Microvascular degeneration in hereditary cystatin C amyloid angiopathy of the brain.

Hereditary cystatin C amyloid angiopathy (HCCAA), an autosomal dominant form of cerebral amyloid angiopathy (CAA) occurring primarily in Iceland, is characterized by a variant cystatin C amyloid deposition in the walls of cerebral parenchymal and leptomeningeal vessels. Cystatin C is also found to colocalize with amyloid beta/A4 protein in cerebral vessel walls of patients with Alzheimer's disease (AD), sporadic CAA, and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D). The abundance of cystatin C deposition in cerebral blood vessel walls suggests that cellular elements of the vessel wall itself may play a role in its deposition. Microvascular changes in the brains of HCCAA patients were investigated by single- and double-label immunohistochemistry. We found that cystatin C amyloid immunoreactivity was present not only in cerebral cortical and leptomeningeal vessels, but also in white matter parenchymal vessels. Cystatin C deposition was more prominent in the media of parenchymal vessels and in the adventitia of leptomeningeal vessels. Smooth muscle (sm) cells were few or could not be identified within vessel walls showing extensive cystatin C deposition, suggesting progressive loss of these cells as cystatin C accumulates. However, in less severely affected vessels, cystatin C was present in cells that also had the phenotype of sm, suggesting that sm cells synthesize or process cystatin C. Cystatin C immunoreactivity was in addition, detected in some neuronal cell bodies throughout the cortex in patients with HCCAA and AD-related CAA. Our results indicate that cellular components of the vessel walls may play an important role in cystatin C deposition, as they do in beta/A4 deposition in AD-related CAA. Cystatin C deposition within the vascular media and adventitia, with associated vessel wall injury as manifested by sm cell loss, represents microvascular degeneration that leads to cerebral hemorrhage.

Immune reactions associated with cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) occasionally coexists with cerebral vasculitis. An immune system may influence deposition or degradation of the amyloid in cerebral blood vessels. The purpose of this study was to elucidate immune reactions associated with CAA. METHODS: In 11 elderly patients with sporadic CAA, 2 patients with Icelandic familial CAA, and 2 patients with CAA and granulomatous angiitis, the cerebrovascular amyloid proteins and infiltrating inflammatory cells were analyzed immunohistochemically. RESULTS: In both sporadic CAA (beta-protein amyloid angiopathy) and Icelandic familial CAA (cystatin C amyloid angiopathy), leptomeningeal and cortical vessels were associated with an increase or activation of monocyte/macrophage lineage cells. In the cases of CAA with granulomatous angiitis, the vascular amyloid was of beta-protein and associated with infiltration of many monocyte/macrophage lineage cells, which included multinucleated giant cells containing the amyloid in the cytoplasm as well as T cells composed of CD4+ and CD8+ subsets. Amyloid P component, which was reported to be a common component of amyloid deposits and to prevent phagocytic proteolysis of amyloid fibrils of beta-protein, was negative for the vascular amyloid in a case of CAA with granulomatous angiitis but positive in the others. CONCLUSIONS: In both the beta-protein and cystatin C amyloid angiopathies, cerebrovascular amyloid deposition was associated with an increase or activation of monocyte/macrophage lineage cells. Prominent reactions of monocyte/macrophage lineage cells admixed with CD4+ and CD8+ T cells (granulomatous angiitis) were occasionally associated with beta-protein angiopathy. In some of these cases, the absence of amyloid P component might be related to pathogenesis of the granulomatous reaction.

The molecular pathology of hereditary cystatin C amyloid angiopathy causing brain hemorrhage.

Knowledge about molecular pathology of hereditary cystatin C amyloid angiopathy (HCCAA), also called hereditary cerebral hemorrhage with amyloidosis, Icelandic type, has increased greatly in the last decade. The disorder has an autosomal dominant mode of inheritance and causes fatal brain hemorrhage in normotensive young adults. It is due to a mutation in the gene encoding the cysteine proteinase inhibitor, cystatin C.A single nucleotide is substituted, A for T, in the codon 68, resulting in glutamine replacing leucine in the protein sequence. This variant protein has an increased tendency to aggregate and forms heavy depositions of amyloid in the walls of the small arteries and arterioles of the brain. The amyloid deposition leads to arterial damage with single or multiple strokes. In the following review the clinical features, family studies, pathology, biochemistry and molecular genetics of HCCAA are addressed.

