(Stroke. 1995;26:2190-2193.)
© 1995 American Heart Association, Inc.
Articles |
Cystatin C Mutation in an Elderly Man With Sporadic Amyloid Angiopathy and Intracerebral Hemorrhage
From the Division of Neurology, Department of Medicine (C.G., M.H.H., D.E.S., A.D.R., M.J.A.) and the Stroke Acute Care Unit (M.J.A.), Duke University Medical Center, Durham, NC; and the Department of Pharmacology, New York University Medical Center, New York, NY (E.L.).
Correspondence to Mark J. Alberts, MD, PO Box 3392, Duke University Medical Center, Durham, NC 27710.
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Abstract |
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Background Cerebral amyloid angiopathy (CAA) with intracerebral hemorrhage (ICH) occurs both sporadically and as a result of mutations in either cystatin C or the amyloid precursor protein. ICH due to cystatin C mutations typically occurs in young people of Icelandic origin.
Case Description We report a case of sporadic CAA with ICH in an elderly Croatian man with a mutation in cystatin C identical to that found in Icelandic hereditary cerebral hemorrhage with amyloidosis.
Conclusions This is the first case report of sporadic CAA associated with the same mutation causing hereditary cerebral hemorrhage with amyloidosis of the Icelandic type. Sporadic CAA may thus be associated with genetic mutations in some patients. The frequency of these mutations is yet to be determined.
Key Words: amyloid • cystatins • intracerebral hemorrhage • molecular biology • mutation
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Introduction |
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One of the most deadly types of stroke is ICH.1 2 Hypertension3 and advancing age4 are among the most significant risk factors for ICH. It is recognized that CAA is responsible for 15% to 20% of ICHs in patients older than 70 years.5 Immunohistochemical studies of patients with sporadic CAA have demonstrated dual staining of vessels with Aß and/or cystatin C,6 7 suggesting that these proteins may play a role in the pathogenesis of this disease. Patients with Alzheimer's disease, Down's syndrome, the Dutch variant of HCHWA (HCHWA-D), and sporadic CAA have been shown to have Aß deposition in some cerebral vessels.8
The Icelandic variant of HCHWA (HCHWA-I) is a familial CAA9 that differs from HCHWA-D by presenting at a younger age (between 20 and 30 years). In HCHWA-I the amyloid deposited in the vessels is composed of cystatin C, a cysteine protease inhibitor.9 A pathogenic point mutation (adenine for thymine substitution) in exon 2 of cystatin C has been identified that leads to an amino acid substitution of glutamine for leucine.10 11 There are no reports describing mutations in either Aß or cystatin C in association with sporadic CAA. We report a case of a man with sporadic CAA who was found to have a mutation in exon 2 of cystatin C identical to that associated with HCHWA-I.
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Case Report |
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TopAbstractIntroductionCase ReportDiscussionReferences |
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The patient was a white man who was well until 1987, when at the age of 63 years he suffered a minor stroke that began with a bright visual disturbance in his left visual field (scintillations) followed by left hemianopsia and left-hand weakness requiring hospitalization. A CT and MRI of the head did not show any abnormalities. He was started on aspirin and recovered fully within 6 weeks. He remained well until the fall of 1991, when he presented with nausea and projectile vomiting secondary to a left occipital hemorrhage. A hematoma secondary to an arteriovenous malformation was removed. After 6 weeks of rehabilitation he still had a severe residual aphasia and marked confusion. Three months later while at home he suffered another minor left hemispheric ischemic stroke, which was seen on a head CT. He continued to have several transient ischemic attacks over the next 8 months despite the aspirin treatment. In June of 1992 he was found in bed comatose with Cheyne-Stokes respirations and vomitus in his mouth. His pupils were 6 mm on the right and 4 mm on the left and nonreactive. Oculocephalic responses were absent, corneal reflexes were present, and gag response was absent. Painful stimulation produced decerebrate posturing. A CT scan of his head showed a large intraparenchymal hemorrhage involving the right parieto-occipital lobe (Fig 1
). The
patient had a living will, and at the request of his family he was
treated with compassionate care only. Three days after admission he was
declared dead at the age of 68 years.
