Recent Developments In Neurogenetics:
Impact On Clinical Practice

Kevin B. Boylan, M.D.
Kevin B. Boylan, M.D. is a Consultant, Department of Neurology at Mayo Clinic Jacksonville.

Introduction

Inherited neurological disorders have been a recognized part of medical practice for well over a century, and have contributed to the development of neurology as a medical specialty. Unfortunately, these disorders have also contributed to the perception that neurology is devoted mainly to the diagnosis or description of untreatable diseases. While this is no longer true of neurological disorders commonly encountered in daily practice, it remains largely true for inherited neurological disease. It has only been with research advances in molecular genetics in the past decade that neurogenetic diseases are finally being drawn into the realm of manageable disorder.

Until recently, the approach to neurogenetic disorders in practice has generally involved the same diagnostic techniques used for noninherited disorders. Too little was known about disease pathogenesis and how to test for specific gene mutations for this to have much practical value. For many disorders, accurate clinical diagnosis might be possible, but a patient with a mild form of a disorder (perhaps identified through a definitely affected relative) could face uncertainty as to the status of themselves and their at-risk offspring. It is in the areas of diagnosis and recurrence risk estimation that current neurological practice has been most directly influenced by new research findings in neurogenetic disorders.

Many examples exist of the impact of molecular genetics on the evaluation and management of neurogenetic disorders. A National Institutes of Health Conference a few years ago on developments in this area included presentations on more than 30 disorders (Fogarty International Conference on Molecular Genetics in Neurology. National Institutes of Health, April 5-8, 1993). In the examples that follow, given disorders have been selected to illustrate how progress in neurogenetic disease has influenced patient care.

Duchenne Muscular Dystrophy

Success of molecular cloning in identification of the genetic defect causing Duchenne muscular dystrophy is paradigmatic of the way information concerning the defective gene can reveal disease pathogenesis, lead to improved methods of diagnosis, and provide a basis for undertaking experimental therapy.1,2

Duchenne dystrophy is a severe X-linked neuromuscular disorder of childhood onset, usually resulting in death in the second decade. Incidence is approximately 1 in 3,500 live male births, one third being new mutations. As a rule, female carriers are not symptomatic. The disorder is caused by a mutation in the X-chromosome gene for dystrophin, a structural protein found mainly in skeletal muscle.3

An important result of studies of the dystrophin gene has been the realization that mutations in this gene also cause the less severe Becker muscular dystrophy, as well as a spectrum of milder neuromuscular conditions, including quadriceps dystrophy, X-linked myalgia, and myoglobinuria. In Duchenne dystrophy essentially no functional dystrophin is produced, while in Becker dystrophy and other dystrophin related disorders, the mutation results in partial loss of function of dystrophin, with the degree of dysfunction determining clinical manifestations of the disease.

Testing for dystrophin gene mutations and abnormal dystrophin in muscle has become an important part of the clinical evaluation of patients with neuromuscular disorders compatible with Duchenne dystrophy, Becker dystrophy and related disorders. Evaluation for dystrophin mutations can be carried out on DNA, usually obtained from peripheral blood cells, or dystrophin itself may be examined in muscle biopsies by Western blotting or immunostaining of muscle sections. These methods have allowed earlier (presymptomatic) diagnosis in suspected cases, and confirmation of the diagnosis if family history is noncontributory. Determination of carrier status in females and prenatal diagnosis in at-risk pregnancies also is possible.

Direct DNA analysis of the dystrophin gene using polymerase chain reaction (PCR) technology is commercially available as a means of detecting mutations which involve deletion of DNA. This is the case in 55% of Duchenne and 70% of Becker patients. Affected males not detected by this assay can usually be identified by assay of dystrophin in muscle biopsies by Western blot or immunostaining of muscle sections. Identification of carriers remains problematic in those with a positive family history but no detectable deletions by standard assays (occurring in 40% of Duchenne families), although specialized techniques may still allow for accurate diagnosis.

