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Molecular studies of spinal muscular atrophy
Neuromu.gcu/ar Dtsorderj, Vol. I, No. 2. p p 83-85, Iqql Printed tn Great Britain
0t~60-gt~ 91 $ 3 0 0 * 0.00 (~ 1991 Pergamon Press plc
REVIEW ARTICLE MOLECULAR
STUDIES
OF SPINAL MUSCULAR
ATROPHY
KAY E. DAVIES,* tNEIL H. THOMAS,+ §RACHAELJ. DANIELS* and VICTORDuBowtTz~ *Molecular Genetics Group. institute of Molecular Medicine.John Radcliffe Hospital. Headington. Oxford OX3 9DU; and +Department of Paediatrics and Neonatal Medicine. Royal Postgraduate Medical School. Hammersmith Hospital. Du Cane Road. London WI2 0NN, U.K.
(Received 18 April 1991; accepted 13 June 1991)
Abstract--Spinal muscular atrophy (SMA) is inherited as an autosomal recessive disorder which presents as a severe, intermediate or mild condition. The disease selectively affects the alpha motor neuron but nothing is as yet known about the underlying biochemical defect. Recent genetic studies have mapped all three types of SMA to the same region of human chromosome 5 (Sqll.2-q13.3) raising the possibility that the mutations may be allelic. Polymorphic DNA markers have been charaeterised which are suitable for prenatal diagnosis. This is the first step in the isolation of the mutant gene (or genes) involved in this disorder. Key words: Spinal muscular atrophy, gent mutation, linkage, prenatal diagnosis.
INTRODUCTION
led to tile suggestion that SMA is a result of different non-allclic mutations [3] or a different set of mutations within the same gene [4]. With the advent of molecular techniques to map disease genes, it has become possible to resolve this issue.
The most common form of spinal muscular atrophy is inherited in an autosomal recessive manner. The disease varies in severity and one can define a severe, an intermediate and a mild phenotype on the basis of the ability to sit or MAPPING TIlE MUTATION walk unaided [i}. Severe SMA (also known as Werdnig-Hoffmann disease) has an onset before After searching for linkage throughout the birth or within the first few months of life, and whole human genome, linkage was finally estabthe affected child never sits unaided. The proglished between SMA and markers located on the nosis is poor and the child usually dies within the proximal long arm of human chromosome 5. In first two years of life. The intermediate form has the initial linkage studies linkage was found in a later age of onset and the affected child is able families segregating for the milder forms of the to sit but not walk. The life expectancy is disease [5,6]. Werdnig-Hoffmann families were determined by the degree of respiratory muscle not included because of the lack of large multiply weakness. Mild SMA (Kugelberg-Welander affected kindreds. The problem of small families disease) is characterised by the ability to walk was solved by segregation analysis in consanunaided. The onset is usually after the first year. The clinical course is variable but in some may be guinous pedigrees [7] and by the study of a large number of small families [8]. These analyses very mild with a normal life expectancy. demonstrated that the mutation in all three The incidence of SMA has been estimated to be forms of SMA map to the same region of about I in 25,000 live births with a carrier chromosome 5 at 5q11.2-13.3. The most probfrequency of I in 80 [2]. The phenotypic variation able location for the mutation is between loci seen between families but not within families has D5S6 and D5S39 [5-9]. Figure I shows an ideogram of chromosome 5 summarising the * Author to whom correspondence should be addressed. § Present address: Department of Paediatrics, Guy's DNA markers used in the multipoint linkage Hospital, St Thomas Street, London SEI 9RT, U.K. mapping. The position of SMA relative to the 83
84
Review Article
locus D5S112 remains uncertain at present. Initial data placed SMA distal to D5S i 12 but our more recent results with more informative probes favour a location proximal to D5SII2 (unpublished observations). More markers and the pooling of all family analyses will be needed to resolve this map position. The probes used to detect restriction fragment length polymorphisms (RFLPs) are given in Table 1 together with the percentage of individuals informative at these loci (PIC value). Also included is a dinucleotide PCR based repeat
