Molecular Genetic Diagnosis of Neurological Diseases

Molecular Genetics of Neurological Disorders CHAPTER 107

Molecular Genetic Diagnosis of Neurological Diseases Thomas Gasser and N.W. Wood

Recent progress in molecular genetics has greatly improved our understanding of the molecular basis of many inherited neurological diseases. With the Human Genome Project nearing completion, the genomic sequence of a large number of genes which, when mutated, can cause neurological disorders, is now known. This increasing wealth of knowledge has allowed the reclassification of a number of formerly heterogeneous clinical syndromes, opens up novel diagnostic possibilities, and allows characterization of the pathological gene products, thereby providing further insight into the molecular pathogenesis of these disorders. This will eventually lead toward new approaches to therapy and prevention. The following section briefly outlines the molecular genetic basis of inherited diseases, describes some fundamental methods of genetic analysis, and gives an overview of the present role of molecular diagnosis in neurological diseases (see Table I).

M E T H O D S OF GENE MAPPING Genome The human genome consists of 23 pairs of chromosomes (22 autosomes plus the sex chromosomes X and Y). One of each pair is inherited from the mother, the other from the father. The backbone of the chromosome consists of a continuous DNA double-strand, approximately 50-200 million base pairs (bp) in length, depending on the size of the chromosome. The entire genome spans about 3 billion bp. The genetic information is contained within roughly 30-40,000 genes (i.e., DNAsequences coding for a specific protein plus regulatory sequences). The genes themselves have an average length Neurological Disorders: Course and Treatment, Second Edition Copyright 2003, Elsevier Science (USA), All rights reserved.

of several thousand bp each and account for only a small proportion of the entire genomic DNA. They are separated by larger "noncoding" segments, some of which have regulatory functions. There are, however, large regions of the genome for which, at present, no function is known (Figure 1). In a major international collaborative effort, the human genome project (HUGO), the sequence of more than 90% of the human genome, has now been determined. However, it will probably take many more years to unravel its function. Up-to-date information can be obtained through the internet (www.ncbi,gov).

Mutations The genetic information contained in the DNA sequence of a gene is translated into the amino-acid sequence of the corresponding protein (gene product). Mutations (i.e., changes of the DNA sequence of a gene) can result in an altered or absent gene product. These alterations may in turn cause disease. If a mutation occurs within the germ line, it will be passed on from generation to generation. DNA sequence variations outside the coding or regulatory regions of a gene usually have no direct influence on cellular function. They are much more common than functionally relevant mutations, occuring on average once in every 500 bp. They are also transmitted from one generation to the next and can serve as valuable "genetic markers."

Gene Mapping For most inherited neurological diseases, the primary metabolic or structural defect (i.e., the pathological gene 1525

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MOLECULAR GENETICS OF NEUROLOGICAL DISORDERS

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FIGURE 1 Organization of the genetic information. Each chromosome contains a continuous DNA double-strand, approximately 50-200 million basepairs (bp) in length. Genetic linkage analysis allows localization of a gene to a chromosomal region of 1-10 million bp. This region may contain dozens of genes, which themselves contain information-bearing (exons) and intervening (introns) sequences.

product) has escaped detection by biochemical or pathological methods. Most of the disease genes known today have been identified by genetic methods, which allow determination of the chromosomal position of a gene causing an inherited disease without prior knowledge of its gene product. This procedure is called genetic linkage mapping and positional cloning. Because genetic linkage mapping has been by far the most successful strategy in the analysis of hereditary diseases over the past 20 years and because it is of direct importance for molecular genetic diagnosis, the basic concepts of this method will be briefly outlined in the following sections.

