 20 families we have studied with this marker, we have seen five alleles and 17 of the families were informative. This therefore is a valuable new marker for the prenatal diagnosis of the diseaseand should greatly facilitate the fine mapping of the mutation. The families used in this analysis were additional to those previously reported (Brzustowicz et al., 1990; Gilliam et al., 1990) and were predominantly families with two affected children with normal sibs. The clinical details of these new families are given in Table 1. In addition to D5S204, families were typed with the markers D5S6, D5S112, D5S39, and D5S78. Examples of families recombinant with these markers are shown in Fig. 2. These data are a summation of our previous studies with the additional families described here. Because D5S204 was isolated from the same phage as D5S39, it can be assumed to be the same locus for genetic analysis. (There were no recombinants between these two loci in this data set.) The two-point lod scores are given in Ta-
FIG. 1. Analysis of the microsatellite at the D5S204 locus (see Materials and Methods for details). (A) Segregation analysis in a family together with hybrids containing a whole or deleted human chromosome 5. (B) Segregation of D5S204 in a second family. The alleles of the mother were typed on another gel. She passed allele 2 (which came from her mother who is homozygous for allele 2 as shown) to all her children.
ble 2, and the multipoint mapping analysis dividing linked and unlinked families is given in Fig. 3. Multipoint analysis was performed and the resulting multipoint lod scores were used for heterogeneity testing. The heterogeneity test found strong evidence for a mixture of linked and unlinked families (Table 3). The estimate of the proportion of linked families is (Y = 85%. To represent lod scores and support for the map position, X, of the SMA gene under heterogeneity, we calculated Z(X) = max, Z(a,X), where Z(a,X) is the bivariate log,, likelihood with respect to (x and X. In Fig. 3 we plot such lod scores under heterogeneity as well as lod scores under
LINKAGE
ANALYSIS
OF
SPINAL
TABLE Clinical
MUSCULAR
1
Details
of Patients Course
Number of affected individuals
Number of families Severe
1
SMA
Intermediate
Mild
SMA
Families of mixed severity intermediate/ mild SMA
of disease
Mean age of onset (Range)
milestones
16
-
49.6 (4-156) 11.8 (6-17) 16.2 (6-36) 21.6 (12-36)
12 inter 24 12 mild
the assumption of homogeneity (CX= 1). The maximum lod score under heterogeneity Z = 26.44 occurs at the order 5cen-D5S6-SMA-D5S112-D5S39-D5S78 at a distance of 0.02 CM distal to D5S6 (Fig. 3). The 22
(all in months) Mean age of walking (Range)
Mean age of sitting (Range)
(7-910) -
26
13
and motor
Mean age of death (Range)
3
13 12 mutliplex 1 consanguine 6
SMA
337
ATROPHY
-
-
9.8 (4-42) 15.4
9.9 (4-18)
-
(12-M)
7.2
12.8 (mild
(6-10)
(11-18)
support interval for the position of the susceptibility gene for SMA was calculated as defined by Conneally et al. (1985). To refine the mapping of the SMA gene within the
A
D5S6 D5Sl12 D55204 D5S39 D5S76
w
B 2kf%5 52 JK 45 DD FG
I
cc
AC JK 22 EE GF
I
Bc JK 34 DE Gi=
KK 25 ED GF
D5S6 D5Sll2 D5S204 D5S39 D5S76
i 2491 BC JK 42 DE GF
2492 2493 2494 CA BC BA KJ JK JJ 52 42 42 DE DE FG :F’ GF Family 10 (type II).
2516 cc KK 53 DE FF Family
izso 2573
2574 BB KK 22 EE GF Family
2575 BB KK 22 EE FF 22 (type Ill)
FIG.
2.
2517 cc KK 53 DE FG 2 (type II).
2331 BB JJ 41 DE GF Family
2339 2419 BB BB JJ JK 41 41 DE DE GF GF 26 (type ll/lll).
I
Bc KK 43
2343 BC JK 43 DO FG
2352 AB I
44 DE FF
GF
2L!AC KK 13 DD FF
F 2613 PA KK 13 DD FF Family
2RBB KK 43 DE GF
4
27
2614 AA KK 33 DD FG (type 11-111)
(A, B) Examples
of marker
F 2317 BA KK 44 DD GF Family
44 DD FG
2318 BA KJ 41 DE GF 34 (type II).
typings
only)
in SMA
2368 CB 43 DD FG
2344 CB KK 43 DD ffi
2346 BB JJ 44 DD FG
33 DD FG
43 DD FG
2347 BB JJ 44 DD FF
2351 BB JJ 44 DD FF Family
families
where
recombinants
2353 BB JJ 44 DD FF 45
2356 CC KK 33 DD GF (type
were
11-111)
observed.
