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Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.

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Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition.

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Genetic Neurological Disorders

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Correspondence to Kunihiko Suzuki, Neuroscience Center, CB7250, Departments of Neurology and Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.

Traditional molecular biological approaches to genetic neurological disorders, in which the defective gene products responsible for the phenotypes are known, have been well developed and are providing a powerful tool for our understanding and eventual therapy for these disorders. The traditional approach starts with the knowledge of the defective gene products, such as an enzyme deficiency, and reaches the gene and its characterization through purification of the gene products and cloning of the gene. Examples of the approaches and recent advances in such genetic disorders are illustrated elsewhere in this book (see Chap. 41). However, the genetic disorders in this category constitute a relatively small proportion of the over 3,000 known human genetic disorders of Mendelian inheritance [1,2]. Methodological advances have allowed us to approach the genetic causes of the remaining disorders without knowledge as to which genes are responsible for the disease. Since the flow of logic in this approach is in the reverse direction of the traditional approach, it is often termed “reverse genetics” because one first tries to obtain the gene responsible for the disorder without knowing its function and then tries to understand its function and the pathophysiology of the disease. The cloning strategy is also termed “positional cloning” since the genes are cloned on the basis of position in the genome only [3]. The ongoing project of mapping and sequencing the entire human genome is expected to greatly facilitate the search for the genes responsible for all types of genetic disorders [4].

If the present revolutionary advances in our molecular genetic understanding of hereditary neurodegenerative disorders are to be of pragmatic value in the future, strategies to achieve the ultimate gene therapy are of utmost importance. Technological progress in this direction has also been noteworthy. A large number of prokaryotic and eukaryotic systems that allow expression of cloned genes have been developed. Gene-transfer technology is progressing rapidly, either vector-mediated or as transgenes in the whole animal. Furthermore, homologous recombination is being used to generate mouse mutant strains in which specific genes are artificially and intentionally rendered inactive, termed “gene targeting.” This approach allows production of authentic murine models of human genetic disorders when naturally occurring mutants do not exist among the easily manipulated small laboratory animals. Many murine models that are genetic equivalents of known human genetic disorders have already been generated using this approach (See Chap. 41).

Reverse genetics involves linkage analysis, positional cloning and diagnosis using linked markers

Linkage analysis can localize a disease-causing gene to a region of a chromosome without knowledge of its function. When the abnormal gene product responsible for a given genetic disease is not known, one would like to know first the location of the responsible gene within the genome. The strategy of locating the gene to a single chromosome and then to as specific a region as possible within the chromosome is referred to as linkage analysis. The traditional approach for this analysis is based on two principles. First, the primary sequence of the human genome is not fixed but varies in different individuals; this phenomenon is known as polymorphism. Such polymorphisms are statistically more common within introns and other noncoding regions of the genome, which constitute most of the genomic sequences. In addition, there are regions of the human genome which are particularly polymorphic. Since such polymorphisms often either generate a new restriction site or abolish an existing site, they can be identified by digestion with appropriate restriction enzymes and observing the size of the generated fragments with a probe spanning the region, which is known as a restriction fragment length polymorphism (RFLP). Second, during meiosis, two corresponding pairs of chromosomes line up together and then are separated into two daughter cells that eventually generate the germ cells. Thus, two regions of the genome which are on the same chromosome tend to stay together, while those on different chromosomes distribute to the germ cells independently from each other. However, the two corresponding chromosomes do not always separate cleanly from each other after coming together in meiosis. A crossover can occur, and the two chromosomes exchange an equivalent portion, generating a new pair of chromosomes. Therefore, even two regions on the same chromosome can be separated onto two chromosomes if a crossover occurs between the two regions. It then follows that the closer the two regions are on the chromosome, the more likely they are to stay together. In the extreme case when the polymorphic marker site is within the gene responsible for the disease, the marker will always be together with the gene. The strategy is, therefore, to find a polymorphic marker which is closely “linked” to affected individuals within the family being analyzed and, thus, to the disease-causing gene. The physical map of the human genome is being developed rapidly, with locations of known DNA sequences marked [5]. Many of those sequences are polymorphic, such that they provide different RFLPs in different individuals and, thus, can be used as markers for linkage analyses.

A successful linkage analysis requires DNA materials from family members of affected patients, preferably from as many generations as possible. A set of known polymorphic markers are chosen as probes, and genomic DNA is prepared from individuals. The genomic DNA is then digested by appropriate restriction enzymes and the RFLP patterns examined for each of the markers. Any markers which do not segregate with or against the disease state are discarded. In X-linked diseases, the chromosomal localization is known and only the markers on the X-chromosome need to be selected for the study. As should be clear from this description, linkage analysis is essentially a statistical procedure requiring assistance of the computer. Software programs have been developed specifically for analyses of the results of complex linkage studies. The results are expressed as the lod score [6,7], which gives a statistical estimate, in decimal logarithm, of the relative closeness of the given polymorphic marker to the disease-causing gene. A lod score of at least 3, preferably greater, is considered to be an indication that the gene being searched for is “linked” to the marker.

Linkage analysis has been used to map the genes responsible for a number of genetic neurological disorders to specific chromosomes. The first spectacular success of linkage analysis was the localization of the gene responsible for Huntington's disease [8]. The study was successful partly because of the availability of an enormous pedigree in the Lake Maracaibo region of Venezuela. Since then, chromosomal localization of many genes responsible for neurodegenerative disorders have been identified, including familial retinoblastoma, neurofibromatosis I, Charcot-Marie-Tooth disease, myotonic dystrophy, three different forms of Batten disease and ataxia telangiectasia. The catalogue of such diseases continues to expand [9].