Linkage and Recombination During meiosis, the homologous maternal and paternal chromosomes of the stem cell are separated to form the haploid set of chromosomes of the gamete. During this process, corresponding fragments of homologous chromosomes are exchanged (recombination or crossing-over). An average of one to three recombinations occur on each chromosome during meiosis. The recombination breakpoints are, to a large extent, randomly placed along the length of the chromosome. Therefore, genes that are located in proximity to each other on the same chromosome are passed on together to the following generation (i.e., they are genetically linked). On the other hand, if genes are located far apart on a chromosome, they are likely to be separated by recombination events during meiosis

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FIGURE 2 (A) Cosegregation of two inherited phenotypic traits, the ABO-blood group and an inherited disease: all affecteds in this family carry the A-allele. (B) Cosegregation of a DNA marker and an inherited disease: all affecteds in this family carry the marker allele " 1 . "

and are therefore inherited independently of each other. This means that the frequency of observed recombination events between two gene loci is a measure of their distance on a chromosome. On average, a recombination frequency of 1% (1 recombination in 100 observed meioses) corresponds roughly to a physical distance of 1 million bp. If a large number of meioses is studied for different gene loci, the order and distance of these loci can be determined and genetic "maps" can be established. Figure 2 shows an example for genetic linkage of two inherited traits: tuberous sclerosis and the ABO bloodgroup system. The genes for both traits are located on the long arm of chromosome 9. All affected family members, indicated by black symbols, have a blood group allele in common (allele A in this example). This cosegregation of two traits in a small family could well be observed by chance alone. However, if it is found in large families or in a large number of small families, it provides statistical evidence that the two genes are located within a relatively small region of the same chromosome. If the chromosomal position of one of these genes is already known, the position of the linked gene locus can be inferred.

MOLECULAR GENETIC DIAGNOSIS

DNA Polymorphisms Only polymorphic traits (i.e., those that are present in two or more distinguishable forms [alleles]), can be used for linkage mapping. Polymorphic phenotypic traits, which can be detected by biochemical or immunological means and are inherited in a mendelian fashion, such as blood groups or HLA types, are rare. The major contribution of molecular genetics to linkage analysis has been the discovery of polymorphisms that can be distinguished on the DNA level (DNA markers). The most useful polymorphisms are short repetitive DNA motifs, which can be highly variable in length (variable number of tandem repeats = VNTRs or dinucleotide, trinucleotide or tetranucleotide repeats = microsatellites). A large number of these polymorphic DNA sequences has been identified and placed on genetic maps, covering the entire genome. Most of these polymorphisms are situated in noncoding DNA regions (introns) and seem to have no functional significance. The chromosomal segments bearing these DNA markers can be distiguished, and their segregation can be followed through the generations of a family (Figure 2B). In principle, any disease showing mendelian inheritance can be mapped to a chromosomal region by this method, provided that a sufficient number of meioses can be observed.

Linkage Analysis Linkage analysis is the statistical method used to determine the recombination frequency, and consequently the genetic distance, between two (or more) genetic loci (genes or DNA markers). The statistical measure for the reUability of these observations is the lod score (lod = log of the odds). The lod score is calculated as the log of the ratio of the two probabilities that linkage does or does not exist between two gene loci. A lod score of greater than three is generally considered to be positive evidence for linkage, provided that the genetic parameters (mode of inheritance, penetrance, frequency of phenocopies) are known. If this is not the case (as in diseases with presumed polygenic inheritance or multifactorial etiology), lod scores have to be interpreted with caution.

Positional Cloning The approach of linkage lization of a gene locus to million bp. Dozens of genes such a region. To identify the

analysis allows the locaa segment of about 1-2 may be contained within gene under consideration.

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all the genes of this segment may have to be sequenced and searched, base by base, for mutations. After completion, at least in draft form, of the human genome, gene identification is much simpler than it was. There are now a number of search engines available free to anyone who wishes to investiagate a genetic region (e.g., the Human Genome Browser at http://genome.ucsc.edu/goldenPath/septTracks.html). One can now rapidly assimilate a "virtual" contig across the region of interest. This can then be searched for possible candidates, initially on the computer but eventually sequencing in the laboratory is required to identify the pathogenic mutation.