GF
31 DE GF
2354 CC KK 33 DD GF
2345 BA JK 31 DE GF
338
DANIELS
ET
TABLE Two-Point
SMA SMA SMA SMA SMA
vs vs vs vs vs
D5S6 D5S112 D5S39 D5S78 D5S204
Lod
Scores
between
5 Markers
0.10
0.20
0.30
-02 -cr -02 -cc -02
12.88 1.67 3.03 -2.60 4.59
16.73 5.76 7.68 5.49 5.86
14.33 5.07 6.65 5.44 5.31
8.89 2.99 3.95 3.78 3.34
4.27 1.31 1.84 2.01 1.51
TABLE
3
Heterogeneity
Test Estimates
Maximum InL
CY
60.8822 50.7555 (0) Components
H2 vs Hl heterogeneity 0.0000 Hl vs HO linkage 0.0000 H2 vs HO total
and Chromosome
0.05
Previous linkage analyses of chromosome 5 markers have shown that SMA lies in the region close to the loci D5S6, D5S112, and D5S39. Mapping studies using somatic cell hybrids deleted for the region 5q11.2-q13.3 localized D5S6 and D5S39 within the deleted segment (Gilliam et al., 1989). The in situ hybridization result presented here refines the localization of D5S6 to 5q12.2-q13. These data are consistent with those recently found by Mattei et al. (1991) and suggest that the SMA locus probably lies at some distance from the centromere. Thus, this is the region that we are targeting for microdissection experiments (Ludecke et al., 1989). Taking into account heterogeneity places the susceptibility gene between D5S6 and D5S112, with a maximum lod score of Z = 26.44 and a support interval that is entirely confined between D5S6 and D5S112. Pooling of the data in this paper with analysis from other laborato-
Source
SMA
0.001
DISCUSSION
H2: Linkage, heterogeneity Hl: Linkage, homogeneity HO: No linkage
2
0.00
region 5cen-q13, we performed in situ hybridization with the probe pM4, which detects the D5S6 locus. The results, which are illustrated in Fig. 4, show that D5S6 lies at 5q12-q13.1, toward the interface of these two bands.
Hypotheses
AL.
ries, and the use of more informative markers as well as typing more markers proximal to D5S6 will be needed to obtain better confidence of disease location and heterogeneity assessment. In view of the fact that many of the families coming for prenatal diagnosis are affected by Werdnig-Hoffmann disease, the affected child is often dead. A PCRbased polymorphism that can be used on fixed tissue specimens or dried blood spots is therefore essential. The one described here (D5S204) is highly polymorphic in our families and will prove to be a very useful marker for both the prenatal diagnosis of SMA and the refinement of the linkage map. We are currently searching for other PCR-based polymorphisms to develop an accurate set of flanking markers for the disease in the future.
ACKNOWLEDGMENTS We are grateful to Drs. Somer, Louhimo, and Krusus for patient referrals and to Dr. Robin Sherrington for access to unpublished data. We thank Graeme Suthers for helpful comments and Helen Blaber for assistance in the preparation of this manuscript. We also thank the Medical Research Council of Great Britain, the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association of the United States of America, the Finnish Muscular Dystrophy Association, the Deutsche Forschungsgeneinschaft, and the Deutsche Gesellschaft Bekampfung der Muskelkrankheiten, and the U.S. National Center for Human Genome Research (Grant HGOOOOSS) for financial support.
of
0
0.8500 (1) (0)
0.0020 -0.1116 (0.5)
x2
Likelihood ratios
of x2
df 1 1 2
20.254 101.511 121.764
-0.5
25009 l.lE + 22 2.8E + 26
Note. The HOMOG program was used to calculate the log likelihood (1nL) under the appropriate hypotheses, where (Y is the proportion of families with the disease locus linked to the given markers. (Maker D5S6 represents map location 0.00.)
