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Current Concepts of Cerebrovascular Disease and Stroke
Genetic Aspects of Cerebrovascular Disease Mark J. Alberts, MD
I
n the past conventional wisdom held that stroke and other forms of cardiovascular disease were due primarily to environmental factors and that genetic background played a relatively minor role in disease pathogenesis. However, recent discoveries in the fields of molecular and population genetics have suggested the need to reexamine the role of genetic factors in the pathogenesis of vascular disease. Research into the genetics of stroke presents some unique challenges since stroke is a heterogeneous disease with multiple risk factors. However, such studies provide an opportunity to elucidate the etiology of stroke at a molecular level and to attempt true primary prevention. This article reviews some of the principles of inherited disorders and genetic research techniques and applies the concepts to the study of the genetics of specific stroke types. Over 3,000 genetic disorders have been described in man, but the gene defect responsible for a specific disease has been identified for only a handful of diseases. Many genetic diseases are termed "simple" diseases: they follow a clear Mendelian pattern of inheritance (e.g., autosomal dominant, X-luiked recessive) and are due to a defect in only a single gene, and every individual with the gene defect will display the mutant phenotype. Examples of simple neurogenetic diseases include Huntingdon's chorea, Duchenne's muscular dystrophy, and myotonic muscular dystrophy. The "complex" genetic disorders are thought to be due to multiple gene interactions (perhaps influenced by environmental factors), with variable clinical expression of the disease phenotype.1 Examples include diabetes, cancer, and atherosclerosis. The study of complex genetic disorders is complicated by numerous factors: 1) Environmental factors: Extrinsic influences such as diet, smoking, and stress may be responsible for the modified expression of certain genetic traits. For example, an individual with the genetic propensity for hyperlipidemia may reduce serum cholesterol by eating a modified diet. Likewise, genetic factors may have
a role in determining how much an individual eats, how one handles stress, and who chooses to smoke. 2) Risk factors: It is apparent that the development of certain major risk factors such as hypertension and diabetes are under some genetic influence. It is likely that these risk factors are themselves synthetic traits (see below) and are also partially influenced by environmental factors. 3) Synthetic traits: Multiple genes can interact to produce a trait. Atherosclerosis or hypertension may be examples of synthetic traits. Since complex genetic diseases may be due to several synthetic traits acting together, analysis of such interactions becomes exceedingly complex. 4) Polygenic trait: In this case abnormalities of multiple genes are necessary to produce a specific trait. 5) Genetic heterogeneity: Defects at more than one genetic allele can produce similar phenotypic abnormalities. 6) Phenocopy: An environmental factor can produce a mutant phenotype identical to that caused by a defective gene. For example, a dissection of the carotid artery due to neck trauma can produce a stroke with clinical findings similar to atherosclerotic occlusion of the carotid artery. Two genetic terms which are often misunderstood are penetrance and expressivity. Penetrance is an all-or-nothing phenomenon: a genetic disease is completely penetrant if every person carrying the mutant genotype expresses the mutant phenotype and incompletely penetrant if some individuals carrying the mutant genotype do not have the mutant phenotype. Variable expressivity refers to the propensity of some genetic disorders to produce quite varied phenotypic findings. Neuroflbromatosis is an example of a genetic disorder with variable expressivity. The issues described above are important for understanding genetic diseases in populations. The techniques described below are used for studying the molecular genetic abnormalities responsible for these complex diseases.
From the Stroke Acute Care Unit, Division of Neurology, Department of Medicine, Duke University Medical Center, Durham, N.C. This work was supported in part by grants T-32-NS07274 and P-50-NS06233 from the National Institutes of Health, Bethesda, Md. Dr. Alberts is a recipient of a National Institutes of Health Fellowship Training Award in Cerebrovascular Disease. Reprinted from Current Concepts of Cerebrovascular Disease and Stroke 1990;25:25-30.
Molecular Genetic Techniques The goal of molecular genetic research is to identify and characterize the genetic defect responsible for a specific genetic disease. The magnitude of this task can be better understood when it is realized that the human genome has over 3 billion bases of DNA organized into about 100,000 genes. Since only a small number of specific genes have been isolated
Downloaded from http://stroke.ahajournals.org/ by guest on September 30, 2015
Alberts Genetic Aspects of Cerebrovascular Disease and characterized, the genes responsible for most genetic diseases have not been identified. Classic genetic research has attempted first to find the defective protein and then to isolate the abnormal gene. However, since the abnormal protein responsible for most genetic diseases has not been found, a new approach is warranted. This approach, termed reverse genetics, uses genetic linkage analysis to identify chromosomal regions that are close to disease loci. One advantage of reverse genetics is that no a priori information about the location of the mutant gene is necessary. Linkage can be tested with probes (fragments of DNA) that are scattered throughout the human genome. The specific techniques and terminology of reverse genetics have been reviewed recently, and the interested reader is referred to these excellent references for more detailed information.1'2 One of the key steps in studying genetic linkage is to identify sufficiently large families with the disease of interest. If the familial form of the disease is rare or the available families do not have multiple generations or many living members available for testing, the usefulness of classic genetic linkage studies becomes somewhat limited. Because of the likely multigenic pathogenesis of stroke, classic genetic linkage analysis may be insufficient to find linkage. Some of these problems may