MOLECULAR GENETIC DIAGNOSIS In clinical practice, the availability and the limitations of molecular diagnosis depend on our knowledge of the molecular genetic basis of the respective disease but also on the degree of genetic complexity of the disorder under investigation. Some diseases, such as Huntington's disease, are caused by a specific mutation in a single gene (Huntington's Disease Collaborative Research Group, 1993), and routine molecular diagnosis can be provided by a simple polymerase chain reaction (PCR)-based assay. In other cases, however, many different mutations in a given gene may underlie a disorder (allelic heterogeneity). Depending on the size of the gene(s), this may render molecular diagnosis very costly and time-consuming. Molecular diagnosis may be further complicated by the fact that mutations in a number of different genes may cause similar or indistinguishable phenotypes (genetic heterogeneity). For example, tuberose sclerosis may be caused by mutations in a gene either on chrosome 9 or chrosome 16. Clinicaly these disorders are indistinguishable.

Molecular Genetic Diagnosis in Diseases with Known Genetic Defect If the gene causing a neurological disorder is known, direct molecular diagnosis can be performed by mutational analysis. Only DNA from the affected individual is required. Usually, exons that are known to harbor mutations in the particular disorder will be amplified from DNA, which has been extracted from peripheral blood leukocytes by the PCR. Depending on its type, the mutation will then be detected either directly by gel electrophoresis (e.g., in the case of trinucleotide repeat expansions or large deletions) or after digestion by restriction enzymes or by direct sequencing (point mutations, small deletions, or insertions).

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MOLECULAR GENETICS OF NEUROLOGICAL DISORDERS

Diseases Caused by Expansions of Triplet Repeat Sequences An increasing number of inherited neurological disorders are recognized to be caused by expansions of unstable triplet repeat sequences within or adjacent to the coding region of genes, among them Huntington's disease (Huntington's Disease Collaborative Research Group, 1993), the spinocerebellar ataxias (Brice, 1998), X-linked bulbospinal neuronopathy (Kennedy's disease) (La Spada et aL, 1991), as well as myotonic dystrophy (Brook etal., 1992) and Friedreich's ataxia (Campuzano et al, 1996). Most triplet repeat disorders are caused by the expansion of a CAG-repeat sequence within the coding region of the gene, leading to an elongated polyglutamine domain in the encoded protein. These expansions are likely to be "gain of function" mutations, with the mutation causing a novel toxic function that dominates the normal gene/protein function. Friedreich's ataxia is an exception. The expansion of a GAA-repeat in the Frataxin gene leads to a loss of functional protein and consequently to a recessive mode of inheritance. All repeat expansions can be detected by a simple assay based on PCR or Southern blotting. Routine molecular diagnosis is possible for confirmation of a clinical diagnosis or for the determination of genetic status presymptomatically or prenatally. Age at onset of disorders caused by triplet repeat expansions is usually inversely correlated with the length of the triplet repeat. The number of triplets may increase from one generation to the next (dynamic mutation); this observation explains the phenomenon of anticipation (i.e., progressively earlier age of onset of an inherited disease in successive generations), which had been described in Huntington's disease and myotonic dystrophy long before the discovery of triplet repeats. Diseases Caused by Point Mutations, Deletions, and Insertions Most inherited neurological disorders can be caused by a number of different mutations (base exchanges, small deletions, insertions or other alterations of the DNA sequence). This is called "allelic heterogeneity." In principle, these sequence variations can be detected by direct sequencing of the exons of a gene. However, if a gene is very large (genes with more than 30 exons are not uncommon) and mutations are scattered throughout the entire gene, direct mutational analysis can be very costly and time-consuming. In these cases, routine sequence analysis is sometimes offered only for portions of a gene, where mutations may be clustered (e.g., in CADASIL, where 70% of mutations are found in exons 3 and 4 of the Notch3 gene (Joutel et al, 1997).

