EDITORIAL
The Triumph of Lnkage Analysis In the past decade, rapid advances have occurred in OUT understanding of the biochemical basis of inherited neurological disease, primarily resulting from detection of enzyme defects {l,21. In the next decade, the pulse of development in this area will quicken considerably because of the introduction of such recombinant DNA techniques as linkage analysis {3, 41. The number of diseases recognized as inherited in a mendelian form has grown substantially in the past several years, as documented in McKusick‘s catalogs. Between 1983 (sixth edition) and 1988 (eighth edition) {51 the number of documented genetic disorders increased by 976 to 4,344 separate entities. Those inherited autosomal dominant disorders in which the chromosomal locus has been identified now exceed one-half of all mendelian inherited disorders where the loci are known. In genetic disorders of the nervous system, dominant diseases are also in the majority {3]. Paradoxically, however, the greatest degree of molecular understanding of genetic diseases involving the nervous system comes from those disorders that are recessively, not dominantly, inherited. The reason is that the recessive disorders, including the mucopolysaccharidoses, leukodystrophies, aminoacidopathies, and gangliosidoses, are all associated with the accumulation of a metabolite related to a well-established biochemical pathway C21. The problem posed by dominantly inherited diseases of the nervous system, such as the dystonias, neurofibromatosis, myotonic dystrophy, Huntington’s disease, the spinocerebellar degenerations, tuberous sclerosis, and Alzheimer’s disease, is that there is no known primary metabolic clue or primary storage product to indicate a potential molecular basis of disease. In some of these disorders there is usually a patterned neuronal degeneration with variable secondary gliosis. Even the findings of specific protein abnormalities on two-dimensional gels or of specific changes in messenger RNA on Northern blots obtained from brain samples of patients having a dominantly inherited neurological disorder are not sufficiently precise evidence to warrant the conclusion that these products are due to primary gene mutations rather than the result of disease {6, 71. Therefore, new research strategies must be employed to determine the molecular basis of autosomal dominant disorders and the molecular explanation for phenotypic variation within and between families. Linkage analysis has proved to be a powerful tool with which to map the chromosomal location of a series of important, dominantly inherited
neurological diseases for which no biochemical insight was known. It is this approach, as reported in this issue of Annals, that allowed Kramer and colleagues {81 to map the dystonia gene in an Ashkenazi Jewish population to chromosome 9q32-34. Thomas Hunt Morgan established that genes are linked to each other on chromosomes, and he formulated gene maps in the fruit 0y Drosophikz mekznogaster. Genes that are on the same chromosome and are closely associated physically will move together when exchanges of chromosomal segments occur during the meiotic stages of gametogenesis. The closer they are, the tighter the linkage and the lower the likelihood that they will be separated as a result of the exchange (crossing-over) of genetic material that occurs between homologous chromosomes during meiosis. The units of measurement, as worked out by Sturtevant in 1925 {97 are referred to as centimorgans (cM) in honor of Morgan. One centimorgan refers to one map unit, about lo6 base pairs of nucleotides and a 1% recombination frequency. Recombination is defined as the independent segregation of genes that occurs when chromosomal segments exchange. Linkage between two adjacent genes is measured by the percent recombination between loci. For practical clinical purposes, two genes are linked if they are 2 to 5 cM apart. Thus, a percent recombination frequency of between 2 and 5% is acceptable for clinical linkage studies. Complementary D N A (cDNA) radiolabeled probes are used to determine a site that is close (3) as computed by the method of maximum likelihood and the computer program LIPED, then the marker cDNA locus being studied is linked to the disease locus [lo]. It has been estimated that only about 200 polymorphic DNA markers, evenly spaced 10 to 20 million base pairs apart, are required to map the entire human genome. These few cDNA probes would allow chromosomal localization of the majority of mendelian disorders 1101. Recently, the chromosomal map site has been determined by linkage analysis for several important autosomal dominant neurological disorders using RFLPs of patients and normal family members to defined cDNA probe sites, including myotonic dystrophy (19cenq13.2) 111, 121, tuberous sclerosis (9q) 1131, hereditary motor and sensory neuropathy (lq2) 1147, Huntington’s disease (4~16.3)115-171, Alzheimer’s disease (21q11-22) 1181, neurofibromatosis type 