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His only other medical problem before the ICHs was insulin-dependent diabetes mellitus. He was a retired university professor who still kept up with his academic field of interest until the hemorrhage of 1991.
Family History
The family history was obtained from interviews with his wife and
his sister. Because part of his family migrated from Europe, medical
records were not available to confirm causes of death in family
members.
The patient was married and had three sons, all of whom were healthy, as was his only sibling, a sister. His father was of Croatian ancestry and died of lung cancer. The paternal grandfather died suddenly in late life. The underlying etiology is not known. The patient's mother is Anglo-Saxon. She is 93 years old and lives in a nursing home. She has been diagnosed as having Alzheimer's disease on the basis of the presence of a progressive cortical dementia and a head CT scan showing diffuse cortical atrophy without evidence of infarctions. The maternal grandparents died of cancer (grandmother) and trauma (grandfather). There was no other family history of ICH or dementia of which anyone in the family was aware.
Genetic Studies
Genomic DNA was extracted from paraffin-embedded brain, liver,
and kidney tissue saved from the autopsy with published
methods.12 The DNA was further purified using a
phenol/chloroform extraction method.13 Genomic DNA was
extracted from blood (obtained by venipuncture) from a
healthy control volunteer and from the patient's mother and sister
using the Gene 341 Nucleic Acids Purification System, Genepure (Applied
Biosystems). Polymerase chain reaction14 was used to
amplify exon 2 of cystatin C and exons 16 and 17 of the amyloid
precursor protein, and the resulting products were sequenced as has
been previously described.15
Immunocytochemical Methods
Paraffin sections (6 to 8 µm thick) of parietal and frontal lobe
cortex were treated with 90% (wt/wt) formic acid for 3 minutes and
washed.16 Aß was localized by using either monoclonal
antibody 4D12/2/6 (to Aß fragments 8 to 17, courtesy of G. Glenner
and D. Alsop17 18 ) or 10D5 (to Aß [1 to 28], courtesy
of Athena Neuroscience)19 and using the standard ABC
method (Vector Labs). The apoE immunoreactivity was localized by using
monoclonal antibody to human apoE, which recognizes all three apoE
isoforms (courtesy of Ross Milne, University of Ottawa, Canada).
Cystatin C immunoreactivity was localized by using rabbit
anti-human cystatin C (Accurate Chemical and Scientific
Corporation) diluted 1:200 and 1:250.20
Autopsy Results
Gross examination of the brain showed a hemorrhage
involving the right parietal cortex and extending to the occipital
lobe. There was also evidence of an old 2x1.5x2.5-cm infarct in the
left occipital lobe. No residual evidence of the previous vascular
malformation was found.
Microscopic examination of the right parietal cortex showed multiple vessels that were distorted and thickened by homogenous eosinophilic deposits primarily localized to the muscular media. Staining with Congo red and subsequent examination under polarized light confirmed the presence of moderately heavy deposits of amyloid within the distorted vasculature. A section of the left occipital cortex in the region of the remote hemorrhage also showed vessels with similar microscopic pathology. Examination of cortical tissue showed only rare neuritic plaques with no other changes to suggest Alzheimer's disease. No other significant neuropathologic findings were noted.
Molecular Genetic Studies
A heterozygous point mutation was found in exon 2 of cystatin C
that resulted in a substitution of adenosine for thymine
(CAG instead of CTG) (Fig 2). This
mutation was found by forward and reverse sequencing and was confirmed
using manual and automated sequencing protocols. The mutation was found
in the kidney and liver as well as in the brain DNA. The mutation was
not detected in the control subject. Neither the patient's mother nor
the sister carried the mutation. The patient's father had been dead
for many years, and autopsy tissue was not available. We were not able
to determine whether the patient's mutation was inherited from the
father or whether it was a spontaneous mutation.
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Exons 16 and 17 of amyloid precursor protein were sequenced, and no mutations were detected in the patient or his available family members.