In other cases, indirect molecular diagnosis by family studies with linked DNA markers (see later) will remain an important tool for the presymptomatic detection of gene carriers. One example is Duchenne and Becker muscular dystrophy, which are both caused by a variety of mutations in the dystrophin gene on chromosome Xp21.1, only about 60% of all mutations (usually structural alterations such as large deletions or duplications) are detected by routine mutational analysis (PCR amplification of exons or sets of exons). In the remaining cases, which may be caused by more subtle gene defects, such as point mutations, monoclonal antibodies against dystrophin can be used to demonstrate a lack or abnormal size of this structural protein in muscle biopsy specimens by immunohistochemistry or Western blot analysis, thus allowing a diagnosis at the protein level. In such families, studies with linked DNA markers may be used to determine carrier status at the DNA level.

Genetic Diagnosis in Diseases with Known Gene Location ("Indirect" Molecular Diagnosis) Knowledge of the chromosomal position of a disease gene allows molecular support for the diagnosis, even if the disease gene itself is unknown or if analysis is unfeasible. "Indirect" molecular diagnosis is limited to risk determination for an individual in whose family an inherited neurological disease has already been diagnosed clinically. The method is based on the analysis of DNA markers known to be closely linked to the disease under investigation. Determination of these marker alleles in healthy and affected family members allows the identification of the disease-gene-bearing chromosome in this particular family. It must be emphasized that accurate clinical diagnosis in at least one affected family member is an absolute prerequisite for this type of molecular diagnosis, and there is a small but definite error rate with this sort of linkage analysis. Until the gene for Huntington's disease was identified in 1993, predictive testing for Huntington's disease was the most widely used application for indirect molecular diagnosis. As more and more disease genes are identified, direct mutational analysis will become increasingly important.

Genetic Heterogeneity Many hereditary neurological disorders are genetically heterogeneous (i.e., the disease can be caused by mutations in a number of different genes). This may complicate molecular genetic diagnosis in individuals who do not carry one of the frequent mutations.

MOLECULAR GENETIC DIAGNOSIS

The most common form of inherited neuropathy, Charcot-Marie-Tooth disease (CMT, formerly called hereditary motor and sensory neuropathies, HMSN), is an example (Pareyson, 1999). Most cases of the demyelinating form of the disease, CMT I, are caused by the duplication of a large segment (approximately 1.5 megabases) of chromosome 17 (CMT type la). The duplicated DNA segment contains the gene for a component of the myelin sheath of peripheral nerves, peripheral myelin protein-22 (PMP-22). This duplication can easily be detected by routine molecular methods. In some families, however, a point mutation, rather than a duplication, of PMP-22 is responsible (i.e., allelic heterogeneity, see earlier). In still other families (about 5%-10% of cases), mutations in another component of myelin (PO-Protein) on chromosome 1 cause a clinically indistinguishable disorder (CMT lb). Another form of CMT is due to mutations of the gene for connexin 32, a gap junction protein on the Xchromosome, and other forms, collectively called CMT-lc, exist for which the genetic defects are still unknown (Table I). Another example of genetic heterogeneity are the spinocerebellar ataxias. Although there are group differences between the SCAs with respect to their clinical features, the overlap between the different entities makes prediction of the molecular origin impossible and emphasizes the usefulness of molecular analysis (Stevanin et aL, 2000).

Mitochondrial Diseases In recent years, several syndromes have been described in association with mutations of the mitochondrial genome (mtDNA). Deletions, and occasionally duplications, of mitochondrial DNA are found in most patients with mitochondrial encephalomyopthies, a spectrum of disorders ranging from mild chronic progressive external ophthalmoplegia (CPEO), sometimes with associated neurological symptoms (CPEO-plus), to severe cases of the Kearns-Sayre syndrome (Simon and Johns, 1999). Deletions are sometimes not detectable in DNA prepared from lymphocytes, so that muscle DNA has to be examined. Recurrence risk for relatives, including offspring, is very low in patients with deletions (Klopstock and Gasser, 1999). Point mutations of mitochondrial DNA, the two most common (at positions 3243 and 8344) of which are associated with two well-described maternally inherited syndromes, mitochondrial encephalopathy with lactic acidosis and strokelike episodes (MELAS) and myoclonic epilepsy with ragged red fibers (MERRF), can be detected in a blood sample in most cases. Other mutations have been found to cause Leber's hereditary

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optic neuropathy (LHON), multiple symmetric lipomatosis, and an increasing number of other phenotypes (Scharfe et al, 2000).