1 (von Recklinghausen’s disease) 117ql1.2) 1191, and type 2 (central neurofibromatosis (22q11.1-13.1) 120, 21). As they report in this issue, Kramer and coworkers, using linkage analysis, found the chromosomal map locus for idi9pathic torsion dystonia (ITD) in 12 Ashkenazi Jewish families to be at 9q32-34. It is another major advance in neurogenetics and another triumph for linkage analysis. In a, previous paper, this group headed by Stanley Fahn of Columbia University, The Neurological Institute, New York, showed that ITD in this Jewish population was inherited as an autosomal dominant disorder with a low penetrance of clinical expression, estimated at about 30%. This fact supports the view that in both Jewish and non-Jewish families ITD is dominantly inherited but with variable peneuance 1221. A gene for ITD (DyTI) in a nonJewish kindred was found to be located on chromosome 9q32-34 with tight linkage to the gene encoding gelsolin (GSN) 1231. In the present study by Kramer and associates 181, the ITD gene exhibited tight linkage with the gene encoding argininosuccinate synthetase (ASS) and also mapped to 9q32-34. Thus, their paper strongly suggests that the same gene may be responsible for ITD in both Jewish and non-Jewish kindreds. The differences in penetrance between the Jewish families (30%) and non-Jewish families (75%) may reflect allelic mutations within the same gene or, alternatively, the same gene mutation but segregating in a variable manner with nonallelic modifiers that can 112 Annals of Neurology Vol 27 No 2 February 1990
alter the peneuance and the expressivity of the same primary genetic mutation and produce phenotypic variation 13, 43. Now that the ITD gene has been mapped, it is possible to focus on attempting to isolate, clone, and sequence the mutant gene. Determination of the normal and disease-mutant gene product may help explain the molecular pathogenesis of disease. Knowledge of the mutant gene product offers the possibility of pharmacological or even gene therapy targeting the normal gene into the neuronal-glial genome of the basal ganglia, perhaps with defective retroviral vectors. With linkage analysis it is now possible to decipher the molecular basis of all neurogenetic diseases irrespective of any other biochemical information about them. Beadle and Tanun 1241 proved that one gene equals one protein. Linkage analysis extends that concept to one gene:one protein:one eponym. The triumph of Botstein and co-workers [lo], the pioneers who conceived of linkage analysis to solve genetic disease, and its early proponents, Gusella and associates [l5], has been to provide a technique that finds the genetic needle in a large haystack of DNA. Roger N . Rosenberg, M D Department of Neurology University of Texas Southwestern Medical Center Dallas, T X
References 1. Rosenberg RN. Genetic variation and neurologic disease. Trends Neurosci 1980;3:144-148 2. Rosenberg RN. Biochemical genetics of neurologic disease. N Engl J Med 1981;305:1181-1193 3. Rosenberg RN. Neurogenetics: principles ahd practice. New York Raven Press, 1986. 324 p 4. Rosenberg RN, Harding AE. The molecular biology of neurological disease. London: Butterworths, 1988. 263 p 5. McKusick VA. The mendelian inheritance in man. 8th ed. Baltimore: Johns Hopkins University Press, 1988:xi 6. Morrison MR, Rosenberg RN. Specific messenger RNA changes in Joseph disease cerebella. Ann Neurol 1983;14:7379 7. Rosenberg RN, Ivy N, Kirkpatrick J, et al. Joseph disease and Huntington disease. Protein patterns in fibroblasts and brain. Neurology 1981;3 1: 1003- 1014 8. h e r PL, de Leon D, Ozelius L, et al. Dystonia gene in Ashkenazi Jewish population is located on chromosome 9q3234. Ann Neurol 1990;27:114-120 9. Sturtevant AH. The effects of unequal crossing over at the bar locus in Drosophila Genetics 1925;10:117 10. Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 1980;32:314-331 11. Perick-Vance M, Yamaoka L, Assinder R, et al. Tight linkage of apolipoprotein CZ to myotonic dystrophy on chromosome 19. Neurology 1986;36:1418-1423 12. Bird T, Boehnke M, Schellenberg G, et al. The use of apolipoprotein C2 as a genetic marker for myotonic dystrophy. Arch Neurol 1987;44:273-275
13. Fryer A, Conner J, Povey S, et al. Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet 1987;l (March 21):659-661 14. Bird TD. Hereditary motor-sensory neuropathies. In: Johnson W, ed. Neurogenetic disease. Philadelphia: WB Saunders, 19899-23 15. Gusella JF, Wexler NS, Connedy P, et al. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983;306:234-238 16. Gusella JF. Genetic linkage of the Huntington’s disease gene to a DNA marker. Can J Neurol Sci 1984;11:421-425 17. Wexler N, Young A, Tanzi R, et al. Homozygotes for Huntington’s disease. Nature 1987;326:194-197 18. St George-Hyslop P, Tanzi R, Polinsky R, et al. The genetic defect causing familial Alzheimer’s disease maps on chromosome 21. Science 1987;235:885-890 19. Barker D, Wright E, Nguyen K, et al. Gene for von Reckling-
20.