be overcome by using newer methods of linkage analysis. The affected pedigree member (APM) method, developed by Weeks and Lange,3 uses marker phenotypes for APMs only. This technique is more sensitive for distantly related relatives that are affected and share the same rare allele. Another advantage of the APM method is that no assumptions about the mode of inheritance are required. Another potentially powerful technique is the affected relative pairs (ARP) technique.4 Various combinations of pairs of affected relatives (siblings, first cousins, half sibs, etc.) can be used to identify major or minor disease susceptibility loci. This approach is attractive in cases where large immediate families may not be available but members of extended pedigrees are still alive. Both the APM and ARP methods may be most useful for identifying major gene effects in the pathogenesis of complex diseases rather than identifying every gene involved in an inherited disease. Stroke Genetics Risk Factors The well-documented risk factors described for stroke include hypertension, diabetes, lipid abnormalities, heart disease, advanced age, fibrinogen levels, smoking, and coagulation defects. Many of these risk factors are probably synthetic traits themselves (e.g., hypertension, lipid levels) while others have a stronger environmental component (i.e., smoking). A recent study evaluated the relative importance of genetic and environmental factors in determining
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blood pressure, lipid levels, and body mass index in over 300 twins and 1102 healthy adults. This study found that genetic background had a significantly greater influence on these risk factors than environmental components.5 Another risk factor that is receiving increasing attention is fibrinogen. Several studies have shown a correlation between plasma levels of fibrinogen, stroke risk, and the rate of progression of carotid atherosclerosis.6'7 The control of plasma fibrinogen levels may be under both environmental and genetic control. Since fibrinogen is an acute phase reactant, it increases as a result of various injuries (including stroke or myocardial infarction), inflammation, and cigarette smoking. Genetic studies have shown that plasma fibrinogen levels are significantly elevated in individuals who are homozygous for the rare B2 allele within the fibrinogen gene. Therefore, fibrinogen may represent an important risk factor under both genetic and environmental control. Screening at-risk individuals for the potentially harmful B2 allele might help identify people who should be closely monitored and who should avoid environmental factors that could accelerate atherosclerosis. Various lipid and lipoprotein abnormalities at the protein and gene level have been correlated with the development of cardiovascular disease.8'9 These disorders are also under environmental and genetic control. Familial hypoalphalipoproteinemia is an autosomal dominant trait that is a common cause of high density lipoprotein (HDL) deficiency. It has been associated with premature cardiovascular disease and strokes.10 Other disorders such as familial hypercholesterolemia (homozygous form), type III and type IV hyperlipoproteinemia, and Tangier disease have been associated with premature atherosclerosis and stroke.11-12 As characterization of the complex genetic pathways responsible for lipid metabolism improves, more high-risk alleles and mutations will be identified. Epidemiologic Studies If genetic factors play a role in the etiology of stroke, one might expect epidemiologic studies to identify a family history of stroke as a significant risk factor. Although almost a dozen large-scale studies reported since 1966 have attempted to analyze this issue,13"23 they are limited by some intrinsic weaknesses. Most of the studies did not separate strokes into various subtypes and pathogenic mechanisms, thereby severely limiting any genetic analysis. For example, a large hemispheric stroke could be caused by a cardiac embolism (from atrial fibrillation or valvular heart disease) or by atherosclerotic occlusion of the internal carotid artery. The resulting phenotype is the same, but the underlying etiology is quite different. Likewise, it is unclear if lacunar strokes should be considered etiologically similar to carotid occlusion although they share some of the same risk factors. Some misclassification of stroke type could have occurred since many of the studies
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were done before computed tomographic (CT) scans became widely available. Few of the studies corrected for patients' ages although it is well known that the risk of stroke increases significantly with advancing age. When a similar error was corrected for in the study of families with Alzheimer's disease, a marked familial aggregation was detected, consistent with an autosomal dominant trait. Only four studies used modern statistical multivariate analysis to evaluate the role which standard risk factors played in determining stroke risk.16-18'21'22 Despite these limitations it is interesting to note that three of the four studies using multivariate statistical analysis and correction for age did find a significant contribution of genetic factors to the development of stroke. These findings must be weighed against the results of the Honolulu Heart Study, which evaluated the occurrence of stroke and heart disease in a cohort of men who migrated from Japan to Hawaii. This study found that environmental factors were of primary importance in determining the occurrence of cardiovascular disease.24 However, the results could be interpreted as showing that environmental factors modified the expression of the underlying genetic predisposition for vascular disease by accelerating or retarding the rate of atherosclerosis. Since stroke shares many common epidemiologic and pathogenic features with coronary artery disease (CAD), studies of the genetics of CAD may provide clues about common genetic influences. The epidemiologic study of CAD has been underway for over 40 years with many well-described familial associations.25 The presence of CAD in a first degree relative prior to age 55 increases by tenfold the risk to other family members.26 In several twin studies of CAD, most showed a strong genetic component.27-28 Familial associations of high-risk lipid alleles have also been demonstrated. Although genetic factors appear to play a major role in determining the risk and development of CAD, direct comparisons between CAD and stroke are difficult since CAD is a fairly homogeneous entity and stroke is clinically and etiologically heterogeneous. Familial Stroke An alternative approach to large population-based studies is to identify specific families with particular types of stroke. While such familial aggregations are uncommon, an analysis of such families could provide significant information about the pathogenesis of stroke in sporadic cases. Also, the study of such families could lead to the development of larger pedigrees that could be sufficient for genetic linkage analysis. There have been several reports of well-documented pedigrees with familial cerebral infarction. A French group has described a large family with an apparent autosomal dominant disease that presents with strokes or transient ischemic attacks (TIAs) in the 30s and 40s. These patients appear to have mostly white matter