0
Genetic -
map
Heterogenity
FIG. 3. Multipoint linkage analysis five DNA markers. The program used AGE package (Lathrop et al., 1984). As with the disease locus, they are located order D5S6-D5S112-D5S39-D5S78.
0.5
in Morgans +
Homogeneity
of the SMA disease locus with was LINKMAP of the LINKthe loci all show recombinants at the dip of the curves in the
LINKAGE
ANALYSIS
OF
SPINAL
MUSCULAR
339
ATROPHY
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
15.3 152 15.1
Gilliam, T. C., Freimer, N. B., Kaufmann, C. A., Powchik, P. P., Bassett, A. S., Bengtsson, U., and Wasmuth, J. J. (1989). Deletion mapping of DNA markers to a region of chromosome 5 that cosegregates with schizophrenia. Genomics 5: 940-944.
14 13.3 13.2 13.1 12 11 11.1
Gilliam, T. C., Brzustowicz, L. M., Castilla, L. H., Lehner, T., Penchaszadeh, G. K., Daniels, R. J., Byth, B. C., Knowles, J., Hislop, J. E., Shapira, Y., Dubowitz, V., Munsat, T. L., Ott, J., and Davies, K. E. (1990). Genetic homogeneity between acute and chronic forms of spinal muscular atrophy. Nature 345: 823-825.
112
Kunkel, L. M., Smith, K. D., Boyer, S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, 0. J., Breg, W. R., et al. (1977). Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natl. Acad. Sci. USA 74: 1245-1249.
12 13.1 13.2
Lathrop, G. M., Lalouel, for multilocus linkage USA 81: 3443-3446.
13.3 14
J. M., Julier, C., and Ott, J. (1984). analysis in humans. Proc. Natl.
Strategies Acad. Sci.
Ludecke, H.-J., Senger, G., Claussen, U., and Horsthemke, B. (1989). Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338: 348-350. Mankoo, B. S., Sherrington, R., De La Concha, A., Kalsi, G., Curtis, D., Melmer, G., and Gurling, H. M. D. (1990). Two microsatellite polymorphisms at the D5S39 locus. Nuc&c Acids Rex 19: 1963.
15
21
22 23.1
Mattei, M.-G., Melki, J., Bachelot, M.-F., Abdelhak, S., Burlet, P., Frezal, J., and Munnich, A. (1991). In-situ hybridization of two markers closely flanking the spinal muscular atrophy gene to 5q12q13.3. Cytogenet. Cell Genet. 57: 112-113.
23.2 23.3 31.1
Melki, J., Abdelhak, S., Sheth, P., Bachelot, M. F., Burlet, P., Marcadet, A., Aicardi, J., Barois, A., Carriere, J. P., Fardeau, M., Fontan, D., Ponsot, G., Billette, T., Angelini, C., Barbosa, C., Ferriere, G., Lanzi, G., Ottolini, A., Babton, M. C., Cohen, D., Hanauer, A., Clerget-Darpoux, F., Lathrop, M., Munnich, A., and Frezal, J. (1990a) Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 344: 767-768.
31.2 31.3 32 33.1 33.2 33.3 34
Melki, J., Sheth, P., Abdelhak, S., Burlet, P., Bachelot, Lathrop, M. G., Frezal, J., Munnich, A. (199Ob). Mapping (type 1) spinal muscular atrophy to chromosome 5q12-q14. 336: 271-273.
35.1 35.2 35.3
5 FIG. 4. An ideogram representing the distribution of silver grains, scored over 19 metaphases each with at least one silver grain over chromosome 5.
Munsat, T. L., Skerry, L., Korf, B., Pober, B., Schapira, Y., Gascon, G. G., Al-Rajeh, S. M., Dubowitz, V., Davies, K., Brzustowicz, L. M., Penchaszadeh, G. K., and Gilliam, T. C. (1990). Phenotypic heterogeneity of spinal muscular atrophy mapping to chromosome 5q11.2-13.3 (SMA 5q). Neurology 40: 1831-1836. Ott, J. (1985). “Analysis Univ. Press, Baltimore.