Wilson disease in Iceland: a clinical and genetic study.

A survey of Wilson disease in Iceland has revealed two large kindreds with affected individuals. We have carried out studies of haplotypes of dinucleotide repeat polymorphisms (CA repeats) flanking the Wilson disease gene. The same mutation, a 7-bp deletion, is present in both families, and the clinical features are similar. The haplotype data and nature of the mutation support the existence of a founder chromosome carrying the mutation. This Icelandic mutation was not found in patients of Irish or Scottish origins, who could share some of the Icelandic ancestral genes. Although the protein function is predicted to be completely abolished by the deletion, predicting early-onset liver disease, we find that the patients present with later-onset neurological and psychiatric symptoms. We show that alternative splicing of the transcript in the region of the deletion could contribute to later onset, suggesting that alternative isoforms of the protein might have some functional significance.

Mapping the locus of atrophia areata, a helicoid peripapillary chorioretinal degeneration with autosomal dominant inheritance, to chromosome 11p15.

Atrophia areata (AA) is an early onset autosomal dominant helicoid peripapillary chorioretinal degeneration, which was first demonstrated to be hereditary in an Icelandic family. It is characterized by bilateral wing-shaped atrophic areas of the retina, radiating from the optic disk. Primary complaints of affected individuals are due to refractive errors and scotomata associated with myopia which increases with age. A genome linkage search with 112 microsatellite DNA markers resulted in the highest probability of location for AA on chromosome 11. We genotyped 18 polymorphic markers on chromosome 11 and seven showed significant linkage to AA. The markers D11S1323 and D11S902 on 11p15 flank the region encompassing the gene for AA.

[Mild haemophilia A in Iceland.].

A family survey of mild hemophilia A has been carried out during the last 25 years. Presently these families contain 27 living affected males or approximately twice the number of males with severe form of hemophilia A in Iceland. The aim of the study was to define the phenotype in three families with mild haemophilia A and to determine RFLP (Restriction Fragment Length Polymorphism) polymorphisms, which could support a hypothesis of a common progenitor of the families. Family survey was made and index cases were identified in and outside hospitals and a survey for symptoms and signs of bleeding in family members and sampling for coagulation and RFLP studies were mostly carried out in the field. Family members with and without symptoms of bleeding were selected for investigation and normal spouses and unrelated individuals were investigated for control. Bleeding time, factor VIII activity, quantification of factor VIII Ag, von Wil-lebrand factor Ag and vWF ristocetin assay. Typing of RFLP polymorphisms for genetic homogeneity. Bleeding manifestations are present in both sexes in the three families although more frequent and more severe in the males. Prolonged bleeding time in 6 of 7 affected members in one of the families and subnormal levels in von Willebrand related assays point to an additional abnormality which has not been explained. The level of factor VIII activity is between 10-20% in most affected males whereas 35-60% is found approximately in 2/3 of females carriers and in 1/3 of them factor VIII activity is within the normal range. Major bleeding episodes in family members are associated with accidents, surgical procedures menorrhagia and in some cases provoked by diseases such as intestinal ulceration, kidney stones and abnormalities of vasculature. RFLP polymorphisms were detected, which are common to the families and they support hypothesis of a common progenitor. The genetic studies show relatively high prevalence of this type of mild haemophilia A, which seems to have been present for at least 6-8 generations in Iceland. It is suggested that screening for this mild haemophilia A gene by molecular genetic method would be of clinical value now, when it's mutation has been detected. Transmission of mild haemophilia A through 6-9 generations is demonstrated by the study. Founder effect is confirmed by studies of RFLP polymorphisms. The mild haemophilia A type described is the most prevalent of haemophilia A types in Iceland (population 265,000, 1993).

Familial macroglobulinaemia: hyperactive B-cells but normal natural killer function.