infarcts. There is no significant aggregation of risk factors and no evidence of an inherited coagulopathy. Angiographic studies have been negative. Head CT or magnetic resonance imaging (MRI) showed white matter lucency that in some cases was dramatic. This may be a form of inherited Binswanger's disease or perhaps a defect in white matter metabolism.29 In 1987 Sonninen and Savontaus30 reported a 51-member family (16 affected) with an inherited form of multi-infarct dementia. Examination of 14 patients showed a mean age of onset of 46 years and mean disease duration of 10.6 years. Head CT showed white matter infarcts that were confirmed on postmortem examination of one patient. The familial occurrence of vascular malformations, particularly arteriovenous malformations (AVM) and cavernous angiomas, has been reported. In 1985 Boyd et al31 reported four males in two generations who had AVMs documented by the head CT scan. Several other cases of apparent familial AVMs have been noted.32-33 The pattern of inheritance in this report and others suggests an autosomal dominant trait, perhaps with variable penetrance. Since few studies have evaluated asymptomatic relatives of apparently sporadic cases, the true incidence and hereditary pattern are unclear. A study of cavernous malformations by Rigamonti et al34 found a familial aggregation in 13 of 24 probands. In several asymptomatic at-risk individuals brain MRI showed lesions consistent with cavernous malformations. This familial form may be particularly common among Mexican-American families and appears to be inherited as an autosomal dominant trait, perhaps with incomplete penetrance. Pettigrew et al35 reported another large kindred of 75 individuals with hereditary cavernous hemangiomas. Intracranial aneurysms have been associated with several other inherited disorders such as polycystic kidney disease, Ehlers-Danlos syndrome type IV, and Marian's syndrome. There have been a total of 177 patients from 74 kindreds with familial aneurysms reported in the literature.36 A multivariate analysis showed that intracranial aneurysms in siblings tended to occur at identical or mirror sites and that familial aneurysms tended to rupture at smaller sizes. The exact percentage of cases of aneurysm that are familial is unclear since asymptomatic relatives would not usually be studied if the proband had a negative family history. An autosomal dominant mode of inheritance has been suggested.36 Several candidate genes for familial and sporadic intracranial aneurysms are being studied, including type I and type III collagen. However, in a recent study LeBlanc et al37 failed to identify a collagen defect in some cases of familial intracranial aneurysm. A prime example of the successful interaction of genetic and population research in stroke is the study of Icelandic amyloid angiopathy. This is an autosomal dominant disease with the onset of severe intracerebral hemorrhages before age 40.38-39 The cerebral vasculature shows depositions of cystatin C, an
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Alberts Genetic Aspects of Cerebrovascular Disease
amyloid-like protein. The cystatin C protein and gene have been isolated and sequenced.38 Family studies have shown a specific DNA mutation of the cystatin C gene in every clinically affected family member.39 While the exact pathophysiology of the defect has not been clarified, the finding of a disease-specific mutation will accelerate the understanding of the disease process and offer an accurate tool for genetic counseling. Another recent study by Levy et al40 identified a pathogenic mutation of the amyloid gene presumed responsible for hereditary cerebral hemorrhage, Dutch type. There have been other scattered reports of genetic factors underlying the pathogenesis of moyamoya disease, amyloid angiopathy, and transient global amnesia.41-43 A recent review described many other inherited conditions (coagulopathies, malformations, metabolic disorders) associated with stroke.12 Although the collection of isolated families with specific stroke types may introduce ascertainment and population biases into genetic studies, this approach has the advantage of focusing research on a specific disease while minimizing the influence of a myriad of risk factors. Because stroke is such a complex disorder, population-based genetic studies may not be practical at this time. Directed efforts using families with a specific stroke type or studying various candidate genes in a well-defined population may avoid some of the limitations of past research. Animal studies of stroke genetics have been limited by a paucity of models with inherited stroke. The spontaneously hypertensive stroke-prone rat has been used in most studies, but the overwhelming genetic influence of the hypertension trait is a limitation. Also, anatomic variations may account for some of the genetic influences. Two reports have found evidence for either a recessive trait or a multifactorial gene effect.44-45 Future Directions Large-scale epidemiologic studies of stroke have been attempted in the past with variable results. Perhaps a more efficient approach would be the identification and study of kindreds with a familial aggregation of a particular stroke type. If several families could be developed, genetic linkage studies could begin. More sophisticated methods of linkage analysis such as APM and ARP could be used to isolate major gene effects, even if extended families could not be ascertained. If a major gene effect is identified, apparent sporadic cases could be screened to determine the frequency of these high-risk alleles. Once major deleterious genes or alleles are identified, steps to prevent or alter gene expression could be instituted. If large population-based studies are to be attempted again, several modifications may improve patient ascertainment and classification: 1) Identify patients with precursor lesions prior to becoming symptomatic. For example, the relatives of probands with carotid atherosclerosis could be screened by