REFERENCES Brzustowicz, L. M., Lehner, T., Castilla, L. H., Penchaszadeh, G. K., Wilhelmsen, K. C., Daniels, R., Davies, K. E., Leppert, M., Ziter, F., Wood, D., Dubowitz, V., Zerres, K., Hausmanowa-Petrusewicz, I., Ott, J., Munsat, T. L., and Gilliam, T. C. (1990). Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Nature344: 540-541. Callen, D. F., Hyland, V. J., Baker, E. G., Fratini, A., Simmers, R. N., Mulley, J. C., and Sutherland, G. R. (1988). Fine mapping of gene probes and anonymous DNA fragments to the long arm of chromosome 6. Genomics 2: 144-153. Conneally, P. M., Edwards, J. H., Kidd, K. D., Lalouel, J.-M., Morton, N. E., Ott, J., and White, R. (1985). Report of the committee on methods of linkage analysis and reporting. Cytogenet. Cell Genet. 40: 356-359. Dubowitz, V. (1978). “Muscle Disorders in Childhood,” pp. 146-178, Saunders, London/Philadelphia. Dubowitz, V. (1989). “A Colour Atlas of Muscle Diseases in Childhood,” Wolfe Medical Books, London.
M.-F., of acute Lancet
of Human
Genetic
Linkage,”
Johns
Hopkins
Patterson, M. N., Bell, M. V., Bloomfield, J., Flint, T., Dorkins, H., Thibodeau, S., Schwartz, C., Weiringa, B., Ropers, H.-H., Callen, D. F., Sutherland, G., Froster-Iskenius, U., Vissing, H., and Davies, K. E. (1989). Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome. Genomics 4: 570-578. Rigby, P. W. J., Dieckmann, Labeling deoxyribonucleic nick translation with DNA Sealey, P. G., Whittaker, of repeated sequences 13: 1905-1922.
M., Rhodes, C., and Berg, P. (1977). acid to high specific activity in vitro by polymerase I. J. Mot. Biot. 113: 237-251.
P. A., and Southern, E. M. (1985). Removal from hybridisation probes. Nucleic Acids Res.
Simmers, R. N., Stupans, I., and Sutherland, G. R. (1986). Localization of the human haptoglobin genes distal to the fragile site at 16q22 using in situ hybridization. Cytogenet. Cell Genet. 41: 3&41. Zabel, B. U., Naylor, S. L., Sakaguchi, A. Y., Bell, G. I., and Shows, T. B. (1983). High-resolution chromosome localization of human genes for amylase, proopiomelanocortin, somatostatin and a DNA fragment (D3Sl) by in situ hybridization. Proc. Natt. Acad. Sci. USA 80: 6932-6936.
12,335~3%
(19%)
Linkage Analysis of Spinal Muscular Atrophy R. J. DANIELS,* N. H. THOMAS,t R. N. MACKINNON * T. LEHNER,$ J OTT,* T. J. FLINT,* V. DUBOWlTZ,t J. IGNATIUS,~ M. DONNER,~~ K. ZERRES,~ M. RIETSCHEL,~ W. 0. C. COOKSON,~ L. M. BRZUSTOWICZ,# T. C. GILLIAM,# AND K. E. DAVIES* *Mo/ecu/ar Genetics Group, institute of Molecular Medicine, lohn Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom; tRoyal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, WI2 OHS, United Kingdom; *Department of Psychiatry, Columbia University, 722 West 168th Street, New York, New York 10032; SDepartment of Medical Genetics, Vaestoftitto, Kalevankatu 16, SF-00700 Helsinki, Fin/and; “Gyldenintie 8 8 21, SF-00200 Helsinki, Fin/and; %siitut fur Humangenetik der Universitat Bonn, Wilhelmstrasse 37, 8300 Bonn 1, Federal Republic of Germany;lINufie/d Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom; and #College of Physicians and Surgeons, 722 West 168th Street, Columbia University, New York, New York 10032 Received
June 12, 1991;
revised
Linkage data between four markers on chromosome 5 confirm and extend our previous studies that localized the mutation in spinal muscular atrophy to 5q11.2-q13.3. Localization of D5S6 by in situ hybridization refines the mapping of the defective gene to the region 5q12.2-q13. We also report the use of a highly informative PCR-based polymorphism with five alleles. This RFLP will be particularly useful for prenatal diagnosis where only old tissue samples from af-
fected individuals are available. The high heterozygosity of this locus should also assistin identifying recombinants that will Academic
refine
the genetic
mapping
of the mutation.
o 1992
Press. Inc.
