The Cystatin-C gene is not linked to early onset familial Alzheimer's disease

Neuroscience Letters, 154 (1993) 81-83

81

© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00 NSL 09488

The Cystatin-C gene is not linked to early onset familial Alzheimer's disease M a t t h e w Parfitt "'b, R i c h a r d C r o o k a, P e n e l o p e R o q u e s a, M a r t i n R o s s o r a a n d M a r i e - C h r i s t i n e C h a r t i e r - H a r l i n a'b ~Departments of Biochemistry and Neurology, St. Mary's Hospital Medical School, London ( UK) and bU 156 INSERM, Lille (France)

(Received 16 November 1992; Revised version received 1 February 1993; Accepted 7 February 1993) Key words: Cystatin-C; Alzheimer's disease; Hereditary cerebral haemorrhage with amyloidosis of the Icelandic type (HCHWA-I); Genetics;

Amyloidosis The APP717 mutations discovered in only a few early onset Alzheimer's disease (AD) familieshave confirmed the genetic heterogeneity of this disorder. To identify the other gene(s) involved in the disease we selected the protease inhibitor, Cystatin-C, as a candidate gene. Cystatin-C is an amyloidogenic protein causing hereditary cerebral haemorrhage with amyloidosis Icelandictype (HCHWA-I). It is localised with the fl-amyloid peptide in the arterial walls of AD brains. We have analysed the segregation of a polymorphic marker in this gene in 8 early onset AD families. Two early onset familiesshowed clear non-segregationof the marker with the disease. When the 8 familiesare analysed together (assuming only one other gene is involved), they present exclusion linkage criteria. These data indicate that Cystatin-C is not the site of the defect in 2 families and is not likely to be in the other families analysed. We conclude that the deposition of Cystatin-C in AD is a secondary event in the disease process, and that this gene is not pathogenic in familial AD.

The recent discovery that the amyloid precursor protein (APP) gene on chromosome 21 is the pathogenic locus in some families with the autosomal dominant form of Alzheimer's disease (AD) [1, 7, 14] has greatly enhanced our understanding of the aetiology of the disease. However the same neuropathological picture can be caused by a wide range of contributary elements (reviewed in ref. 25). Most cases of A D are sporadic and occur in the elderly. They have no clear genetic element but might be the result of environmental and genetic influences [4, 5]. O f the remaining familial cases, only a few are due to a pathogenic mutation in the APP gene [3, 6, 22]. Furthermore, the lack of genetic linkage to this locus in m a n y families has been demonstrated [19, 23, 24], and has led to the suggestion that other loci on chromosome 21 or on other chromosomes might be involved [17, 21]. Two strategies have been used to find these other pathogenic loci. When large pedigrees with multiple affected members are available, a genome search using highly polymorphic markers can be undertaken to find regions genetically linked to the disease. This has been termed the positional cloning approach. However the genetic Correspondence: M.-C. Chartier-Harlin, U 156 INSERM, Place de Verdun, 59045 Lille, France.

weakness of most families with A D makes this approach impractical. Thus we have adopted the candidate gene approach in which genes whose products are thought to be involved in the aetiology of A D are examined by genetic linkage analysis. Markers within or very close to the gene are used, so that the presence of just one recombinant between the disease locus and the marker excludes that gene as the site of the defect. In order to utilize the candidate gene approach, families were selected in which the APP gene had been excluded as the pathogenic locus. Eight such families (Table I) were chosen on the basis of three exclusion criteria. Firstly the families were screened for known APP mutations. The APP locus was then examined by genetic linkage. Any families that were not excluded by the linkage data were then examined by SSCP analysis [2, 3, 6] (and unpublished data). Some of the strongest candidate genes for A D are those whose products are involved in regulating APP. In A D the fl-amyloid peptide, an abnormal cleavage product from APP, is deposited at various sites in the brain and in brain blood vessels (reviewed in ref. 20). This and the pathogenic mutations in A P P have led to the suggestion that fl-amyloid deposition is the central event in A D aetiology [10]. Any protein that might be involved in the

82 TABLE l CLINICAL DETAILS OF EARLY ONSET FAMILIES Family Meanage of number onset (range)

Numberof Affecteds generations (path. confirmed)

148 121 134 74 126 84 15 206

4 4 4 4 3 3 6

46 (42 51) 36132 39) 45 141 49) 42 (39 50i 54 (48 63) 62 (59 64) 65 (60 70) 39 (37 42)