DNA within i 5 kb of D5S39 and can therefore be considered as the same genetic locus [ ! 0]. D5S204 is informative in approximately 80% of individuals [I0,1 I]. At present there is no evidence for genetic heterogeneity in the disorder [12,13]. The SM A mutations on chromosome 5q present with a broad continuum of clinical abnormalities. This strongly suggests that the mutations causing the differing severities of the disease may be different mutations at the same locus similar to the Table I. Characteristics of DNA polymorphisms for linkage studies
15.3 15.2
Probe
Locus
15.I
.11)t 10H C L407 M4 JK53 pi05 153 Ra
D5S21 D5S63 D5S6 D5S112 D5S39
---LII55
D5S204 D5S51
pi05 798 Rb
D5S78
14
13.3 13.2 13.1 12 11 11.1
D5S21 D5,$63
11.2
Enzyme Msp I Taq I Barn HI Pvu II Msp I Xba I Pst 1 PCR based Taq I Hint II Msp I
PIC Value 0 4t 033 0.52 0.48 0.34 033 04,~ 078 036 0 34 037
D..,~S6
12
D.5~112
13.1
DSS39/D,5~204
13.2
D,5S,51
13.3
DSS78
J sMA
14
15 21 22 23.1 23.2 23.3 31.1
31.2 31,3 32 33.1 33.2 33.3
situation observed for Duchenne and Becker muscular dystrophy [14,15]. Since there is no evidence for significant genetic heterogeneity for autosomal rect~ssive SMA, it is possible to use the markers for prenatal diagnosis. We are performing diagnoses where both parents are informative for the flanking markers D5S6 and D5S39/D5S204 (Daniels and Davies, in preparation). In many cases, however, diagnosis is not possible because the affected child is dead. We are currently developing other PCR based markers in addition to D5S204 which can be typed on tissue samples such as muscle biopsies and Guthrie spots. Since variable number microsatellite repeats occur frequently in the human genome (once in every 60 kb [16]), it is likely that such highly informative markers will be available in the near future. FUTURE PROSPECTS
34 35.1 35.2 35.3
5 Fig. I. Ideogram of chromosome 5 showing the localisation
of the SMA mutation,
polymorphism (D5S204). This was derived from
The prospects for the future of SMA research look very promising. The mutation causing the disease has been narrowed down to 20-30 million base pairs of DNA. Although this is still a large stretch of sequence, it is clear that new more closely linked markers will shortly be identified refining this localisation to 2-5 million base pairs of DNA. Recent methods of chromosome mapping such as the cloning of large pieces of DNA into yeast (yeast artificial chromosomes,
Review Article
YACs [17]) and the use of microdissection techniques [18] should permit the rapid isolation of the region of chromosome 5 containing the disease locus. Progress at this stage will then depend on how readily the gene can be recognised. If the mutation turns out to be a deletion on chromosome 5, the gene will be relatively easy to identify. The situation might be very similar to that observed for Duchenne and Becker muscular dystrophy, for example. Werdnig-Hoffmann disease may be the result of deletions in the gene whereas the milder form of the disease may result from point mutations or non-frameshift mutations in the gene [19]. Whatever the mutation turns out to be, the identification of the gene will be an important step in the understanding and eventual treatment of SMA. Acknowledgements--We thank Helen Blaber for the typing of the manuscript. We are grateful to the Muscular Dystrophy Association, U.S.A., the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Medical Research Council for financial support for this work. REFERENCES
I. 2.
3. 4. 5.
Dubowitz V. Muscle Disorder.v in Chihlhood. Philadelphia: Saunders, 1978. Pcarn J tl. The gcne frequency of acutc WcrdnigIloffmann di,~asc (SMA type I): a total population survey in north-east-England. J Med Genet 1973; I0: 260-265. Pearn J II. Infantile motor neuron diseases. In: Rowland L P, ed. Human Motor Neuron Diseases. New York: Raven Press. 1978: 227-248. Dubowitz V. Infantile muscular atrophy: a prospective study with particular reference to a slowly progressive variety. Bruin 1964; 87: 707-718. Brustowicz L M, Lehner T, Castilla L H, et al. Genetic mapping of childhood-onset-spinal musuclar atrophy to chromosome 5(111.2-13.3. Nature 1990; 344: 540-541.