INTRODUCTION The childhood spinai muscular atrophies (SMA) are a group of disorders inherited through an autosomal recessive mechanism and characterized by degeneration of the anterior horn cells and symmetrical weakness and wasting of voluntary muscles. Three broad groups can be defined on the basis of their clinical severity but these merge with and overlap each other (Dubowitz, 1978, 1989). Severe SMA (type I; acute SMA; Werdnig-Hoffmann disease) has an onset before birth or in early infancy, the affected child is severely paralyzed and never achieves the ability to sit or stand unaided and normally dies within the first few years of life. Individuals affected by the intermediate form (type II) achieve the ability to sit but not to walk unaided. Their prognosis depends on the degree of respiratory involvement but they may survive into adolescence and adulthood. Mild SMA (type III; Kugelberg-Welander disease; chronic SMA) has a later onset, patients achieve the ability to walk and usually have a good long-term prognosis, dependent on the degree of muscle weakness and of associated respiratory involvement,
October
1, 1991
We have demonstrated that all three types of SMA map to the same region of chromosome 5 at 5q11.2-q13.3 (Brzustowicz et al., 1990; Gilliam et aZ., 1990). A similar localization has been found by other investigators (Melki et al., 1990a,b). Here, we present an extension of these analyses in 31 more families and the fine mapping of one of the closely linked loci, and we report the use of a highly informative polymorphism (D5S204) that should be very helpful in the prenatal diagnosis of this disorder. MATERIALS
AND METHODS
Clinical details. The families were studied by clinical teams in the United Kingdom, Germany, Finland, and the United States. The diagnosis was established on the basis of clinical examination showing symmetrical proximal muscle weakness with absence of upper cranial nerve involvement. Hype- or areflexia was present. Patients with unusual distribution of weakness were excluded as were patients with other central nervous system abnormalities. The affected individuals were classified into severe, intermediate, and mild on the basis of maximal motor performance as outlined above. The diagnosis was confirmed by electromyography and/or muscle biopsy in at least one affected member of the family. Thirty-one families containing 69 affected individuals were studied. One family had 3 children affected by severe SMA. Thirteen families were affected by intermediate SMA and 6 by mild SMA, and 11 families had affected children of both intermediate and mild severity within the same sibship. The ages of onset and motor milestones are summarized in Table 1. The clinical details of the 16 families (40 affected children) used in our previous studies are described elsewhere (Munsat et ai, 1990). In situ hybridization. pM4 DNA was labeled with L3H]dCTP by nick-translation (Rigby et al., 1977), using a kit (Amersham), to a specific activity of 5 X 10’ cpm/gg. A slight human repetitive element in the insert was competed out with sonicated human DNA (Sealey et al., 1985) as described in Callen et al. (1988). Competed probe was hybridized in situ to normal male prometaphase chromosomes, at 0.2 ng/).d, as described in Simmers et al. (1986), except that denatured chromosomes were first quenched in ice-cold 2X SSC, pH 7.0, before dehydration in an ice-cold graded ethanol series. Slides were dipped in Ilford L4 photographic emulsion diluted 1:l with water and exposed for 8 days. Chromosome spreads were banded after in situ hybridization by the method of Zabel et al. (1983).
335 All
OSSS-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. rights of reproduction in any form reserved.
336
DANIELS
ET
AL.