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carotid ultrasound. 2) Reclassify stroke types so that similar phenotypes and pathologic lesions are grouped together. Patients with CAD may be considered similar to stroke patients with carotid disease. 3) Ascertain families with early onset disease since they are least likely to be significantly influenced by the standard risk factors. In conclusion, the study of genetically complex diseases such as CAD, cancer, hypertension, and stroke is now beginning. Rapid advances in molecular genetic techniques and genetic epidemiology have provided the tools to identify and isolate major gene effects responsible for these disorders. Understanding these major genes will provide new avenues for the prevention and treatment of stroke. Acknowledgments The author wishes to thank Dr. Lawrence Brass for assistance in obtaining some reference material and Dr. Margaret Pericak-Vance for advice in population genetic studies. References 1. Lander E: Mapping complex genetic traits in humans, in Davies K (ed): Genome Analysis: A Practical Approach. Oxford, IRL Press, 1988, pp 171-187 2. Payne CS, Roses AD: The molecular genetic revolution: Its impact on clinical neurology./)rc/i Neurol 1988;45:1366-1375 3. Weeks D, Lange KJ The affected-pedigree-member method of linkage analysis. Am J Hum Genet 1988;42:315-326 4. Risch N: Linkage strategies for genetically complex traits: II. The power of affected relative pairs. Am J Hum Genet 1990; 46:229-241 5. Hunt SC, Hasstedt SJ, Kuida H, Stults BM, Hopkins PN, Williams RR: Genetic heritability and common environmental components of resting and stressed blood pressures, lipids, and body mass index in Utah pedigrees and twins. Am J Epidemiol 1989;129:625-638 6. Grotta J, Yatsu F, Pettigrew L, Rhoades H, Bratina P, Vital D, Alam R, Earls R, Picone C: Prediction of carotid stenosis progression by lipid and hematologic measurements. Neurology 1989;39:1325-1331 7. Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K, Larson B, Welin L, Tibblin G: Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984;311:501-505 8. Steinberg D: Lipoproteins and the pathogenesis of atherosclerosis. Circulation 1987;76:508-514 9. Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson G, Boerwinkle E, Unterman G: Relation of serum lipoprotein (a) concentration and apolipoprotein (a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. AT Engl J Med 1990;322:1494-1499 10. Third J, Montag J, Flynn M, Freidel J, Laskarzewski P, Glueck CJ: Primary and familial hypoalphalipoproteinemia. Metabolism 1984^3:136-146 11. Brown M, Goldstein J, Fredrickson D: Familial type 3 hyperlipoproteinemia, in Stanbury J, Wyngaarden J, Frederickson D (eds): The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1983, pp 655-671 12. Natowicz M, Kelley RI: Mendelian etiologies of stroke. Ann Neurol 1987;22:175-192 13. Alter M: Genetic factors in cerebrovascular accidents. Trans Am Neurol Assoc 1967;92:205-208 14. Alter M, Kluznik J: Genetics of cerebrovascular accidents. Stroke 1972;3:41-48 15. Gifford AJ: An epidemiological study of cerebrovascular disease. Am J Public Health 1966^6:452-461 16. Herman B, Schmitz PI, Leyten AC, Van LJH, Frenken CW, Op de Coul AAW, Schulte BP: Multivariate logistic analysis of
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risk factors for stroke in Tilburg, The Netherlands. Am J Epidemiol 1983;118:514-525 Heyden S, Heyman A, Camplong L: Mortality patterns among parents of patients with atherosclerotic cerebrovascular disease. J Chronic Dis 1969;22:105-110 Khaw KT, Barrett CE: Family history of stroke as an independent predictor of ischemic heart disease in men and stroke in women. Am J Epidemiol 1986;123:59-66 Marshall J: Familial incidence of cerebrovascular disease. J Med Genet 1971;8:84-89 Marshall J: Familial incidence of cerebral hemorrhage. Stroke 1973;4:38-41 Welin L, Svardsudd K, Wilhelmsen L, Larsson B, Tibblin G: Analysis of risk factors for a stroke cohort of men born in 1913. NEnglJMed 1987;317:521-526 Howard G, Evans G, Toole JF, Tell J, Rose L, Espeland M, Truscott B: Characteristics of stroke victims associated with early cardiovascular mortality in their children. / Clin Epidemiol 1990;43:49-54 Diaz JF, Hachinski VC, Pederson LL, Donald A: Aggregation of multiple risk factors for stroke in siblings of patients with brain infarction and transient ischemic attacks. Stroke 1986; 17:1239-1242 Kagan A, Popper JS, Rhoads GG: Factors related to stroke incidence in Hawaii Japanese men: The Honolulu Heart Study. Stroke 1980;ll:14-21 Deutscher S, Epstein F, Kjelsberg M: Familial aggregation of coronary heart disease. Circulation 1966;33:911—924 Nora J, Lortscher R, Spangler R, Nora A, Kimberling W: Genetic-epidemiological study of early-onset ischemic heart disease. Circulation 1980;61:503-508 Berg K: Twin research in coronary heart disease, in Parisi GL, Nance WE (eds): Twin Research 3: Epidemiological and Clinical Studies. New York, Alan R Liss, 1981, pp 117-130 de Faire U: Ischaemic heart disease in death discordant twins: A study on 205 male and female pairs. Ada Med Scand Suppl 1974;568:1-109 Bousser M-G, Tournier-Lasserve E, Aylward R, Tourbah A, Dormont D, Romero N, Cabanis E: Recurrent strokes in a family with diffuse white matter abnormalities and muscular lipiosis —A new mitochondrial cytopathy? / Neurol 1988; 235(suppl):S4-S5 Sonninen V, Savontaus ML: Hereditary multi-infarct dementia. Eur Neurol 1987;27:209-215