Immunocytochemistry
Congo red–stained sections showed moderately severe amyloid
angiopathy. Vessels in semiadjacent sections showed immunoreactivity to
Aß, cystatin C, and apoE (Fig 3).
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Discussion |
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TopAbstractIntroductionCase ReportDiscussionReferences |
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Although inherited forms of ICH such as HCHWA-D and HCHWA-I are rare in the United States, sporadic CAA is being recognized increasingly as a significant cause of ICH, particularly in the elderly,21 in those with lobar hemorrhages,22 and in patients with ICH related to the use of anticoagulants or thrombolytic agents.23 24
We have demonstrated that sporadic CAA with an ICH in an elderly man not of Icelandic origin was associated with a mutation in cystatin C. This case raises several important questions. Typically, HCHWA-I presents in patients of a much younger age and is usually fatal before the sixth decade.25 This man was functioning at a high intellectual and physical level until his sixties, yet he carried the same mutation as the Icelandic patients. It has been demonstrated recently that in some genetic diseases different clinical presentations can be seen in patients with identical mutations. For example, patients with fatal familial insomnia have the same mutation as some patients with Creutzfeldt-Jacob disease.26 The different clinical presentations may in some cases depend on the presence of a coding polymorphism elsewhere in the gene.26 Polymorphisms in or near the cystatin C gene may be important in the clinical expression of the cystatin C mutation in some cases of CAA. Other inherited or environmental factors may be important in producing the early age of onset for HCHWA-I. Although we did not find a similar mutation in any of the family members tested, we were extracting leukocyte DNA in their case. The mutation in cystatin C that we are reporting was found in DNA extracted from the patient's brain as well as kidney and liver. It is most likely therefore that he also would have carried this mutation in the DNA of his leukocytes, making blood screening a reliable method of detecting the mutation reported.
It is not known what proportion of patients with late-onset sporadic CAA have this mutation. We have not detected this mutation in a population of 48 patients with sporadic ICH, in whom the majority of bleeds were deep basal ganglionic ICHs15 ; however, the recognition of a genetic etiology for some cases of late-onset sporadic CAA is significant. It has been demonstrated that the prevalence of CAA found at autopsy increases significantly with advancing age.21 22 As the population continues to age, the prevalence of CAA will likewise increase, making ICH due to CAA increasingly common.
Recent studies have shown that the risk of ICH in patients treated with anticoagulants or thrombolytic drugs increases with increasing age.27 28 29 It has been demonstrated that in some of these cases the underlying pathology was CAA. To date, there has been no way of screening for the presence of late-onset amyloid angiopathy in the premorbid state. It is now possible to screen high-risk individuals in the premorbid state for the presence of the cystatin mutation. This might be most useful before the institution of thrombolytic therapy for myocardial infarction or of long-term warfarin therapy, as this would put patients with such a mutation at high risk for fatal hemorrhages. Likewise, patients with significant hypertension and amyloid angiopathy would also be at higher risk for ICH, perhaps justifying more aggressive control of blood pressure in such patients.
Because CAA is a diagnosis usually made postmortem, genetic studies of proven cases have been limited. New techniques have allowed the isolation and amplification of genomic DNA from postmortem paraffin-embedded tissue.12 Studies are in progress to determine the prevalence of this mutation among autopsy-proven cases of sporadic CAA with ICH. If this mutation or other mutations are found to be frequently associated with sporadic CAA, then analysis of genomic DNA may prove useful for identifying patients at risk for developing CAA and ICH. The identification of such high-risk patients would have significant implications in terms of risk-factor control and the use of certain concomitant medications.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a fellowship training grant from the Canadian Heart and Stroke Foundation (Dr Graffagnino) and by a Grant-in-Aid from the American Heart Association (Dr Alberts). We wish to thank Ann Saunders, PhD, for her help reviewing this manuscript and Jackie Davis, Rohan De Silva, PhD, and Mark Rubino for their technical assistance in this study. We also wish to thank Dr C.T. Durham and Dr R.L. Landau for their assistance and support.
Received June 12, 1995; revision received August 15, 1995; accepted August 15, 1995.
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