DNA analysis. DNA was prepared from lymphocytes or EBVtransformed cell lines by methods described previously (Kunkel et al., 1977; Patterson et aZ., 1989). After digestion with the appropriate restriction enzyme, samples were electrophoresed in 0.8% agarose gels and Southern blotted onto Hybond N (Amersham International). Probes were randomly primed according to the method of Feinberg and Vogelstein (1983) and hybridized to the filters in 50% formamide, 4X SSC, 50 &sodium pyrophosphate, 8% dextran sulfate, 10X Denhardt’s at 42°C. Filters were washed in 1X SSC, 0.1% SDS at 65°C and autoradiographed for l-3 days. The probes used were pM4 (D5S6), JK53 (D5S112), p105-153Ra (D5S39), and p105-798Rb (D5S78) (Brzustowicz et aZ., 1990; Gilliam et al., 1990). PCR analysis. One 21-mer and one 22-mer primers flanking a region of 212-220 bp from the D5S204 locus were used in the amplification (Mankoo et al., 1990). The conditions were as follows: 50 ng DNA, 5 pmol of each primer, 1.5 mM MgCl,, 10 n&f Tris-HCl, pH 8.3, 50 mM KCl, 200 aM dNTPs, 1 unit Toq polymerase (Perkin-Elmer Cetus), 0.01% gelatin, and 1 ~1 of [?S]dATP at >lOOO Ci/mmol (Amersham) in a 25-~1 reaction. The template was amplified for 30 cycles with denaturation at 94’C, annealing at 6O”C, and extension at 72°C all for 1 min each, with one further cycle that included a 4-min 72°C extension. Half of 6-pl reaction volumes were run on 6% denaturing acrylamide gels at 50 W for 4 h and autoradiographed overnight or longer at room temperature. Linkage analysis. Linkage analysis including both two-point and multipoint analysis was carried out using the LINKAGE (Version 5.1) group of programs (Lathrop et al., 1984). For homogeneity testing the HOMOG program was used (Ott, 1985). The gene frequency estimate was used as previously published (Brzustowicz et al., 1990). RESULTS
Recent mapping in the SMA region of chromosome 5 has shown that there is a highly informative microsatellite polymorphism closely linked to D5S39 (Mankoo et al., 1990). This locus, D55204, maps in the same bacteriophage clone as D5S39. The products of the PCR amplification across this region were analyzed on a 6% acrylamide gel. A typical run is shown in Fig. 1. PCR products are only seen when the region of chromosome 5 containing this locus is present. The lane labeled chr5del contains the hybrid deleted for the region 5q11.2-q13.3 (Gilliam et aZ., 1989). Although there are a large number of background bands, the alleles can be clearly typed. In the 20 families we have studied with this marker, we have seen five alleles and 17 of the families were informative. This therefore is a valuable new marker for the prenatal diagnosis of the diseaseand should greatly facilitate the fine mapping of the mutation. The families used in this analysis were additional to those previously reported (Brzustowicz et al., 1990; Gilliam et al., 1990) and were predominantly families with two affected children with normal sibs. The clinical details of these new families are given in Table 1. In addition to D5S204, families were typed with the markers D5S6, D5S112, D5S39, and D5S78. Examples of families recombinant with these markers are shown in Fig. 2. These data are a summation of our previous studies with the additional families described here. Because D5S204 was isolated from the same phage as D5S39, it can be assumed to be the same locus for genetic analysis. (There were no recombinants between these two loci in this data set.) The two-point lod scores are given in Ta-
FIG. 1. Analysis of the microsatellite at the D5S204 locus (see Materials and Methods for details). (A) Segregation analysis in a family together with hybrids containing a whole or deleted human chromosome 5. (B) Segregation of D5S204 in a second family. The alleles of the mother were typed on another gel. She passed allele 2 (which came from her mother who is homozygous for allele 2 as shown) to all her children.
ble 2, and the multipoint mapping analysis dividing linked and unlinked families is given in Fig. 3. Multipoint analysis was performed and the resulting multipoint lod scores were used for heterogeneity testing. The heterogeneity test found strong evidence for a mixture of linked and unlinked families (Table 3). The estimate of the proportion of linked families is (Y = 85%. To represent lod scores and support for the map position, X, of the SMA gene under heterogeneity, we calculated Z(X) = max, Z(a,X), where Z(a,X) is the bivariate log,, likelihood with respect to (x and X. In Fig. 3 we plot such lod scores under heterogeneity as well as lod scores under
LINKAGE
ANALYSIS
OF
SPINAL
TABLE Clinical
MUSCULAR
1
Details
of Patients Course
Number of affected individuals
Number of families Severe
1
SMA
Intermediate
Mild
SMA
Families of mixed severity intermediate/ mild SMA
of disease
Mean age of onset (Range)
milestones
16
-
49.6 (4-156) 11.8 (6-17) 16.2 (6-36) 21.6 (12-36)
12 inter 24 12 mild
the assumption of homogeneity (CX= 1). The maximum lod score under heterogeneity Z = 26.44 occurs at the order 5cen-D5S6-SMA-D5S112-D5S39-D5S78 at a distance of 0.02 CM distal to D5S6 (Fig. 3). The 22
(all in months) Mean age of walking (Range)
Mean age of sitting (Range)
(7-910) -
26
13
and motor
Mean age of death (Range)
3
13 12 mutliplex 1 consanguine 6
SMA
337
ATROPHY
-
-
9.8 (4-42) 15.4
9.9 (4-18)
-
(12-M)
7.2
12.8 (mild
(6-10)
(11-18)
support interval for the position of the susceptibility gene for SMA was calculated as defined by Conneally et al. (1985). To refine the mapping of the SMA gene within the
A
D5S6 D5Sl12 D55204 D5S39 D5S76
w
B 2kf%5 52 JK 45 DD FG
I
cc
AC JK 22 EE GF
I
Bc JK 34 DE Gi=
KK 25 ED GF
D5S6 D5Sll2 D5S204 D5S39 D5S76
i 2491 BC JK 42 DE GF
2492 2493 2494 CA BC BA KJ JK JJ 52 42 42 DE DE FG :F’ GF Family 10 (type II).