An Icelandic family with two cases of benign monoclonal gammopathy and one case each of Waldenström's macroglobulinaemia, histiocytic lymphoma and multiple myeloma was first described in 1978. Nine family members had then shown raised values for se-IgM. Of these one has since died and another was not available for testing. In four of the remaining seven se-IgM had returned to normal; the three subjects who still showed raised se-IgM included the case of multiple myeloma diagnosed in 1985. Baseline production of IgM, IgG and Ig in vitro was normal in the 35 family members studied compared with 13 healthy control subjects, but the mean production of all immunoglobulin classes in response to minimal stimulation with PWM (1 microgram/ml) was significantly increased (P < 0.05). Ten family members showed markedly increased production of all three immunoglobulin classes (> 3 x SD above mean for controls). Raised production of IgM never occurred alone, indicating intact class switching. One family member showed extremely high values: IgA: 5.15 micrograms/ml, IgG: 16.3 micrograms/ml, IgM: 24.8 micrograms/ml (means for controls: 0.066, 0.123, 0.185 respectively). These 10 family members were of both sexes, ranged in age from 16 to 84 years and were clustered mainly in three distinct groups within the pedigree suggesting heredity. Proliferative responses to PWM were not significantly increased. Serum levels of interleukin-4 were tested in the patient with multiple myeloma and the family member with highest Ig production and found to be normal. We found no evidence for depressed NK function. Thus, in this family with a tendency for macroglobulinaemia and B cell derived malignancies B cell hyperreactivity was detectable by in vitro testing in several asymptomatic family members, of both sexes and all ages. No evidence was obtained for defects in regulatory mechanisms.

Co-localization of beta/A4 and cystatin C in cortical blood vessels in Dutch, but not in Icelandic hereditary cerebral hemorrhage with amyloidosis.

Based on the recent discovery of co-localization of beta/A4 and cystatin C in cortical blood vessels of patients with cerebral hemorrhages due to sporadic amyloid angiopathy and patients with Alzheimer's disease we investigated the presence of these two proteins in the cortical blood vessels of patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch (n = 11) and the Icelandic (n = 2) type. The brains of three patients with sporadic cerebral amyloid angiopathy were also investigated. Blood vessels of the Dutch patients clearly showed immunostaining with beta/A4 as well as with cystatin C antibodies, whereas the blood vessels of Icelandic patients showed only staining with cystatin C. In one of the three sporadic amyloid angiopathy patients co-localization was shown as well. The co-localization of mutated beta/A4 with normal cystatin C in the Dutch patients suggests that cystatin C deposition occurs secondarily to beta/A4 deposition. This is probably also the case in sporadic amyloid angiopathy and Alzheimer's disease. Cystatin C deposition may play a role in the development of cerebral hemorrhages and leukoencephalopathy.

Linkage analysis of 62 X-chromosomal loci excludes the X chromosome in an Icelandic family showing apparent X-linked recessive inheritance of neural tube defects.

We report here the findings of a linkage analysis, involving numerous markers from the human X chromosome, in an attempt to localise a putative gene causing apparent X-linked spina bifida and anencephaly (SBA) in a large Icelandic pedigree. Two-point linkage analysis was performed using markers from 62 informative loci in this family. Although small positive lod scores were found at a number of these loci, none reached the significance level for linkage. Haplotypes were extensively analysed and found to exclude linkage to the X chromosome.

Mild haemophilia A in Iceland: clinical genetic studies of three families with the same mutation.

OBJECTIVES: The aim of the study was to define the phenotype in three families with mild haemophilia A and to determine restriction fragment length polymorphisms (RFLP), which could support a hypothesis of a common progenitor of the families. DESIGN: Family survey. SETTING: Index cases were identified in and outside hospital and a family survey for symptoms and signs of bleeding in family members and sampling for coagulation and RFLP studies were mostly carried out in the field. SUBJECT: Family members with and without symptoms of bleeding were selected for investigation and normal spouses and unrelated individuals were investigated for control. INTERVENTIONS: Medical advice regarding affected family members were given to the families and their physicians. MAIN OUTCOME MEASURES: Bleeding time, factor VIII activity, quantification of factor VIII:Ag, von Willebrand factor (vWF) Ag and vWF ristocetin assay. Typing of RFLP polymorphisms for genetic homogeneity. RESULTS: Bleeding manifestations are present in both sexes in the three families although more frequent and more severe in the males. The level of factor VIII activity is between 10 and 20% in most affected males whereas 35-60% is found approximately in 2/3 of female carriers and in 1/3 of them factor VIII activity is within the normal range. It is suggested that screening for this mild haemophilia A gene by a molecular genetic method would be of clinical value now, its mutation having been detected. CONCLUSIONS: Transmission of mild haemophilia A through six to seven generations is demonstrated by the study. The mild haemophilia A type described is the most prevalent of haemophilia A types in Iceland (population 260,000, 1992). The founder effect was confirmed by studies of RFLP polymorphisms.