A few caveats must be kept in mind. Linkage analyses are effective for Mendelian disorders where single genes are responsible for the disease states. This is not because the principle of linkage analysis is not applicable for multigenic disorders but, rather, that the increase in the number of genes that must be taken into consideration logarithmically increases the complexity of the linkage analysis. Another potentially complicating factor is that genetically different diseases can manifest themselves with similar clinical phenotypes. For example, what had been classified as Sanfilippo's syndrome has turned out to consist of four distinct genetic diseases caused by genetic defects in four different genes (see Chap. 41). If families gathered for a linkage analysis of what is considered to be one genetic disease in fact represent more than one genetically distinct disorder, the results of the linkage analysis may obscure the location of the gene. It is essential, therefore, to ascertain that all individuals included in a linkage analysis be within a single genetic complementation group. The complementation test is based on the principle that if the genetic cause of two patients with a similar phenotype lies in the same gene, fusion of the cells from these patients will show the phenotype. Furthermore, the disease phenotype should disappear in the fused cells if defects in two different genes are the causes of the similar phenotype. Despite these theoretical constraints, linkage analysis is being applied to potentially highly complex genetic disorders, such as manic-depressive disorder and schizophrenia.

In some instances, cytologically identifiable deletions or other chromosomal abnormalities associated with the disease can provide the crucial clue as to the location of the responsible gene. Such abnormalities were used in the positional cloning of the dystrophin gene, which is responsible for Duchenne's and Becker's muscular dystrophy [10]. Association of the disease with another phenotype due to a known gene can provide a useful hint at the location of the disease-causing gene. For example, the frequent association of X-linked adrenoleukodystrophy with color blindness indicated the close proximity of the two genes. More recently, the chromosomal localization of the gene responsible for Niemann-Pick type C disease was determined by taking advantage of the fact that an equivalent disease exists in both humans and mice. In this approach, artificially constructed mouse microcells, each containing a single copy of a human chromosome, were used as the human chromosome donor. When fused with cells from affected mice, the biochemical phenotype of the disease could be corrected only when human chromosome 18 was introduced. The gene localization could be narrowed down further because those cells that lost a portion of chromosome 18 in subsequent subcloning reverted back to the Niemann-Pick type C phenotype. Finally, the gene responsible for the major complementation group of Niemann-Pick type C disease was cloned, several mutations were identified and the homology search suggests that it is related to known cholesterol homeostasis genes [11].

Positional cloning. Localizing the gene to a chromosome, even to a specific region of a chromosome, is obviously the first step for cloning and characterization of the gene responsible for the disorder. It is generally desirable to obtain more than one linked marker, particularly two markers that flank the target gene. While a variety of ways to reach the target gene have been devised, the basic principle is to extend the DNA sequence information from the identified linked marker region toward the gene, either contiguously or discontinuously. These procedures are often referred to as “chromosomal walking” and “jumping.” Genomic DNA libraries in the λ phage, cosmids and, more recently, in the form of the yeast artificial chromosome (YAC) are commonly used to track down the target gene. Human chromosome-specific YAC clones that collectively give the complete contiguous chromosomal DNA sequence have been constructed. In the primitive “walking,” a segment of genomic DNA containing the identified marker is sequenced from the marker region. A new primer is made from the newly sequenced region and used for further sequencing, slowly extending along the stretch of DNA toward the target. For this strategy to be effective, the marker must be reasonably close to the target gene because the number of nucleotides that can be sequenced in one step is still limited. Another variation is “jumping,” in which the genomic DNA containing the marker sequence is circularized with the marker region on one end and sequenced across the ligated region, thus obtaining the sequence information some distance away from the marker for the next “jumping.”

These procedures are slow and tedious as attested to by the search for the Huntington's disease gene, which took 10 years to be cloned after it was localized to a chromosome [12]. The positional cloning approach has been successful in many disorders when no other approaches were feasible. Some notable examples of neuromuscular disorders for which the responsible gene has been cloned by this strategy include familial retinoblastoma, myotonic dystrophy, fragile X syndrome, neuronal ceroid-lipofuscinosis, Duchenne/Becker muscular dystrophy and neurofibromatosis-I, also known as von Recklinghausen's disorder. Although not a neurological disorder, the cloning of the gene responsible for cystic fibrosis also provides an excellent example of how the positional cloning strategy has been successfully employed [13].

The final step of the positional cloning strategy for genetic diseases is elucidation of the cloned gene function and mutational analysis in affected individuals. When the cloned gene is homologous to genes with known functions, elucidation of its function is aided enormously. Otherwise, the search for the function of the cloned gene can be as tedious as the cloning process itself. It is a priori evident that if the correct gene has been obtained, the gene structure and the function of its product should be abnormal in affected patients.

Diagnosis. When the marker is sufficiently close to the target gene, the polymorphism of the marker sequence itself can be used for diagnosis of affected individuals. This is true even when the gene responsible for the disease has not been identified because essentially no crossover occurs on the chromosome between the disease-causing gene and the marker. As in DNA diagnosis based on the disease-causing mutation within the responsible gene, knowledge of the nature of the polymorphism in the family is a prerequisite. Conceptually, the disease-causing mutation is merely a polymorphism within the gene. It is no different from a polymorphism in the marker sequence which is so close to the gene that no crossover occurs.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 1999, American Society for Neurochemistry.
Bookshelf ID: NBK27953

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