21.
22.
23.
24.
hausen neurofibromatosis is in the pericentromeric region of chromosome 17. Science 1987;236:1100-1102 Rouleau G, Wenelecki W, Haines J, et al. Genetic linkage of bilateral acoustic neurofibromatosis to a DNA marker on chromosome 22. Nature 1987;329:246-248 Wenelecki W, Rodeau G, Superneau D, et al. Neurofibromatosis 2: clinical and DNA linkage studies of a large kindred. N Engl J Med 1988;319:278-283 Bressman S, de Leon D, Brin M, et al. Idiopathic dystonia among Ashkenazi Jews: evidence for autosomal dominant inheritance. Ann Neurol 1989;26:612-620 Ozelius L, Kramer P, Moskowitz C, et al. Human gene for torsion dystonia located on chromosome 9q32-34. Neuron 1989;2:1427-1434 Beadle GW, Tatum EL. Genetic control of biochemical reactions in neurospora Proc Natl Acad Sci USA 1941;27:499506
Editorial: Rosenberg: Triumph of Linkage Analysis 113
The Triumph of Lnkage Analysis In the past decade, rapid advances have occurred in OUT understanding of the biochemical basis of inherited neurological disease, primarily resulting from detection of enzyme defects {l,21. In the next decade, the pulse of development in this area will quicken considerably because of the introduction of such recombinant DNA techniques as linkage analysis {3, 41. The number of diseases recognized as inherited in a mendelian form has grown substantially in the past several years, as documented in McKusick‘s catalogs. Between 1983 (sixth edition) and 1988 (eighth edition) {51 the number of documented genetic disorders increased by 976 to 4,344 separate entities. Those inherited autosomal dominant disorders in which the chromosomal locus has been identified now exceed one-half of all mendelian inherited disorders where the loci are known. In genetic disorders of the nervous system, dominant diseases are also in the majority {3]. Paradoxically, however, the greatest degree of molecular understanding of genetic diseases involving the nervous system comes from those disorders that are recessively, not dominantly, inherited. The reason is that the recessive disorders, including the mucopolysaccharidoses, leukodystrophies, aminoacidopathies, and gangliosidoses, are all associated with the accumulation of a metabolite related to a well-established biochemical pathway C21. The problem posed by dominantly inherited diseases of the nervous system, such as the dystonias, neurofibromatosis, myotonic dystrophy, Huntington’s disease, the spinocerebellar degenerations, tuberous sclerosis, and Alzheimer’s disease, is that there is no known primary metabolic clue or primary storage product to indicate a potential molecular basis of disease. In some of these disorders there is usually a patterned neuronal degeneration with variable secondary gliosis. Even the findings of specific protein abnormalities on two-dimensional gels or of specific changes in messenger RNA on Northern blots obtained from brain samples of patients having a dominantly inherited neurological disorder are not sufficiently precise evidence to warrant the conclusion that these products are due to primary gene mutations rather than the result of disease {6, 71. Therefore, new research strategies must be employed to determine the molecular basis of autosomal dominant disorders and the molecular explanation for phenotypic variation within and between families. Linkage analysis has proved to be a powerful tool with which to map the chromosomal location of a series of important, dominantly inherited