Exclusion mapping of the gene for X-linked neural tube defects in an Icelandic family.

Various polymorphic markers with a random distribution along the X chromosome were used in a linkage analysis performed on a family with apparently X-linked recessive inheritance of neural tube defects (NTD). The lod score values were used to generate an exclusion map of the X chromosome; this showed that the responsible gene was probably not located in the middle part of Xp or in the distal region of Xq. A further refining of these results was achieved by haplotype analysis, which indicated that the gene for X-linked NTD was located either within Xp21.1-pter, distal from the DMD locus, or in the region Xq12-q24 between DXS106 and DXS424. Multipoint linkage analysis revealed that the likelihood for gene location is highest for the region on Xp. The region Xq26-q28, which has syntenic homology with the segment of the murine X chromosome carrying the locus for 'bent tail' (Bn), a mouse model for X-linked NTD, is excluded as the location for the gene underlying X-linked NTD in the present family. Thus, the human homologue of the Bn gene and the present defective gene are not identical, suggesting that more than one gene on the X chromosome plays a role in the development of the neural tube.

[Transfusion medicine--Scandinavian recommendations]. / Transfusionsmedicin--nordiske rekommandationer.

At the 14th Nordic Congress on Transfusion Medicine, held in Reykjavik in 1993, it was recommended that every blood bank should have an established programme for the management of screening-positive donors and blood components deriving from them. Other topics discussed at the congress were the education of registrars in transfusion medicine, new screening methods and questions of quality assurance.

Icelandic families with autosomal dominant polycystic kidney disease: families unlinked to chromosome 16p13.3 revealed by linkage analysis.

We have mainly used 3 highly polymorphic DNA markers, 3'HVR (D16S85), 16AC2.5 (D16S291) and SM7 (D16S283), flanking the PKD1 region on chromosome 16p13.3 to establish linkage status in seven Icelandic families with autosomal dominant polycystic kidney disease (ADPKD). In four families, the disease locus is in the PKD1 region, and three families are "unlinked" to chromosome 16p13.3. In one of the "unlinked" families, the disease locus is excluded from a part of the long arm of chromosome 2, and we support a theory of more than 2 loci being responsible for ADPKD. Our data confirm the location of the locus YNH24 (D2S44) to chromosome 2q13-q24.

Hereditary cystatin C amyloid angiopathy: identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease characterized by amyloidosis, dementia and fatal cerebral hemorrhage of young adults. A method for rapid and simple diagnosis of HCCAA is described. It is based upon oligonucleotide-directed enzymatic amplification of a 275-bp genomic DNA segment containing exon 2 of the cystatin C gene from a blood sample, followed by digestion of the amplification product with AluI. Loss of an AluI recognition site in the amplified DNA segment from HCCAA patients results in a deviating band-pattern at agarose gel electrophoresis, compared with that obtained from normal subjects or unaffected HCCAA family members. In a population of 9 patients with manifest HCCAA, 14 patients with other causes of brain hemorrhage and 16 healthy individuals, the diagnostic procedure displayed a sensitivity and specificity for HCCAA of 100%. Amplified DNA segments from 4 HCCAA patients of four different families were analyzed by nucleotide sequencing; the HCCAA-causing mutation in all families was found to be a single T----A substitution in the codon for amino acid residue 68 of cystatin C.

On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy.