31. Boyd MC, Steinbok P, Paty DW: Familial arteriovenous malformations: Report of four cases in one family. / Neurosurg 1985;62:597-599 32. Snead O, Acker J, Morawetz R: Familial arteriovenous malformation. Ann Neurol 1979;5:585-587 33. Aberfeld DC, Rao KR: Familial arteriovenous malformation of the brain. Neurology 1981;31:184-186 34. Rigamonti D, Hadley MN, Drayer BP, Johnson PC, Hoenig RK, Knight JT, Spetzler RF: Cerebral cavernous malformations: Incidence and familial occurrence. N Engl J Med 1988; 319:343-347 35. Pettigrew L, Immken L, Hejtmancik J: Natural history of familial cavernous hemangioma (abstract). Stroke 1989;20:139A 36. Lozano AM, Leblanc R: Familial intracranial aneurysms. / Neurosurg 1987;66;522-528 37. Leblanc R, Lozano AM, van der Rest M, Guttmann RD: Absence of collagen deficiency in familial cerebral aneurysms. J Neurosurg 1989;70:837-840 38. Levy E, Lopez OC, Ghiso J, Geltner D, Frangione B: Stroke in Icelandic patients with hereditary amyloid angiopathy is related to a mutation in the cystatin C gene, an inhibitor of cysteine proteases. / Exp Med 1989;169:1771-1778 39. Palsdottir A, Abrahamson M, Thorsteinsson L, Arnason A, Olafsson I, Grubb A, Jensson O: Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet 1988;2:603-604 40. Levy E, Carman M, Fernandez-Madrid I, Power M, Lieberburg I, van Duinen SG, Bots GTAM, Luyendik W, Frangione B: Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 1990;248: 1124-1126 41. Kitahara T, Okumara K, Semba A, Yamaura A, Makino H: Genetic and immunologic analysis on Moya-Moya. / Neurol Neurosurg Psychiatry 1982;45:1048-1052 42. Wattendorff A, Bots G, Went L, Endtz L: Familial cerebral amyloid angiopathy presenting as recurrent cerebral hemorrhage. J Neurol Sci 1982;55:121-135 43. Munro J: Transient global amnesia —Familial incidence. J Neurol Neurosurg Psychiatry 1982;45:1070 44. Coyle P, Odenheimer DJ, Sing CF: Cerebral infarction after middle cerebral artery occlusion in progenies of spontaneously stroke-prone and normal rats. Stroke 1984;15:711-716 45. Yamori Y: Importance of genetic factors in stroke: An evidence obtained by selective breeding of stroke-prone and -resistant SHR. Jpn Circ J 1974;38:1095-1100 KEY WORDS • cerebrovascular disorders • genetics
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Genetic aspects of cerebrovascular disease. M J Alberts Stroke. 1991;22:276-280 doi: 10.1161/01.STR.22.2.276 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628
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Current Concepts of Cerebrovascular Disease and Stroke
Genetic Aspects of Cerebrovascular Disease Mark J. Alberts, MD
I
n the past conventional wisdom held that stroke and other forms of cardiovascular disease were due primarily to environmental factors and that genetic background played a relatively minor role in disease pathogenesis. However, recent discoveries in the fields of molecular and population genetics have suggested the need to reexamine the role of genetic factors in the pathogenesis of vascular disease. Research into the genetics of stroke presents some unique challenges since stroke is a heterogeneous disease with multiple risk factors. However, such studies provide an opportunity to elucidate the etiology of stroke at a molecular level and to attempt true primary prevention. This article reviews some of the principles of inherited disorders and genetic research techniques and applies the concepts to the study of the genetics of specific stroke types. Over 3,000 genetic disorders have been described in man, but the gene defect responsible for a specific disease has been identified for only a handful of diseases. Many genetic diseases are termed "simple" diseases: they follow a clear Mendelian pattern of inheritance (e.g., autosomal dominant, X-luiked recessive) and are due to a defect in only a single gene, and every individual with the gene defect will display the mutant phenotype. Examples of simple neurogenetic diseases include Huntingdon's chorea, Duchenne's muscular dystrophy, and myotonic muscular dystrophy. The "complex" genetic disorders are thought to be due to multiple gene interactions (perhaps influenced by environmental factors), with variable clinical expression of the disease phenotype.1 Examples include diabetes, cancer, and atherosclerosis. The study of complex genetic disorders is complicated by numerous factors: 1) Environmental factors: Extrinsic influences such as diet, smoking, and stress may be responsible for the modified expression of certain genetic traits. For example, an individual with the genetic propensity for hyperlipidemia may reduce serum cholesterol by eating a modified diet. Likewise, genetic factors may have
a role in determining how much an individual eats, how one handles stress, and who chooses to smoke. 2) Risk factors: It is apparent that the development of certain major risk factors such as hypertension and diabetes are under some genetic influence. It is likely that these risk factors are themselves synthetic traits (see below) and are also partially influenced by environmental factors. 3) Synthetic traits: Multiple genes can interact to produce a trait. Atherosclerosis or hypertension may be examples of synthetic traits. Since complex genetic diseases may be due to several synthetic traits acting together, analysis of such interactions becomes exceedingly complex. 4) Polygenic trait: In this case abnormalities of multiple genes are necessary to produce a specific trait. 5) Genetic heterogeneity: Defects at more than one genetic allele can produce similar phenotypic abnormalities. 6) Phenocopy: An environmental factor can produce a mutant phenotype identical to that caused by a defective gene. For example, a dissection of the carotid artery due to neck trauma can produce a stroke with clinical findings similar to atherosclerotic occlusion of the carotid artery. Two genetic terms which are often misunderstood are penetrance and expressivity. Penetrance is an all-or-nothing phenomenon: a genetic disease is completely penetrant if every person carrying the mutant genotype expresses the mutant phenotype and incompletely penetrant if some individuals carrying the mutant genotype do not have the mutant phenotype. Variable expressivity refers to the propensity of some genetic disorders to produce quite varied phenotypic findings. Neuroflbromatosis is an example of a genetic disorder with variable expressivity. The issues described above are important for understanding genetic diseases in populations. The techniques described below are used for studying the molecular genetic abnormalities responsible for these complex diseases.
From the Stroke Acute Care Unit, Division of Neurology, Department of Medicine, Duke University Medical Center, Durham, N.C. This work was supported in part by grants T-32-NS07274 and P-50-NS06233 from the National Institutes of Health, Bethesda, Md. Dr. Alberts is a recipient of a National Institutes of Health Fellowship Training Award in Cerebrovascular Disease. Reprinted from Current Concepts of Cerebrovascular Disease and Stroke 1990;25:25-30.