85
6. Melki J, Abdclhak S, Shcth P, et al. Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 1990; 344: 767-768. 7. Gilliam T C. Brzustowicz L M, Castilla L H, et al. Genetic homogeneity between acute (SMA i) and chronic (SMA It & Ill) forms of spinal muscular atrophy. Nature 1990; 345: 823-825. 8. Melki J. Sheth P, Abdelhak S. et al. Mapping of acute (type I) spinal muscular atrophy to chromosome 5qI2q14. Lancer 1990; 336: 271-273. 9. Sheth P. Abdelhak S, Bachelor M F, et al. Linkage analysis in spinal musuclar atrophy, by six closely flanking markers on chromosome 5. Am J Hum Genet 1991; 48: 764-768. 10. Sherrington R, Mankoo B S, Kalsi G. Curtis D, Melmer G, Gurling H M D. Dinucleotide repeat polymorphism at the D5S204 locus. Nucl Acid Res 1991; (in press). I I. Danicls R J, Thomas N H, MacKinnon R N, et al. Linkage analysis of spinal muscular atrophy 1991: submitted. 12. Munsat T L, Skerry L, Korf B, et al. Phenotypic heterogeneity of spinal muscular atrophy mapping to chromosome 5ql 1.2-13.3 (SMA 5q). Neurology 1990: 40: 1831-1836. 13. SMA Workshop, New York, December 1990. 14. Kocnig M, Bcggs A H, Moycr M, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989; 45: 498-506. 15. Love D R. Forrest S M, Smith T J. et al. Molecular analysis of Duchcnnc and Beckcr muscular dystrophies. Br Med Bull 1989; 45: 659-680. 16. Weber J L. Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: Davies K E, ed. Genetic and Physical Mapping. Genome Analysis, Volume I. Cold Spring Harbor Laboratory Press, U.S.A.. 1990: 159-181. 17. Burke D T, Carlc G T. OIson M V. Cloning of large segments of cxogcnous DNA into yeast by means of artificial chromosome vectors. Science 1987; 236: 806-812. 18. Ludccke tl-J, Scngcr G, Claussen U, Horsthemke B. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 1989; 338: 348-350. 19. Monaco A P, Bcrtelson C J, Liechti-Gallati S. Moscr H. Kunkcl L M. An explanation for the phcnotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90-95.
0t~60-gt~ 91 $ 3 0 0 * 0.00 (~ 1991 Pergamon Press plc
REVIEW ARTICLE MOLECULAR
STUDIES
OF SPINAL MUSCULAR
ATROPHY
KAY E. DAVIES,* tNEIL H. THOMAS,+ §RACHAELJ. DANIELS* and VICTORDuBowtTz~ *Molecular Genetics Group. institute of Molecular Medicine.John Radcliffe Hospital. Headington. Oxford OX3 9DU; and +Department of Paediatrics and Neonatal Medicine. Royal Postgraduate Medical School. Hammersmith Hospital. Du Cane Road. London WI2 0NN, U.K.
(Received 18 April 1991; accepted 13 June 1991)
Abstract--Spinal muscular atrophy (SMA) is inherited as an autosomal recessive disorder which presents as a severe, intermediate or mild condition. The disease selectively affects the alpha motor neuron but nothing is as yet known about the underlying biochemical defect. Recent genetic studies have mapped all three types of SMA to the same region of human chromosome 5 (Sqll.2-q13.3) raising the possibility that the mutations may be allelic. Polymorphic DNA markers have been charaeterised which are suitable for prenatal diagnosis. This is the first step in the isolation of the mutant gene (or genes) involved in this disorder. Key words: Spinal muscular atrophy, gent mutation, linkage, prenatal diagnosis.
INTRODUCTION
led to tile suggestion that SMA is a result of different non-allclic mutations [3] or a different set of mutations within the same gene [4]. With the advent of molecular techniques to map disease genes, it has become possible to resolve this issue.