4

7 (1) 6 6 (2) 7 (2) 4 3 2 (1} 8

Individuals genotyped

17 8 15 9 5 15 8 20

expression, processing or degradation of APP derivatives is thus a candidate gene in AD. Cystatin-C is an amyloid protein that is deposited in meningeal and grey matter blood vessels in a cerebral amyloid angiopathy known as hereditary cerebral haemorrhage with amyloidosis of the Icelandic type (HCHWA-I) [9]. The disease is rare and inherited as an autosomal dominant disorder. A pathogenic point mutation in codon 68 of the Cystatin-C gene on chromosome 20, abolishes an AluI restriction site enabling the detection of the disease [15]. In the amyloid fibrils the first ten amino acids of Cystatin-C are missing. Deposition of fibrils in the blood vessels leads to multiple and often fatal cerebral haemorrhages. Cystatin-C was chosen as a candidate gene for A D for a number of reasons: (i) Cystatin-C and the fl-amyloid peptide are colocalized within arteriolar walls in A D brains [26]. (ii) Cystatin-C is a cysteine-protease inhibitor [8] which might be involved in controlling the processing of the fl-amyloid peptide. Mismetabolism of APP by proteases is thought to cause fl-peptide deposition [20]. (iii) H C H W A - I is very similar to H C H W A of the Dutch type (HCHWA-D), in which the fl-peptide, rather than Cystatin-C, is the amyloidogenic protein [18]. In both A D and H C H W A - D the APP gene is the site of a pathogenic mutation [7, 13]. The similarity between AD, H C H W A D and H C H W A - I suggests that there may be a biochemical mechanism leading to amyloidosis that is c o m m o n to all three. A polymorphic dinucleotide (AC) repeat within intron 2 of the Cystatin-C gene was amplified by PCR, as described by Hughes et al. [11]. This four allele system was used to type individuals from the families. Linkage analysis was performed using the M L I N K program from the Linkage Package [12]. Due to the heterogeneity of the disease data from the pedigrees were first analysed individually and then pooled together

(Table II). In the eight early onset families tested tbr linkage no cosegregation of the marker with the disease was found. Clear recombinants between the locus and the disease occur in families 74 and 84. Calculation of the likelihood of linkage between the marker locus and AD failed to yield significant exclusion results in the remaining families (in 5 families the Lod scores were negative, but not below -2). However when making the assumption of only one other major defective locus (combined data from the eight families), linkage exclusion criteria were obtained up to a distance of 3 cM from the Cystatin-C locus. Significant evidence of linkage exclusion was found between the marker and the AD locus in families 74 and 84. Therefore the Cystatin-C gene is not the defective site in these 2 families. Due to the genetic heterogeneity of the disease, one cannot state the number of non- APP loci involved in the pathogenesis of AD. Nevertheless, taking all the data into consideration, we suggest that it is very unlikely that the Cystatin-C gene is the primary cause of the disease in the 6 other families. These results are in agreement with the data obtained by Palsdottir et al. [16]. O f thirty-three AD and aged Down's syndrome patients who were examined for the H C H W A - I Cystatin-C mutation by restriction enzyme analysis, none were found to have the mutation. The colocalisation of Cystatin-C and the fl-amyloid peptide in A D patients, mainly in blood vessels, but occasionally in senile plaques, has led to the suggestion that Cystatin-C may also be involved in A D brain degeneration [26]. However, our data suggest that the involvement of Cystatin-C in A D is a secondary event in the pathogenesis of the disease. It may contribute to the vas-

TABLE iI LINKAGE ANALYSIS BETWEEN CYSTATIN-C MARKER AND ALZHEIMER'S DISEASE IN THE EIGHT EARLY ONSET FAMILIES Lod scores were calculated using 7 liability classes modelling age-dependent penetrances. Pedigree