Molecular Genetic Techniques The goal of molecular genetic research is to identify and characterize the genetic defect responsible for a specific genetic disease. The magnitude of this task can be better understood when it is realized that the human genome has over 3 billion bases of DNA organized into about 100,000 genes. Since only a small number of specific genes have been isolated
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Alberts Genetic Aspects of Cerebrovascular Disease and characterized, the genes responsible for most genetic diseases have not been identified. Classic genetic research has attempted first to find the defective protein and then to isolate the abnormal gene. However, since the abnormal protein responsible for most genetic diseases has not been found, a new approach is warranted. This approach, termed reverse genetics, uses genetic linkage analysis to identify chromosomal regions that are close to disease loci. One advantage of reverse genetics is that no a priori information about the location of the mutant gene is necessary. Linkage can be tested with probes (fragments of DNA) that are scattered throughout the human genome. The specific techniques and terminology of reverse genetics have been reviewed recently, and the interested reader is referred to these excellent references for more detailed information.1'2 One of the key steps in studying genetic linkage is to identify sufficiently large families with the disease of interest. If the familial form of the disease is rare or the available families do not have multiple generations or many living members available for testing, the usefulness of classic genetic linkage studies becomes somewhat limited. Because of the likely multigenic pathogenesis of stroke, classic genetic linkage analysis may be insufficient to find linkage. Some of these problems may be overcome by using newer methods of linkage analysis. The affected pedigree member (APM) method, developed by Weeks and Lange,3 uses marker phenotypes for APMs only. This technique is more sensitive for distantly related relatives that are affected and share the same rare allele. Another advantage of the APM method is that no assumptions about the mode of inheritance are required. Another potentially powerful technique is the affected relative pairs (ARP) technique.4 Various combinations of pairs of affected relatives (siblings, first cousins, half sibs, etc.) can be used to identify major or minor disease susceptibility loci. This approach is attractive in cases where large immediate families may not be available but members of extended pedigrees are still alive. Both the APM and ARP methods may be most useful for identifying major gene effects in the pathogenesis of complex diseases rather than identifying every gene involved in an inherited disease. Stroke Genetics Risk Factors The well-documented risk factors described for stroke include hypertension, diabetes, lipid abnormalities, heart disease, advanced age, fibrinogen levels, smoking, and coagulation defects. Many of these risk factors are probably synthetic traits themselves (e.g., hypertension, lipid levels) while others have a stronger environmental component (i.e., smoking). A recent study evaluated the relative importance of genetic and environmental factors in determining
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blood pressure, lipid levels, and body mass index in over 300 twins and 1102 healthy adults. This study found that genetic background had a significantly greater influence on these risk factors than environmental components.5 Another risk factor that is receiving increasing attention is fibrinogen. Several studies have shown a correlation between plasma levels of fibrinogen, stroke risk, and the rate of progression of carotid atherosclerosis.6'7 The control of plasma fibrinogen levels may be under both environmental and genetic control. Since fibrinogen is an acute phase reactant, it increases as a result of various injuries (including stroke or myocardial infarction), inflammation, and cigarette smoking. Genetic studies have shown that plasma fibrinogen levels are significantly elevated in individuals who are homozygous for the rare B2 allele within the fibrinogen gene. Therefore, fibrinogen may represent an important risk factor under both genetic and environmental control. Screening at-risk individuals for the potentially harmful B2 allele might help identify people who should be closely monitored and who should avoid environmental factors that could accelerate atherosclerosis. Various lipid and lipoprotein abnormalities at the protein and gene level have been correlated with the development of cardiovascular disease.8'9 These disorders are also under environmental and genetic control. Familial hypoalphalipoproteinemia is an autosomal dominant trait that is a common cause of high density lipoprotein (HDL) deficiency. It has been associated with premature cardiovascular disease and strokes.10 Other disorders such as familial hypercholesterolemia (homozygous form), type III and type IV hyperlipoproteinemia, and Tangier disease have been associated with premature atherosclerosis and stroke.11-12 As characterization of the complex genetic pathways responsible for lipid metabolism improves, more high-risk alleles and mutations will be identified. Epidemiologic Studies If genetic factors play a role in the etiology of stroke, one might expect epidemiologic studies to identify a family history of stroke as a significant risk factor. Although almost a dozen large-scale studies reported since 1966 have attempted to analyze this issue,13"23 they are limited by some intrinsic weaknesses. Most of the studies did not separate strokes into various subtypes and pathogenic mechanisms, thereby severely limiting any genetic analysis. For example, a large hemispheric stroke could be caused by a cardiac embolism (from atrial fibrillation or valvular heart disease) or by atherosclerotic occlusion of the internal carotid artery. The resulting phenotype is the same, but the underlying etiology is quite different. Likewise, it is unclear if lacunar strokes should be considered etiologically similar to carotid occlusion although they share some of the same risk factors. Some misclassification of stroke type could have occurred since many of the studies