Recognition that lack of functional dystrophin was the cause of Duchenne dystrophy led to a groundbreaking attempt at human gene therapy several years ago to introduce normal dystrophin into muscle of boys with Duchenne dystrophy. Muscle cells (myoblasts) from normal donors were injected into selected muscles of patients with Duchenne dystrophy. It was hoped that the normal myoblasts would fuse with the patients' dystrophin deficient fibers, and lead to diffusion of dystrophin from the donor myoblasts throughout the host fibers, resulting in improved contractile function. Unfortunately, prospects for this approach dimmed considerably with the discovery that human myoblast transfers yielded no meaningful "take" of the transplanted muscle cells, and resulted in no significant clinical improvement.

Recent studies suggest that packaging the dystrophin gene in an adenovirus may allow direct introduction of the gene into skeletal muscle fibers, circumventing problems encountered with myoblast transfer. The virus, genetically modified to neutralize pathogenicity, is able to transfer the gene into muscle fibers. A multicenter study to investigate this treatment in Duchenne boys is in the final stages of review by the Food and Drug Administration.

In X-Linked Conditions Who Is At
Risk For Having An Affected Son?

When considering the gene studies for x-linked disorders the woman is not always the carrier. All too often the females are told that they are carriers. For Duchenne's muscular dystrophy and Becker's muscular dystrophy (as well as others) both caused by dystrophin gene mutations the gene is on the X chromosome. However, if a family has only one affected individual the one that is affected (son, uncle) may be the only one with this disease gene.

Dystrophin is a very large gene. It has a very high spontaneous mutation rate. Therefore, it is not automatic that ALL of the women in the family are carriers. Women have a 1 in 3 chance of being a somatic carrier, 1 in 3 chance of having a gonadal mutation, and a 1 in 3 chance of having other relatives that are also carriers. If a woman is a carrier she may have received the gene from her mother OR father. The advanced paternal age of the maternal grand-father is a significant factor in the spontaneous change of the normal gene into a disease gene. (Advanced maternal age is also a factor for spontaneous mutation.)

Whenever possible the dystrophin gene should be analyzed in the individual that is affected with the disease. The current genetic testing can only identify about 70% of the males affected with muscular dystrophy. If this is `falsely negative' then there is no easy way to test the others in the family. Only family DNA marker studies can be done, and this requires cooperation of multiple individuals both affected and unaffected to participate in testing.

Myotonic Dystrophy

Myotonic dystrophy is an autosomal dominant multi-systemic disorder resulting in skeletal muscular dystrophy, and may include cataracts, cardiac conduction defects, and bowel dysmotility among its manifestations. Incidence is 1 in 8,000, with prevalence of approximately 5 per 100,000. Men and women are at equal risk of being affected. The mutation causing this disease arises from a short nucleotide triplet repeat (the same three nucleotide bases occurring as a repeating unit) within the gene for the enzyme myotonin protein kinase, located on chromosome 19. The triplet repeats mutation is an example of a type increasingly identified in genetic disorders.4-7

The number of triplet repeats in normals varies from 5 to 37 triplet units. The number of repeats is unstable, however, and may contract or expand during transmission from parent to child. Expansion of this region of the gene, with repeat numbers in the range of 50 to several thousand triplet units, is associated with occurrence of myotonic dystrophy, apparently because of disruption of gene expression. Disease severity is correlated with the degree of expansion, with higher numbers of triplet repeats being associated with more severe disease.

Discovery of the myotonic dystrophy mutation provided an explanation for two aspects of the disease that complicate genetic counseling. The first of these is the phenomenon of anticipation, a term that refers to the tendency for disease severity to increase, and age of onset to decrease, in subsequent generations of an affected family. Anticipation in myotonic dystrophy had been attributed to ascertainment bias, but is now recognized to occur because of progressive expansion of the myotonic dystrophy triplet repeat from generation to generation in some families.