2516 cc KK 53 DE FF Family
izso 2573
2574 BB KK 22 EE GF Family
2575 BB KK 22 EE FF 22 (type Ill)
FIG.
2.
2517 cc KK 53 DE FG 2 (type II).
2331 BB JJ 41 DE GF Family
2339 2419 BB BB JJ JK 41 41 DE DE GF GF 26 (type ll/lll).
I
Bc KK 43
2343 BC JK 43 DO FG
2352 AB I
44 DE FF
GF
2L!AC KK 13 DD FF
F 2613 PA KK 13 DD FF Family
2RBB KK 43 DE GF
4
27
2614 AA KK 33 DD FG (type 11-111)
(A, B) Examples
of marker
F 2317 BA KK 44 DD GF Family
44 DD FG
2318 BA KJ 41 DE GF 34 (type II).
typings
only)
in SMA
2368 CB 43 DD FG
2344 CB KK 43 DD ffi
2346 BB JJ 44 DD FG
33 DD FG
43 DD FG
2347 BB JJ 44 DD FF
2351 BB JJ 44 DD FF Family
families
where
recombinants
2353 BB JJ 44 DD FF 45
2356 CC KK 33 DD GF (type
were
11-111)
observed.
GF
31 DE GF
2354 CC KK 33 DD GF
2345 BA JK 31 DE GF
338
DANIELS
ET
TABLE Two-Point
SMA SMA SMA SMA SMA
vs vs vs vs vs
D5S6 D5S112 D5S39 D5S78 D5S204
Lod
Scores
between
5 Markers
0.10
0.20
0.30
-02 -cr -02 -cc -02
12.88 1.67 3.03 -2.60 4.59
16.73 5.76 7.68 5.49 5.86
14.33 5.07 6.65 5.44 5.31
8.89 2.99 3.95 3.78 3.34
4.27 1.31 1.84 2.01 1.51
TABLE
3
Heterogeneity
Test Estimates
Maximum InL
CY
60.8822 50.7555 (0) Components
H2 vs Hl heterogeneity 0.0000 Hl vs HO linkage 0.0000 H2 vs HO total
and Chromosome
0.05
Previous linkage analyses of chromosome 5 markers have shown that SMA lies in the region close to the loci D5S6, D5S112, and D5S39. Mapping studies using somatic cell hybrids deleted for the region 5q11.2-q13.3 localized D5S6 and D5S39 within the deleted segment (Gilliam et al., 1989). The in situ hybridization result presented here refines the localization of D5S6 to 5q12.2-q13. These data are consistent with those recently found by Mattei et al. (1991) and suggest that the SMA locus probably lies at some distance from the centromere. Thus, this is the region that we are targeting for microdissection experiments (Ludecke et al., 1989). Taking into account heterogeneity places the susceptibility gene between D5S6 and D5S112, with a maximum lod score of Z = 26.44 and a support interval that is entirely confined between D5S6 and D5S112. Pooling of the data in this paper with analysis from other laborato-
Source
SMA
0.001
DISCUSSION
H2: Linkage, heterogeneity Hl: Linkage, homogeneity HO: No linkage
2
0.00
region 5cen-q13, we performed in situ hybridization with the probe pM4, which detects the D5S6 locus. The results, which are illustrated in Fig. 4, show that D5S6 lies at 5q12-q13.1, toward the interface of these two bands.