neurological diseases for which no biochemical insight was known. It is this approach, as reported in this issue of Annals, that allowed Kramer and colleagues {81 to map the dystonia gene in an Ashkenazi Jewish population to chromosome 9q32-34. Thomas Hunt Morgan established that genes are linked to each other on chromosomes, and he formulated gene maps in the fruit 0y Drosophikz mekznogaster. Genes that are on the same chromosome and are closely associated physically will move together when exchanges of chromosomal segments occur during the meiotic stages of gametogenesis. The closer they are, the tighter the linkage and the lower the likelihood that they will be separated as a result of the exchange (crossing-over) of genetic material that occurs between homologous chromosomes during meiosis. The units of measurement, as worked out by Sturtevant in 1925 {97 are referred to as centimorgans (cM) in honor of Morgan. One centimorgan refers to one map unit, about lo6 base pairs of nucleotides and a 1% recombination frequency. Recombination is defined as the independent segregation of genes that occurs when chromosomal segments exchange. Linkage between two adjacent genes is measured by the percent recombination between loci. For practical clinical purposes, two genes are linked if they are 2 to 5 cM apart. Thus, a percent recombination frequency of between 2 and 5% is acceptable for clinical linkage studies. Complementary D N A (cDNA) radiolabeled probes are used to determine a site that is close (3) as computed by the method of maximum likelihood and the computer program LIPED, then the marker cDNA locus being studied is linked to the disease locus [lo]. It has been estimated that only about 200 polymorphic DNA markers, evenly spaced 10 to 20 million base pairs apart, are required to map the entire human genome. These few cDNA probes would allow chromosomal localization of the majority of mendelian disorders 1101. Recently, the chromosomal map site has been determined by linkage analysis for several important autosomal dominant neurological disorders using RFLPs of patients and normal family members to defined cDNA probe sites, including myotonic dystrophy (19cenq13.2) 111, 121, tuberous sclerosis (9q) 1131, hereditary motor and sensory neuropathy (lq2) 1147, Huntington’s disease (4~16.3)115-171, Alzheimer’s disease (21q11-22) 1181, neurofibromatosis type 1 (von Recklinghausen’s disease) 117ql1.2) 1191, and type 2 (central neurofibromatosis (22q11.1-13.1) 120, 21). As they report in this issue, Kramer and coworkers, using linkage analysis, found the chromosomal map locus for idi9pathic torsion dystonia (ITD) in 12 Ashkenazi Jewish families to be at 9q32-34. It is another major advance in neurogenetics and another triumph for linkage analysis. In a, previous paper, this group headed by Stanley Fahn of Columbia University, The Neurological Institute, New York, showed that ITD in this Jewish population was inherited as an autosomal dominant disorder with a low penetrance of clinical expression, estimated at about 30%. This fact supports the view that in both Jewish and non-Jewish families ITD is dominantly inherited but with variable peneuance 1221. A gene for ITD (DyTI) in a nonJewish kindred was found to be located on chromosome 9q32-34 with tight linkage to the gene encoding gelsolin (GSN) 1231. In the present study by Kramer and associates 181, the ITD gene exhibited tight linkage with the gene encoding argininosuccinate synthetase (ASS) and also mapped to 9q32-34. Thus, their paper strongly suggests that the same gene may be responsible for ITD in both Jewish and non-Jewish kindreds. The differences in penetrance between the Jewish families (30%) and non-Jewish families (75%) may reflect allelic mutations within the same gene or, alternatively, the same gene mutation but segregating in a variable manner with nonallelic modifiers that can 112 Annals of Neurology Vol 27 No 2 February 1990