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were done before computed tomographic (CT) scans became widely available. Few of the studies corrected for patients' ages although it is well known that the risk of stroke increases significantly with advancing age. When a similar error was corrected for in the study of families with Alzheimer's disease, a marked familial aggregation was detected, consistent with an autosomal dominant trait. Only four studies used modern statistical multivariate analysis to evaluate the role which standard risk factors played in determining stroke risk.16-18'21'22 Despite these limitations it is interesting to note that three of the four studies using multivariate statistical analysis and correction for age did find a significant contribution of genetic factors to the development of stroke. These findings must be weighed against the results of the Honolulu Heart Study, which evaluated the occurrence of stroke and heart disease in a cohort of men who migrated from Japan to Hawaii. This study found that environmental factors were of primary importance in determining the occurrence of cardiovascular disease.24 However, the results could be interpreted as showing that environmental factors modified the expression of the underlying genetic predisposition for vascular disease by accelerating or retarding the rate of atherosclerosis. Since stroke shares many common epidemiologic and pathogenic features with coronary artery disease (CAD), studies of the genetics of CAD may provide clues about common genetic influences. The epidemiologic study of CAD has been underway for over 40 years with many well-described familial associations.25 The presence of CAD in a first degree relative prior to age 55 increases by tenfold the risk to other family members.26 In several twin studies of CAD, most showed a strong genetic component.27-28 Familial associations of high-risk lipid alleles have also been demonstrated. Although genetic factors appear to play a major role in determining the risk and development of CAD, direct comparisons between CAD and stroke are difficult since CAD is a fairly homogeneous entity and stroke is clinically and etiologically heterogeneous. Familial Stroke An alternative approach to large population-based studies is to identify specific families with particular types of stroke. While such familial aggregations are uncommon, an analysis of such families could provide significant information about the pathogenesis of stroke in sporadic cases. Also, the study of such families could lead to the development of larger pedigrees that could be sufficient for genetic linkage analysis. There have been several reports of well-documented pedigrees with familial cerebral infarction. A French group has described a large family with an apparent autosomal dominant disease that presents with strokes or transient ischemic attacks (TIAs) in the 30s and 40s. These patients appear to have mostly white matter
infarcts. There is no significant aggregation of risk factors and no evidence of an inherited coagulopathy. Angiographic studies have been negative. Head CT or magnetic resonance imaging (MRI) showed white matter lucency that in some cases was dramatic. This may be a form of inherited Binswanger's disease or perhaps a defect in white matter metabolism.29 In 1987 Sonninen and Savontaus30 reported a 51-member family (16 affected) with an inherited form of multi-infarct dementia. Examination of 14 patients showed a mean age of onset of 46 years and mean disease duration of 10.6 years. Head CT showed white matter infarcts that were confirmed on postmortem examination of one patient. The familial occurrence of vascular malformations, particularly arteriovenous malformations (AVM) and cavernous angiomas, has been reported. In 1985 Boyd et al31 reported four males in two generations who had AVMs documented by the head CT scan. Several other cases of apparent familial AVMs have been noted.32-33 The pattern of inheritance in this report and others suggests an autosomal dominant trait, perhaps with variable penetrance. Since few studies have evaluated asymptomatic relatives of apparently sporadic cases, the true incidence and hereditary pattern are unclear. A study of cavernous malformations by Rigamonti et al34 found a familial aggregation in 13 of 24 probands. In several asymptomatic at-risk individuals brain MRI showed lesions consistent with cavernous malformations. This familial form may be particularly common among Mexican-American families and appears to be inherited as an autosomal dominant trait, perhaps with incomplete penetrance. Pettigrew et al35 reported another large kindred of 75 individuals with hereditary cavernous hemangiomas. Intracranial aneurysms have been associated with several other inherited disorders such as polycystic kidney disease, Ehlers-Danlos syndrome type IV, and Marian's syndrome. There have been a total of 177 patients from 74 kindreds with familial aneurysms reported in the literature.36 A multivariate analysis showed that intracranial aneurysms in siblings tended to occur at identical or mirror sites and that familial aneurysms tended to rupture at smaller sizes. The exact percentage of cases of aneurysm that are familial is unclear since asymptomatic relatives would not usually be studied if the proband had a negative family history. An autosomal dominant mode of inheritance has been suggested.36 Several candidate genes for familial and sporadic intracranial aneurysms are being studied, including type I and type III collagen. However, in a recent study LeBlanc et al37 failed to identify a collagen defect in some cases of familial intracranial aneurysm. A prime example of the successful interaction of genetic and population research in stroke is the study of Icelandic amyloid angiopathy. This is an autosomal dominant disease with the onset of severe intracerebral hemorrhages before age 40.38-39 The cerebral vasculature shows depositions of cystatin C, an
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Alberts Genetic Aspects of Cerebrovascular Disease
amyloid-like protein. The cystatin C protein and gene have been isolated and sequenced.38 Family studies have shown a specific DNA mutation of the cystatin C gene in every clinically affected family member.39 While the exact pathophysiology of the defect has not been clarified, the finding of a disease-specific mutation will accelerate the understanding of the disease process and offer an accurate tool for genetic counseling. Another recent study by Levy et al40 identified a pathogenic mutation of the amyloid gene presumed responsible for hereditary cerebral hemorrhage, Dutch type. There have been other scattered reports of genetic factors underlying the pathogenesis of moyamoya disease, amyloid angiopathy, and transient global amnesia.41-43 A recent review described many other inherited conditions (coagulopathies, malformations, metabolic disorders) associated with stroke.12 Although the collection of isolated families with specific stroke types may introduce ascertainment and population biases into genetic studies, this approach has the advantage of focusing research on a specific disease while minimizing the influence of a myriad of risk factors. Because stroke is such a complex disorder, population-based genetic studies may not be practical at this time. Directed efforts using families with a specific stroke type or studying various candidate genes in a well-defined population may avoid some of the limitations of past research. Animal studies of stroke genetics have been limited by a paucity of models with inherited stroke. The spontaneously hypertensive stroke-prone rat has been used in most studies, but the overwhelming genetic influence of the hypertension trait is a limitation. Also, anatomic variations may account for some of the genetic