Hypotheses
AL.
ries, and the use of more informative markers as well as typing more markers proximal to D5S6 will be needed to obtain better confidence of disease location and heterogeneity assessment. In view of the fact that many of the families coming for prenatal diagnosis are affected by Werdnig-Hoffmann disease, the affected child is often dead. A PCRbased polymorphism that can be used on fixed tissue specimens or dried blood spots is therefore essential. The one described here (D5S204) is highly polymorphic in our families and will prove to be a very useful marker for both the prenatal diagnosis of SMA and the refinement of the linkage map. We are currently searching for other PCR-based polymorphisms to develop an accurate set of flanking markers for the disease in the future.
ACKNOWLEDGMENTS We are grateful to Drs. Somer, Louhimo, and Krusus for patient referrals and to Dr. Robin Sherrington for access to unpublished data. We thank Graeme Suthers for helpful comments and Helen Blaber for assistance in the preparation of this manuscript. We also thank the Medical Research Council of Great Britain, the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Muscular Dystrophy Association of the United States of America, the Finnish Muscular Dystrophy Association, the Deutsche Forschungsgeneinschaft, and the Deutsche Gesellschaft Bekampfung der Muskelkrankheiten, and the U.S. National Center for Human Genome Research (Grant HGOOOOSS) for financial support.
of
0
0.8500 (1) (0)
0.0020 -0.1116 (0.5)
x2
Likelihood ratios
of x2
df 1 1 2
20.254 101.511 121.764
-0.5
25009 l.lE + 22 2.8E + 26
Note. The HOMOG program was used to calculate the log likelihood (1nL) under the appropriate hypotheses, where (Y is the proportion of families with the disease locus linked to the given markers. (Maker D5S6 represents map location 0.00.)
0
Genetic -
map
Heterogenity
FIG. 3. Multipoint linkage analysis five DNA markers. The program used AGE package (Lathrop et al., 1984). As with the disease locus, they are located order D5S6-D5S112-D5S39-D5S78.
0.5
in Morgans +
Homogeneity
of the SMA disease locus with was LINKMAP of the LINKthe loci all show recombinants at the dip of the curves in the
LINKAGE
ANALYSIS
OF
SPINAL
MUSCULAR
339
ATROPHY
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13.
15.3 152 15.1
Gilliam, T. C., Freimer, N. B., Kaufmann, C. A., Powchik, P. P., Bassett, A. S., Bengtsson, U., and Wasmuth, J. J. (1989). Deletion mapping of DNA markers to a region of chromosome 5 that cosegregates with schizophrenia. Genomics 5: 940-944.
14 13.3 13.2 13.1 12 11 11.1
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112
Kunkel, L. M., Smith, K. D., Boyer, S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, 0. J., Breg, W. R., et al. (1977). Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natl. Acad. Sci. USA 74: 1245-1249.
12 13.1 13.2
Lathrop, G. M., Lalouel, for multilocus linkage USA 81: 3443-3446.
13.3 14
J. M., Julier, C., and Ott, J. (1984). analysis in humans. Proc. Natl.
Strategies Acad. Sci.
Ludecke, H.-J., Senger, G., Claussen, U., and Horsthemke, B. (1989). Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338: 348-350. Mankoo, B. S., Sherrington, R., De La Concha, A., Kalsi, G., Curtis, D., Melmer, G., and Gurling, H. M. D. (1990). Two microsatellite polymorphisms at the D5S39 locus. Nuc&c Acids Rex 19: 1963.
15
21
22 23.1
Mattei, M.-G., Melki, J., Bachelot, M.-F., Abdelhak, S., Burlet, P., Frezal, J., and Munnich, A. (1991). In-situ hybridization of two markers closely flanking the spinal muscular atrophy gene to 5q12q13.3. Cytogenet. Cell Genet. 57: 112-113.
23.2 23.3 31.1
Melki, J., Abdelhak, S., Sheth, P., Bachelot, M. F., Burlet, P., Marcadet, A., Aicardi, J., Barois, A., Carriere, J. P., Fardeau, M., Fontan, D., Ponsot, G., Billette, T., Angelini, C., Barbosa, C., Ferriere, G., Lanzi, G., Ottolini, A., Babton, M. C., Cohen, D., Hanauer, A., Clerget-Darpoux, F., Lathrop, M., Munnich, A., and Frezal, J. (1990a) Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 344: 767-768.
31.2 31.3 32 33.1 33.2 33.3 34
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35.1 35.2 35.3
5 FIG. 4. An ideogram representing the distribution of silver grains, scored over 19 metaphases each with at least one silver grain over chromosome 5.
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