The pathogenesis of the deposition of a variant cystatin C as amyloid in hereditary cystatin C amyloid angiopathy (HCCAA) is not known. To address this question the synthesis and secretion of cystatin C in cultured monocytes from 9 carriers of the mutated cystatin C gene (5 symptomatic and 4 asymptomatic) was examined. The quantity of cystatin C in cells and supernatants was determined by the ELISA method, Western blots were done and selected samples immunostained for cystatin C. Monocytes from individuals carrying the gene defect synthesized cystatin C that was apparently not truncated, a form found in the cerebral amyloid deposits in HCCAA, but showed a distinctly lower rate of cystatin C synthesis than monocytes from healthy controls. The main difference was that the quantity of cystatin C was significantly lower in the supernatants in monocyte cultures from carriers of the gene defect than from healthy controls, possibly due to a partial block in its secretion. This abnormal processing of the cystatin C could explain the low cerebrospinal fluid levels of cystatin C in HCCAA and might be a part of the pathogenetic pathway of amyloid deposition. Furthermore it could, through a lower extracellular concentration of this inhibitor of cysteine proteinases, contribute to destruction of the amyloidotic blood vessels, leading to the most serious clinical manifestation in HCCAA, intracerebral hemorrhage.

Natural killer cell function and malignant cell phenotype in hairy cell leukaemia.

We have followed for 33 months the changes that occurred in natural killer (NK) cell numbers and activity in a patient (A) with hairy cell leukaemia (HCL), using a single cell assay and a microcytotoxicity assay. The composition of the peripheral blood mononuclear cell population and malignant cell phenotype were also analysed. During this period he received treatment with interferon and his grossly enlarged spleen was removed. Four further patients were also studied, two were splenectomized and all had received treatment with interferon. In four of the five patients studied there was an apparent link between low NK activity and presence of a tumour-infiltrated spleen, and in the fifth patient, who was aleukemic and had no splenomegaly, NK function was related to disease activity. There was no correlation between NK activity and the number of target binding (TB) cells in these five patients. IFN had little direct effect on overall NK activity, but the proportion of killing cells among TB cells was increased. Three patients showed binding of several cells to a single target. Further analysis revealed that in the patients most of the TB cells were not CD56-positive NK cells, in contrast to TB cells from normal subjects. In patient A a large proportion (84%) of TB cells were identified as malignant cells and in patient E 15% of TB cells were malignant cells. The phenotype of the malignant cells was: CD19+, HLA-DR+ and CD25(Tac)+, except for patient A. In this patient the hairy cells were positive for the NK marker CD56 as well as the monocyte marker CD14. Furthermore, a change occurred in phenotype as only later samples carried CD25. It is concluded that the level of NK function correlates closely with disease activity in HCL and that competitive target cell binding by malignant cells may be one cause of depressed NK-cell function in hairy cell leukaemia.

Localization of a mutant gene for cleft palate and ankyloglossia in an X-linked Icelandic family.

Common congenital malformations such as cleft lip and cleft palate are in most cases multifactorial in origin, involving both environmental and genetic components. Molecular biology techniques have enabled the successful chromosomal localization of many mutant genes from disorders that exhibit simple Mendelian segregation, whether autosomally dominant (e.g., Huntington's disease), autosomal recessive (e.g., cystic fibrosis), or X-linked (e.g., Duchenne muscular dystrophy). Studying the genetic aspect of multifactorial disorders is more complex. It requires a model family or families within which the common multifactorial phenotype is displayed as a single gene defect. Such a model has been recently exploited in the form of a large Icelandic family (over 280 members) exhibiting X-linked secondary cleft palate (CP) and ankyloglossia (A) (tongue-tied) as a single gene mutation. Using this family and the large bank of well-characterized DNA probes available for the human X chromosome, the gene for CP + A was localized by linkage analysis to Xq13-q21.1 (LOD score = 3.07, linked to anonymous probe DXYS1). Further fine mapping, using other X probes from this region (confirmed by analysis of DNA from a deletion cell-line) has placed the gene between markers DXYS12 and DXS17 (LOD score = 4.1) at Xq21.3-q22. The approximate distance between these two probes is 5 centimorgans (cM), equivalent to approximately 5 million base pairs. Now that the limits of genetic linkage have been fully tested and there are two markers flanking the defect locus, strategies are being pursued to clone the gene responsible.(ABSTRACT TRUNCATED AT 250 WORDS)

[Scandinavian recommendations for transfusion medicine]. / Nordiska rekommendationer om transfusionsmedicin.

A central issue at the 13th Scandinavian Congress on Blood Transfusion Medicine was national self-sufficiency in plasma and plasma products, as outlined in the resolution adopted by the Council of Europe in 1990. Delegates also discussed the serological screening of donors to prevent the transmission of infection, new developments in the production of blood components, leukocyte filtration and serological technology.