influences. Two reports have found evidence for either a recessive trait or a multifactorial gene effect.44-45 Future Directions Large-scale epidemiologic studies of stroke have been attempted in the past with variable results. Perhaps a more efficient approach would be the identification and study of kindreds with a familial aggregation of a particular stroke type. If several families could be developed, genetic linkage studies could begin. More sophisticated methods of linkage analysis such as APM and ARP could be used to isolate major gene effects, even if extended families could not be ascertained. If a major gene effect is identified, apparent sporadic cases could be screened to determine the frequency of these high-risk alleles. Once major deleterious genes or alleles are identified, steps to prevent or alter gene expression could be instituted. If large population-based studies are to be attempted again, several modifications may improve patient ascertainment and classification: 1) Identify patients with precursor lesions prior to becoming symptomatic. For example, the relatives of probands with carotid atherosclerosis could be screened by
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carotid ultrasound. 2) Reclassify stroke types so that similar phenotypes and pathologic lesions are grouped together. Patients with CAD may be considered similar to stroke patients with carotid disease. 3) Ascertain families with early onset disease since they are least likely to be significantly influenced by the standard risk factors. In conclusion, the study of genetically complex diseases such as CAD, cancer, hypertension, and stroke is now beginning. Rapid advances in molecular genetic techniques and genetic epidemiology have provided the tools to identify and isolate major gene effects responsible for these disorders. Understanding these major genes will provide new avenues for the prevention and treatment of stroke. Acknowledgments The author wishes to thank Dr. Lawrence Brass for assistance in obtaining some reference material and Dr. Margaret Pericak-Vance for advice in population genetic studies. References 1. Lander E: Mapping complex genetic traits in humans, in Davies K (ed): Genome Analysis: A Practical Approach. Oxford, IRL Press, 1988, pp 171-187 2. Payne CS, Roses AD: The molecular genetic revolution: Its impact on clinical neurology./)rc/i Neurol 1988;45:1366-1375 3. Weeks D, Lange KJ The affected-pedigree-member method of linkage analysis. Am J Hum Genet 1988;42:315-326 4. Risch N: Linkage strategies for genetically complex traits: II. The power of affected relative pairs. Am J Hum Genet 1990; 46:229-241 5. Hunt SC, Hasstedt SJ, Kuida H, Stults BM, Hopkins PN, Williams RR: Genetic heritability and common environmental components of resting and stressed blood pressures, lipids, and body mass index in Utah pedigrees and twins. Am J Epidemiol 1989;129:625-638 6. Grotta J, Yatsu F, Pettigrew L, Rhoades H, Bratina P, Vital D, Alam R, Earls R, Picone C: Prediction of carotid stenosis progression by lipid and hematologic measurements. Neurology 1989;39:1325-1331 7. Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K, Larson B, Welin L, Tibblin G: Fibrinogen as a risk factor for stroke and myocardial infarction. N Engl J Med 1984;311:501-505 8. Steinberg D: Lipoproteins and the pathogenesis of atherosclerosis. Circulation 1987;76:508-514 9. Seed M, Hoppichler F, Reaveley D, McCarthy S, Thompson G, Boerwinkle E, Unterman G: Relation of serum lipoprotein (a) concentration and apolipoprotein (a) phenotype to coronary heart disease in patients with familial hypercholesterolemia. AT Engl J Med 1990;322:1494-1499 10. Third J, Montag J, Flynn M, Freidel J, Laskarzewski P, Glueck CJ: Primary and familial hypoalphalipoproteinemia. Metabolism 1984^3:136-146 11. Brown M, Goldstein J, Fredrickson D: Familial type 3 hyperlipoproteinemia, in Stanbury J, Wyngaarden J, Frederickson D (eds): The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1983, pp 655-671 12. Natowicz M, Kelley RI: Mendelian etiologies of stroke. Ann Neurol 1987;22:175-192 13. Alter M: Genetic factors in cerebrovascular accidents. Trans Am Neurol Assoc 1967;92:205-208 14. Alter M, Kluznik J: Genetics of cerebrovascular accidents. Stroke 1972;3:41-48 15. Gifford AJ: An epidemiological study of cerebrovascular disease. Am J Public Health 1966^6:452-461 16. Herman B, Schmitz PI, Leyten AC, Van LJH, Frenken CW, Op de Coul AAW, Schulte BP: Multivariate logistic analysis of
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31. Boyd MC, Steinbok P, Paty DW: Familial arteriovenous malformations: Report of four cases in one family. / Neurosurg 1985;62:597-599 32. Snead O, Acker J, Morawetz R: Familial arteriovenous malformation. Ann Neurol 1979;5:585-587 33. Aberfeld DC, Rao KR: Familial arteriovenous malformation of the brain. Neurology 1981;31:184-186 34. Rigamonti D, Hadley MN, Drayer BP, Johnson PC, Hoenig RK, Knight JT, Spetzler RF: Cerebral cavernous malformations: Incidence and familial occurrence. N Engl J Med 1988; 319:343-347 35. Pettigrew L, Immken L, Hejtmancik J: Natural history of familial cavernous hemangioma (abstract). Stroke 1989;20:139A 36. Lozano AM, Leblanc R: Familial intracranial aneurysms. / Neurosurg 1987;66;522-528 37. Leblanc R, Lozano AM, van der Rest M, Guttmann RD: Absence of collagen deficiency in familial cerebral aneurysms. J Neurosurg 1989;70:837-840 38. Levy E, Lopez OC, Ghiso J, Geltner D, Frangione B: Stroke in Icelandic patients with hereditary amyloid angiopathy is related to a mutation in the cystatin C gene, an inhibitor of cysteine proteases. / Exp Med 1989;169:1771-1778 39. Palsdottir A, Abrahamson M, Thorsteinsson L, Arnason A, Olafsson I, Grubb A, Jensson O: Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet 1988;2:603-604 40. Levy E, Carman M, Fernandez-Madrid I, Power M, Lieberburg I, van Duinen SG, Bots GTAM, Luyendik W, Frangione B: Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral hemorrhage, Dutch type. Science 1990;248: 1124-1126 41. Kitahara T, Okumara K, Semba A, Yamaura A, Makino H: Genetic and immunologic analysis on Moya-Moya. / Neurol Neurosurg Psychiatry 1982;45:1048-1052 42. Wattendorff A, Bots G, Went L, Endtz L: Familial cerebral amyloid angiopathy presenting as recurrent cerebral hemorrhage. J Neurol Sci 1982;55:121-135 43. Munro J: Transient global amnesia —Familial incidence. J Neurol Neurosurg Psychiatry 1982;45:1070 44. Coyle P, Odenheimer DJ, Sing CF: Cerebral infarction after middle cerebral artery occlusion in progenies of spontaneously stroke-prone and normal rats. Stroke 1984;15:711-716 45. Yamori Y: Importance of genetic factors in stroke: An evidence obtained by selective breeding of stroke-prone and -resistant SHR. Jpn Circ J 1974;38:1095-1100 KEY WORDS • cerebrovascular disorders • genetics
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Genetic aspects of cerebrovascular disease. M J Alberts Stroke. 1991;22:276-280 doi: 10.1161/01.STR.22.2.276 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628
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