The most common form of spinal muscular atrophy is inherited in an autosomal recessive manner. The disease varies in severity and one can define a severe, an intermediate and a mild phenotype on the basis of the ability to sit or MAPPING TIlE MUTATION walk unaided [i}. Severe SMA (also known as Werdnig-Hoffmann disease) has an onset before After searching for linkage throughout the birth or within the first few months of life, and whole human genome, linkage was finally estabthe affected child never sits unaided. The proglished between SMA and markers located on the nosis is poor and the child usually dies within the proximal long arm of human chromosome 5. In first two years of life. The intermediate form has the initial linkage studies linkage was found in a later age of onset and the affected child is able families segregating for the milder forms of the to sit but not walk. The life expectancy is disease [5,6]. Werdnig-Hoffmann families were determined by the degree of respiratory muscle not included because of the lack of large multiply weakness. Mild SMA (Kugelberg-Welander affected kindreds. The problem of small families disease) is characterised by the ability to walk was solved by segregation analysis in consanunaided. The onset is usually after the first year. The clinical course is variable but in some may be guinous pedigrees [7] and by the study of a large number of small families [8]. These analyses very mild with a normal life expectancy. demonstrated that the mutation in all three The incidence of SMA has been estimated to be forms of SMA map to the same region of about I in 25,000 live births with a carrier chromosome 5 at 5q11.2-13.3. The most probfrequency of I in 80 [2]. The phenotypic variation able location for the mutation is between loci seen between families but not within families has D5S6 and D5S39 [5-9]. Figure I shows an ideogram of chromosome 5 summarising the * Author to whom correspondence should be addressed. § Present address: Department of Paediatrics, Guy's DNA markers used in the multipoint linkage Hospital, St Thomas Street, London SEI 9RT, U.K. mapping. The position of SMA relative to the 83
84
Review Article
locus D5S112 remains uncertain at present. Initial data placed SMA distal to D5S i 12 but our more recent results with more informative probes favour a location proximal to D5SII2 (unpublished observations). More markers and the pooling of all family analyses will be needed to resolve this map position. The probes used to detect restriction fragment length polymorphisms (RFLPs) are given in Table 1 together with the percentage of individuals informative at these loci (PIC value). Also included is a dinucleotide PCR based repeat
DNA within i 5 kb of D5S39 and can therefore be considered as the same genetic locus [ ! 0]. D5S204 is informative in approximately 80% of individuals [I0,1 I]. At present there is no evidence for genetic heterogeneity in the disorder [12,13]. The SM A mutations on chromosome 5q present with a broad continuum of clinical abnormalities. This strongly suggests that the mutations causing the differing severities of the disease may be different mutations at the same locus similar to the Table I. Characteristics of DNA polymorphisms for linkage studies
15.3 15.2
Probe
Locus
15.I
.11)t 10H C L407 M4 JK53 pi05 153 Ra
D5S21 D5S63 D5S6 D5S112 D5S39
---LII55
D5S204 D5S51
pi05 798 Rb
D5S78
14
13.3 13.2 13.1 12 11 11.1
D5S21 D5,$63
11.2
Enzyme Msp I Taq I Barn HI Pvu II Msp I Xba I Pst 1 PCR based Taq I Hint II Msp I
PIC Value 0 4t 033 0.52 0.48 0.34 033 04,~ 078 036 0 34 037
D..,~S6
12
D.5~112
13.1
DSS39/D,5~204
13.2
D,5S,51
13.3
DSS78
J sMA
14
15 21 22 23.1 23.2 23.3 31.1
31.2 31,3 32 33.1 33.2 33.3
situation observed for Duchenne and Becker muscular dystrophy [14,15]. Since there is no evidence for significant genetic heterogeneity for autosomal rect~ssive SMA, it is possible to use the markers for prenatal diagnosis. We are performing diagnoses where both parents are informative for the flanking markers D5S6 and D5S39/D5S204 (Daniels and Davies, in preparation). In many cases, however, diagnosis is not possible because the affected child is dead. We are currently developing other PCR based markers in addition to D5S204 which can be typed on tissue samples such as muscle biopsies and Guthrie spots. Since variable number microsatellite repeats occur frequently in the human genome (once in every 60 kb [16]), it is likely that such highly informative markers will be available in the near future. FUTURE PROSPECTS
34 35.1 35.2 35.3
5 Fig. I. Ideogram of chromosome 5 showing the localisation
of the SMA mutation,
polymorphism (D5S204). This was derived from
The prospects for the future of SMA research look very promising. The mutation causing the disease has been narrowed down to 20-30 million base pairs of DNA. Although this is still a large stretch of sequence, it is clear that new more closely linked markers will shortly be identified refining this localisation to 2-5 million base pairs of DNA. Recent methods of chromosome mapping such as the cloning of large pieces of DNA into yeast (yeast artificial chromosomes,
Review Article
YACs [17]) and the use of microdissection techniques [18] should permit the rapid isolation of the region of chromosome 5 containing the disease locus. Progress at this stage will then depend on how readily the gene can be recognised. If the mutation turns out to be a deletion on chromosome 5, the gene will be relatively easy to identify. The situation might be very similar to that observed for Duchenne and Becker muscular dystrophy, for example. Werdnig-Hoffmann disease may be the result of deletions in the gene whereas the milder form of the disease may result from point mutations or non-frameshift mutations in the gene [19]. Whatever the mutation turns out to be, the identification of the gene will be an important step in the understanding and eventual treatment of SMA. Acknowledgements--We thank Helen Blaber for the typing of the manuscript. We are grateful to the Muscular Dystrophy Association, U.S.A., the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Medical Research Council for financial support for this work. REFERENCES