Congenital myotonic dystrophy is a second feature of the disease explained by the nature of the mutation, and is the most severe form of the disease, presenting as congenital weakness and hypotonia. Children of affected mothers are the only offspring at risk for this condition; it essentially does not occur if the father is the transmitting parent. Most cases are associated with very large triplet repeat regions. Females have an increased likelihood of transmitting large

 

expansions; hence the relationship of congenital myotonic dystrophy with affected mothers. This phenomenon does not entirely explain the association of maternal transmission and congenital myotonic dystrophy, because some patients with congenital myotonic dystrophy have only moderate (as opposed to large) triplet expansions. The basis for this additional maternal risk factor or factors in congenital myotonic dystrophy is presently unknown. The size of the mutation predicts the severity, but only in a very loose or generalized way. Tables compiled show ample areas of overlap for a given repeat size. For the moderate expansion, the size of the triplet repeat may be a little different, and yet the stage of the child may be worse than the parent, and siblings may differ by 10 years the age of onset. This is true within and between families.

Clinical diagnosis of myotonic dystrophy often is straightforward, but uncertainty may arise in mildly affected patients. It is in these patients, and in the case of prenatal diagnosis, that direct DNA testing for the mutation is especially useful. Testing for the chromosome 19 mutation in peripheral blood has become standardized using both PCR and Southern transfer techniques, and is commercially available. The myotonic dystrophy mutation is specific for the disease, and more than 99% of patients have been found to have this triplet expansion mutation. The disorder in the remaining 1% of patients is caused by mutations other than the chromosome 19 triplet expansion; one of these is linked to chromosome 3q. 8

Huntington Chorea

Huntington chorea is an autosomal dominant disorder presenting in adult life with chorea, personality and mood changes, and eventual dementia. The course, with a mean duration of 18 years, is progressively downhill leading to death. Prevalence is approximately 5 per 100,000 in the United States, Canada, the United Kingdom, and Australia, and is less than this elsewhere. The mutation, like that in myotonic dystrophy, is an unstable nucleotide triplet repeat. The mutation is found on chromosome 4 in the gene for the protein huntingtin. Huntingtin bearing the expanded triplet mutation appears to be toxic to neurons, although the exact mechanism of the disease is not yet worked out. 9-12

Clinical diagnosis is not difficult in fully developed cases with a positive family history, but identification of mildly affected or presymptomatic cases is problematic. It was therefore of great clinical interest that a triplet expansion of 10-29 triplet repeats was found in normal subjects, while Huntington patients show an expansion in the range of 36-121 repeats. Expansion in this range is essentially 100% specific for Huntington chorea, and nearly 100% of patients will have the mutation. In other words, if Huntington chorea is suspected clinically and the mutation is not found, then the diagnosis is unlikely. A recent study found triplet expansion in the "Huntington" range in less than 1% of controls, making assay for the mutation a highly accurate means of diagnosis, even in presymptomatic patients.

The reason why a small number of Huntington chorea, patients in the published data lack the triplet mutation is unknown. These patients may have Huntington chorea, caused by a mutation in the Huntington gene other than the triplet expansion, or may have a mutation in a different gene and not actually have Huntington chorea. Similarly, the significance in normal subjects of triplet expansion in the 36-121 repeat range, although extremely rare, is of unclear significance. Follow-up data and accumulation of more control data may resolve this.

The availability of a diagnostic test for Huntington chorea performed on peripheral blood DNA is potentially of great value in the evaluation of patients in whom the diagnosis is suspected. Persons at risk for Huntington chorea can, in nearly all cases, be counseled based on results of this test, assuming the family member is interested in obtaining this information. Presympomatic testing of minors is not done.