Neurological Diseases with Complex (Polygenic or Multifactorial) Inheritance Neurological disorders exhibiting monogenic mendelian inheritance are relatively rare. In a number of much more common diseases, such as Alzheimer's disease, Parkinson's disease, the epilepsies, or multiple sclerosis, genetic factors seem to contribute to the etiology, but only a small minority of cases (usually 5% or less) are inherited in a clear mendelian fashion. Several genes for rare mendelian subgroups of these disorders have been identified, and their analysis has provided important insight into their molecular pathogenesis. For example, the crucial role of amyloid-p protein and P-secretase in the formation of amyloid plaques and neurodegeneration in Alzheimer's disease (Hardy et ai, 1998), or of alpha-synuclein in Parkinson's disease (Farrer et aL, 1999), has been defined an the basis of the analysis of monogenic variants of these common diseases. Nevertheless, the etiology in most sporadic cases is still unknown. This may be because, in addition to genetic susceptibilty, other nongenetic factors (e.g., toxic, infectious, or immunological factors) may play a role in the etiology of the disorder (multifactorial etiology). Alternatively, several inherited traits may be required to act together to determine a particular phenotype (polygenic inheritance). A number of difficulties limit the applicability of finkage analysis to these disorders. A major prerequisite for "classical" linkage analysis is that the genetic parameters of the disease under investigation, such as the mode of inheritance, penetrance, and gene frequency, are known. For diseases with complex inheritance, these parameters usually cannot be determined with any degree of confidence. Furthermore, a clear distinction between affected and unaffected individuals, which is the basis for all forms of linkage analysis, may not always be made. For example, it is still unclear whether asymptomatic individuals with some degree of neuropathological changes suggestive of Alzheimer's disease or Parkinson's disease ("incidental Lewy-body brains") should be considered as presymptomatically affected. Similarly, it is not known whether certain EEC changes in unaffected relatives of patients with epilepsy should be considered as a sign of the disease. Despite these difficulties, recent advances in our understanding of disorders with complex inheritance show that they are in principle amenable to molecular

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MOLECULAR GENETICS OF NEUROLOGICAL DISORDERS

genetic analysis. The E4-allele of the gene for apoiipoprotein E has been identified as the first major risk factor for sporadic late-onset Alzheimer's disease (Strittmatter et ai, 1993) and more are likely to follow (Du et al., 2000). Out of the heterogeneous clinical syndromes, more and more genetically homogeneous subgroups will be isolated, and their study will provide an increasing understanding of the cascade of molecular and cellular events leading to neuronal dysfunction and disease.

voluntary consent of the patient. Therefore, the neurologist should establish that a patient or lawful surrogate is capable of comprehending relevant information and capable of exercising informed choices. Molecular genetic diagnostic tests should not be performed at the request of members of the patients' families or other third parties (e.g., insurers, employers) without the express written consent of the patient.

Confidentiality

The primary goal of molecular diagnosis is to provide help for the individual patient, client, and/or their families. Reducing the prevalence of inherited disorders in a population or in subsequent generations may be a secondary effect but must never be allowed to guide the process of genetic counseling (Pulst, 2000).

Test results suggesting that patients or family members carry mutations that indicate or predict a major neurological disorder or a susceptibility to a neurological disorder are highly sensitive. Therefore, rigorous measures to ensure confidentiality should be taken. Test results should never be disclosed to a third party without explicit written consent from the patient or their lawful surrogates.