I. 2.
3. 4. 5.
Dubowitz V. Muscle Disorder.v in Chihlhood. Philadelphia: Saunders, 1978. Pcarn J tl. The gcne frequency of acutc WcrdnigIloffmann di,~asc (SMA type I): a total population survey in north-east-England. J Med Genet 1973; I0: 260-265. Pearn J II. Infantile motor neuron diseases. In: Rowland L P, ed. Human Motor Neuron Diseases. New York: Raven Press. 1978: 227-248. Dubowitz V. Infantile muscular atrophy: a prospective study with particular reference to a slowly progressive variety. Bruin 1964; 87: 707-718. Brustowicz L M, Lehner T, Castilla L H, et al. Genetic mapping of childhood-onset-spinal musuclar atrophy to chromosome 5(111.2-13.3. Nature 1990; 344: 540-541.
85
6. Melki J, Abdclhak S, Shcth P, et al. Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 1990; 344: 767-768. 7. Gilliam T C. Brzustowicz L M, Castilla L H, et al. Genetic homogeneity between acute (SMA i) and chronic (SMA It & Ill) forms of spinal muscular atrophy. Nature 1990; 345: 823-825. 8. Melki J. Sheth P, Abdelhak S. et al. Mapping of acute (type I) spinal muscular atrophy to chromosome 5qI2q14. Lancer 1990; 336: 271-273. 9. Sheth P. Abdelhak S, Bachelor M F, et al. Linkage analysis in spinal musuclar atrophy, by six closely flanking markers on chromosome 5. Am J Hum Genet 1991; 48: 764-768. 10. Sherrington R, Mankoo B S, Kalsi G. Curtis D, Melmer G, Gurling H M D. Dinucleotide repeat polymorphism at the D5S204 locus. Nucl Acid Res 1991; (in press). I I. Danicls R J, Thomas N H, MacKinnon R N, et al. Linkage analysis of spinal muscular atrophy 1991: submitted. 12. Munsat T L, Skerry L, Korf B, et al. Phenotypic heterogeneity of spinal muscular atrophy mapping to chromosome 5ql 1.2-13.3 (SMA 5q). Neurology 1990: 40: 1831-1836. 13. SMA Workshop, New York, December 1990. 14. Kocnig M, Bcggs A H, Moycr M, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989; 45: 498-506. 15. Love D R. Forrest S M, Smith T J. et al. Molecular analysis of Duchcnnc and Beckcr muscular dystrophies. Br Med Bull 1989; 45: 659-680. 16. Weber J L. Human DNA polymorphisms based on length variations in simple-sequence tandem repeats. In: Davies K E, ed. Genetic and Physical Mapping. Genome Analysis, Volume I. Cold Spring Harbor Laboratory Press, U.S.A.. 1990: 159-181. 17. Burke D T, Carlc G T. OIson M V. Cloning of large segments of cxogcnous DNA into yeast by means of artificial chromosome vectors. Science 1987; 236: 806-812. 18. Ludccke tl-J, Scngcr G, Claussen U, Horsthemke B. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 1989; 338: 348-350. 19. Monaco A P, Bcrtelson C J, Liechti-Gallati S. Moscr H. Kunkcl L M. An explanation for the phcnotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90-95.