Von Recklinghausen Neurofibromatosis

Von Recklinghausen neurofibromatosis (NF1) is a multisystemic autosomal dominant disorder of relatively high prevalence (1 in 4,000), and for which the mutation rate of 1 in 10,000 is the highest known for human genetic diseases. Central and peripheral nervous system tumors are common manifestations, as are skin pigmentary changes, NFI serves as an example of how, despite identification of the gene responsible, the complexities of the mutation process and its effects have limited the practical application of what has been learned about the disease.13-17

Mutations causing NFI occur in the gene for the protein neurofibromin located on chromosome 17. Neurofibromin appears to function as a tumor suppressor, with at least one functioning copy of the gene needed by cells to inhibit spontaneous tumor formation. Genetic transmission of NF1 involves the inheritance by affected offspring of a mutation in the neurofibromin gene. Loss of function (by somatic mutation) of the normal neurofibromin gene, inherited from the unaffected parent, is thought to result in the clinical manifestations of the disease. This may involve cutaneous pigmentation changes (axillary freckling or cafe-au-lait spots), iris hamartomas (Lisch nodules), and neurofibrosarcomas or neurofibromas (depending on the tissue involved).

The neurofibromin gene is relatively large and is subject to a variety of types of mutations at various points along the gene. This variability of mutation has greatly limited the use of molecular genetic techniques for identification of mutations in individual cases, since unlike Duchenne dystrophy, which also involves mutation of a very large gene, the NFI mutations do not tend to occur in a small enough number of gene locations to allow these to be "screened" with high likelihood of identifying a mutation. As a result "the diagnosis of NFI relies almost exclusively on clinical criteria with an occasional linkage analysis for prenatal testing."14 Direct testing for the mutation, in affected or at-risk subjects is not generally available; a recently reported technique that can identify abnormal neurofibromin produced in vitro from peripheral blood seems promising, but is not yet widely available.15-16 Only one lab offers this test, and it is has a 30% false negative result. This test is unable to predict severity, seizures, age of onset, nor degree of mental deficit. Since, spontaneous somatic mutations have been found in the tumors of affected individuals the inherited mutation is less a predictor than the somatic cell line that is undergoing transformation and growth. Little has been learned to predict the types of outcomes that are so inherent in this neurocutaneous syndrome. Why one child is born with severe involvement and a sibling is thirty years old before signs show up is not known from the DNA or RNA analysis. At this time, there is no mutation analysis that is commercially available for prenatal testing.

Discovery of the neurofibromin mutation and elucidation of the mechanism of tumor formation in NF1 does offer some insight into potential therapies, either by attempting to inhibit neoplastic transformation, or by compensating pharmacologically for deficiency of neurofibromin. Much progress has been made in studies of the genetic basis of NFI, but there is much more to be learned before the impact of these advances can be applied in clinical practice.

Insights From Neurogenetics Into Noninherited Disorders

An important development in neurogenetic research has been in the area of diseases that are generally not considered inherited disorders, but for which inherited forms exist. Clinical similarity between sporadic and inherited forms of these diseases suggests that they may have features of disease pathogenesis in common, and that understanding of the inherited form might be relevant to determining the cause of the sporadic condition; Amyotrophic lateral sclerosis and Alzheimer disease provide examples of this line of research.

Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a progressive disorder of adult life in which degeneration of motor neurons eventually results in generalized paralysis and respiratory Failure. The best current treatments are of marginal benefit, and mean survival is four years. Approximately 10% of cases show autosomal dominant inheritance, but otherwise are similar to the sporadic condition.18-20

Mutations in the gene for a form of superoxide dismutase (SOD) on chromosome 21 cause an autosomal dominant form of ALS in 20% of families with the disease.21 SOD1 is important in scavenging free radicals, and this finding supports the hypothesis that neuronal loss in ALS may result at least in part from the damaging effect of excess free radicals.

Detection of a SOD1 mutation can now help in establishing a cause for ALS in some families, and in appropriate circumstances, in identifying presymptomatic family members at risk for the disease. These presymptomatic individuals may have great impact on ALS research as a well-defined patient group for evaluation of new therapies. Identification of the mutations in other ALS families not linked to superoxide dismutase may also prove beneficial in this regard.