alter the peneuance and the expressivity of the same primary genetic mutation and produce phenotypic variation 13, 43. Now that the ITD gene has been mapped, it is possible to focus on attempting to isolate, clone, and sequence the mutant gene. Determination of the normal and disease-mutant gene product may help explain the molecular pathogenesis of disease. Knowledge of the mutant gene product offers the possibility of pharmacological or even gene therapy targeting the normal gene into the neuronal-glial genome of the basal ganglia, perhaps with defective retroviral vectors. With linkage analysis it is now possible to decipher the molecular basis of all neurogenetic diseases irrespective of any other biochemical information about them. Beadle and Tanun 1241 proved that one gene equals one protein. Linkage analysis extends that concept to one gene:one protein:one eponym. The triumph of Botstein and co-workers [lo], the pioneers who conceived of linkage analysis to solve genetic disease, and its early proponents, Gusella and associates [l5], has been to provide a technique that finds the genetic needle in a large haystack of DNA. Roger N . Rosenberg, M D Department of Neurology University of Texas Southwestern Medical Center Dallas, T X
References 1. Rosenberg RN. Genetic variation and neurologic disease. Trends Neurosci 1980;3:144-148 2. Rosenberg RN. Biochemical genetics of neurologic disease. N Engl J Med 1981;305:1181-1193 3. Rosenberg RN. Neurogenetics: principles ahd practice. New York Raven Press, 1986. 324 p 4. Rosenberg RN, Harding AE. The molecular biology of neurological disease. London: Butterworths, 1988. 263 p 5. McKusick VA. The mendelian inheritance in man. 8th ed. Baltimore: Johns Hopkins University Press, 1988:xi 6. Morrison MR, Rosenberg RN. Specific messenger RNA changes in Joseph disease cerebella. Ann Neurol 1983;14:7379 7. Rosenberg RN, Ivy N, Kirkpatrick J, et al. Joseph disease and Huntington disease. Protein patterns in fibroblasts and brain. Neurology 1981;3 1: 1003- 1014 8. h e r PL, de Leon D, Ozelius L, et al. Dystonia gene in Ashkenazi Jewish population is located on chromosome 9q3234. Ann Neurol 1990;27:114-120 9. Sturtevant AH. The effects of unequal crossing over at the bar locus in Drosophila Genetics 1925;10:117 10. Botstein D, White RL, Skolnick M, Davis RW. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 1980;32:314-331 11. Perick-Vance M, Yamaoka L, Assinder R, et al. Tight linkage of apolipoprotein CZ to myotonic dystrophy on chromosome 19. Neurology 1986;36:1418-1423 12. Bird T, Boehnke M, Schellenberg G, et al. The use of apolipoprotein C2 as a genetic marker for myotonic dystrophy. Arch Neurol 1987;44:273-275
13. Fryer A, Conner J, Povey S, et al. Evidence that the gene for tuberous sclerosis is on chromosome 9. Lancet 1987;l (March 21):659-661 14. Bird TD. Hereditary motor-sensory neuropathies. In: Johnson W, ed. Neurogenetic disease. Philadelphia: WB Saunders, 19899-23 15. Gusella JF, Wexler NS, Connedy P, et al. A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 1983;306:234-238 16. Gusella JF. Genetic linkage of the Huntington’s disease gene to a DNA marker. Can J Neurol Sci 1984;11:421-425 17. Wexler N, Young A, Tanzi R, et al. Homozygotes for Huntington’s disease. Nature 1987;326:194-197 18. St George-Hyslop P, Tanzi R, Polinsky R, et al. The genetic defect causing familial Alzheimer’s disease maps on chromosome 21. Science 1987;235:885-890 19. Barker D, Wright E, Nguyen K, et al. Gene for von Reckling-
20.
21.
22.
23.
24.
hausen neurofibromatosis is in the pericentromeric region of chromosome 17. Science 1987;236:1100-1102 Rouleau G, Wenelecki W, Haines J, et al. Genetic linkage of bilateral acoustic neurofibromatosis to a DNA marker on chromosome 22. Nature 1987;329:246-248 Wenelecki W, Rodeau G, Superneau D, et al. Neurofibromatosis 2: clinical and DNA linkage studies of a large kindred. N Engl J Med 1988;319:278-283 Bressman S, de Leon D, Brin M, et al. Idiopathic dystonia among Ashkenazi Jews: evidence for autosomal dominant inheritance. Ann Neurol 1989;26:612-620 Ozelius L, Kramer P, Moskowitz C, et al. Human gene for torsion dystonia located on chromosome 9q32-34. Neuron 1989;2:1427-1434 Beadle GW, Tatum EL. Genetic control of biochemical reactions in neurospora Proc Natl Acad Sci USA 1941;27:499506
Editorial: Rosenberg: Triumph of Linkage Analysis 113