Genetic Counseling

Presymptomatic Diagnosis

It must always be kept in mind that the molecular genetic diagnosis of an inherited disorder affects not only the patient but also the entire family. Therefore, genetic counseling is an essential component of diagnosis of inherited disorders. Sensitive and informed counseling provides patients and families a foundation for decisions about testing. Patients should be counseled as to the clinical features and course of the respective disease, as well as to potential consequences for the family, taking into consideration the most important genetic parameters such as mode of inheritance and penetrance. If the treating neurologist does not have thorough experience with inherited disorders, counseling by an experienced counselor from a department of human genetics or another genetic counseling unit is strongly advised. In most situations, genetic testing should not be performed until adequate counseling has been provided (The Practice Committee Genetics Testing Task Force of the American Academy of Neurology, 1996; The American College of Medical Genetics/American Society of Human Genetics Huntington Disease Genetic Testing Working Group, 1998). In the case of predictive testing (see later), psychological counseling by appropriately trained persons is essential and mandatory before testing and after results have been disclosed.

The identification of disease genes allows presymptomatic (predictive) diagnosis in many cases. Guidelines for presymptomatic diagnosis have been issued for Huntington's disease by The World Federation of Neurology Research Group on Huntington's Disease (1993). These guidelines, which include extensive pretest and posttest counseling and thorough psychological support during the extensive process, should be followed in all cases of presymptomatic diagnosis. Generally, presymptomatic diagnosis should be provided within the setting of a department of Human Genetics. If no clear therapeutic consequences can be envisioned, presymptomatic diagnosis should not be performed on minors. Molecular genetic diagnosis allows accurate presymptomatic and prenatal determination of an individual's disease-carrying status. It does not indicate whether a person will necessarily have the disease develop. This is governed by the penetrance of the mutation. For example, Huntington's disease is virtually 100% penetrant; that is, all gene mutation carriers will have the disease develop should they live long enough. Whereas in the case of primary generalized dystonia caused by a GAG deletion in the DYTl gene, the penetrance is reduced to 30%. Therefore even carrying the mutation in this case means that only a minority will have the disease develop. There is an age dependency to this penetrance such that older than the age of 28 years the chance of being affected with dystoina is very small. The underlying mechanisms behind reduced penetrance are not clear. Predictive diagnosis is especially important if it allows early therapeutic intervention. For example, early initiation of therapy in Wilson's disease can

ETHICAL CONSIDERATIONS

Informed Consent As is true for all diagnostic procedures, the essential prerequisite for molecular diagnosis is the informed and

MOLECULAR GENETIC DIAGNOSIS

prevent serious neurological complications. Most inherited neurological diseases, however, are still not amenable to specific therapy. In these instances, the decision to determine the risk of an individual has to be considered carefully in each case. In diseases with onset during adult life, such as Huntington's disease, the reason for presymptomatic testing is usually the planning of partnership, family, and career. However, a presymptomatic diagnosis may mean that the individual has to spend many years of productive life with the knowledge of an incurable disease developing later in life. The experience of genetic counselling centers shows that a significant proportion of at-risk individuals seeking presymptomatic testing will change their mind during the counseling process. The initiative for presymptomatic testing should always arise from the proband, not from the counselor, and informed consent must be given. Extensive genetic counseling is required before the test and support should be available afterwards. Molecular genetic techniques have opened up a new approach to many scientific questions and have improved our diagnostic repertoire, but they also pose new problems and dilemmas. The diagnostic approach in an individual patient should always be guided by a careful deliberation of the possible consequences for the patient and his or her relatives. Individual discussion with a physician who is knowledgable about the possibilities and limitations of genetic diagnosis is of paramount importance. In summary, it can be seen that progress in our understanding of molecular pathogenesis is already having a major impact on the clinical neurologist with improved diagnosis and counseling of families. However, this progress will be dwarfed by the changes over the next few years. One can foresee not only improved understanding of the genetic factors involved in relatively rare mendelian disorders but also a much deeper knowledge of those factors involved in common disorders, such as stroke, multiple sclerosis and Parkinson's disease. Pharmacogenomics is an emerging field that will produce major benefits in terms of drug discovery, side effect management, and drug responsiveness. In the coming 5-10 years there will be no neurological field that will not be affected by these developments.

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