Alzheimer Disease

Alzheimer disease results in progressive cognitive decline, usually beginning at age 65 or older, and is the most common cause of dementia in the elderly. Although a positive family history is a significant risk factor for developing Alzheimer disease, late age of onset has complicated attempts to identify an inherited component of the disease, since affected family members may die of unrelated causes before developing signs of dementia, and available families may be too small for meaningful analysis. Familial Alzheimer disease with autosomal dominant inheritance nevertheless has been identified in a small minority of Alzheimer patients. Disease in several of the published families differs from the sporadic form because of early onset (before the sixth decade), but otherwise the disorder in familial and sporadic cases is similar. 22

Development of Alzheimer brain pathology has been studied intensively in search of clues to disease pathogenesis, particularly with respect to the deposition of the amyloid precursor protein in amyloid plaques. This process is considered to be central to disease pathogenesis, and an early question in familial Alzheimer disease was whether inherited forms involved a mutation of the amyloid precursor protein (APP), which might lead to amyloid deposition. This is now proven, with mutation in the gene for the amyloid precursor protein on chromosome 21 having been found in a small number of Alzheimer families, all with early onset. Two other genes are linked to familial Alzheimer disease; one encoding the protein Presenilin 1 on chromosome 14, and the other encoding Presenilin 2 on chromosome 1. Mutations in these genes probably cause about half of all cases of early onset Familial Alzheimer disease, possibly by increasing production of APP.16 As a result of these findings, there is hope that developing a better understanding of the process of amyloid deposition will lead to effective therapy for both sporadic and familial Alzheimer disease.

A key result of research on genetic factors in Alzheimer disease is the identification of an allele of apolipoprotein E, Apo-E4, as a risk factor for Alzheimer disease in familial and sporadic cases with late onset.24, 25 Apo-E4 has not been established as a primary cause of Alzheimer disease in familial cases, but clearly is a genetic factor denoting increased risk for Alzheimer disease, apparently by contributing to amyloid deposition. In a recent study, 36% of sporadic Alzheimer patients carried the Apo-E4 allele compared to 16% of controls. In a separate study, the odds ratio of Apo-E4 for association with Alzheimer disease was 17.9 in homozygotes and 4.2 for heterozygotes. It has been suggested that the role of Apo-E4 in Alzheimer disease is an example of how multiple genetic risk factors may contribute to the pathogenesis of the disease. It seems likely that other such risk factors will be identified, and lead to development of specific treatments.

Apo-E4 assay of peripheral blood is commercially available and is marketed as being of value in the diagnosis of Alzheimer disease. Although Apo-E4 clearly is an Alzheimer's risk factor, nonspecificity in patients with dementia, and limited predictive value in presymptomatic testing limit its value in current practice.

Conclusions

The study of inherited diseases in neurology (and medicine generally) may be thought of as comprising three stages. The first involves clinical description of the disorder, the second, genetic and biochemical characterization of the mutation or mutations, and the third, treatment of the disorder based on what is learned about its molecular basis. Research on Duchenne dystrophy has reached the stage of attempted therapy; for other disorders (i.e., myotonic dystrophy and Huntington chorea), the first stage has led to the second, with research ongoing to determine the molecular pathogenesis of the disease. Expanding knowledge of inherited disorders continues to add to our understanding of the genetically based features of even complex disorders such as Alzheimer's disease.

Basic and clinical research on inherited neurological disorders has already provided important information regarding disease pathogenesis, and allowed refinement of diagnostic techniques for many disorders, several of which have been discussed. The pace of advancement in molecular genetic research is rapid, and offers hope that treatment of genetic diseases and the genetic aspects of multifactorial disorders will some day become a part of standard management of these conditions.

REFERENCES

The material in this paper summarizes an extensive literature that is well reviewed in the relatively small number of references cited, many of these being review articles. The references provide a more detailed discussion than was possible within the scope of this paper, and for interested readers, include citations for the original research.

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February, 1999/ Jacksonville Medicine

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