Theta 0.00

148 121 134 74 126 84 15 206 Totals

-0.035 0.302 -0.099 -3.233 -0.128 -7.766 -0.115 -0.105

0.01

I).02

0.03

0.04

-0.035 0.297 -0.097 -1.291 -0.119 -1.659 -0.110 -0.100

-0.034 0.293 -0.095 -0.995 -0.110 -1.285 -0.106 -0.096

-0.033 0.288 -0.092 -0.824 -0.102 -1.053 -0.101 -0.092

-0.033 0.282 -0.090 -0.703 -0.094 -0.884 -0.096 -0.089

-2.428

-2.009

-1.707

-11.179 -3.113

83

cular deposits in AD, but is certainly not the trigger of ,8-amyloid deposition. This work was supported by a grant from the Medical Research Council. We wish to thank Dr. J. Hardy and Dr. J. Brown for encouragement and advice, and the Centre Hospitalier R6gional de Lille for additional assistance. M.P. is a recipient of a Wellcome Trust Grant. 1 Chartier-Harlin, M.C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, P., Hardy, J. and Mullan, M., Early-onset Alzheimer's disease caused by mutations at codon 717 of the fl-amyloid precursor protein gene, Nature, 353 (1991) 844-846. 2 Chartier-Harlin, M.C., Crawford, F., Hammandi, K., Mullan, M., Goate, A., Hardy, J.H.M., Backhovens, J.-J. and Van Broeckhoven, C., Screening for the fl-amyloid precursor protein mutation (APP717:Val ~ lle) in extended pedigrees with early onset Alzheimer's disease, Neurosci. Lett., 129 (1991) 134--135. 3 Crawford, F., Hardy, J., Mullan, M., Goate, A., Hughes, D., Fidani, L., Roques, P., Rossor, M. and Chartier-Harlin, M.C., Sequencing of exons 16 and 17 of the fl-amyloid precursor protein gene in families with early onset Alzheimer's disease fails to reveal mutations in the fl-amyloid sequence, Neurosci. Lett., 133 (1991) 1-2. 4 Farrer, L.A., Myers, R.H., Connor, L., Cupples, L.A. and Growdon, J.H., Segregation analysis reveals evidence of a major gene for Alzheimer's disease, Am. J. Hum. Genet., 48 (1991) 102(~103Y 5 Farrer, L.A., Myers, R.H., Cupples, L.A., St. George-Hyslop, P.H., Bird, T.D., Rossor, M.N., Mullah, M.J., Polinsky, R., Nee, I. and Heston, L., Transmission and age at onset patterns in familial Alzheimer's disease: evidence for heterogeneity, Neurology, 48 (1990) 395-403. 6 Fidani, L., Rooke, K., Chartier-Harlin, M.C., Hughes, D., Tanzi, R.. Mullan, M., Roques, P., Rossor, M., Hardy, J. and Goate, A., Screening for mutations in the open reading frame and promoter of the fl-amyloid precursor protein gene in familial Alzheimer's disease: identification of a further family with APP717 Val ~ Ile, Human Mol. Genet., 1 (1992) 65-168. 7 Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F, Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M. and Owen, M., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nature, 349 (1991) 704-706. 8 Grubb, A., Jensson, O., Gudmundsson, G., Arnasson, A., Lofberg, H. and Maim, J., Abnormal metabolism of gamma-trace alkaline microprotein. The basic defect in hereditary cerebral haemorrhage with amyloidosis, N. Engl. J. Med., 311 (1984) 1547-1549. 9 Gudmundsson, G., Hallgrimsson, J., Jonasson, T.A. and Bjarnason, O., Hereditary cerebral haemorrhage with amyloidosis, Brain, 95 (1972) 387-404. 10 Hardy, J. and Allsop, D., Amyloid deposition as the central event in the aetiology of Alzheimer's disease, TIPS, 12 (1991) 383-388. 11 Hughes, D., Brown, J., Hardy, J. and Chartier-Harlin, M.C., A polymorphic dinucleotide repeat in intron 2 of the human cystatinC gene, Human Mol. Genet., 1 (1992) 143. 12 Lathrop, G., Lalouel, J.M., Julier, C. and Ott, J., Strategies for multilocus linkage analysis in humans, Proc. Natl. Acad. Sci. USA, 81 (1981) 3443 3446.

13 Levy, E., Carman, M., Fernandez-Madrid, I., Power, M., Lieberburg, I., van Duinen, S., Bots, G., Luyendijk, W. and Frangione, B., Mutation of the Alzheimer's disease amyloid gene in hereditary cerebral haemorrhage, Dutch type, Science, 248 (1990) 1124-1126. 14 Murrell, J., Farlow, M., Bernadino, G. and Benson, M.D., A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease, Science, 253 (1991) 95-98. 15 Palsdottir, A., Abrahamson, M., Thorsteinsson, L., Arnason, A., Olafrson, I., Grubb, A. and Jensson, O., Mutation on Cystatin-C gene causes hereditary brain haemorrhage, Lancet, ii (1988) 603604. 16 Palsdottir, A., Jonsdottir, S., Arnason, A., Snaedal, J., Magnusson, T., Snorrason, E., Ingibergsdottir, R., Abrahamson, M. and Olafsson, I., Study of restriction fragment length polymorphism in the Cystatin-C gene of elderly patients with dementia and aged Down's syndrome patients, Prog. Clin. Biol. Res., 317 (1989) 135-139. 17 Pericak-Vance, M., Bebout, J., Gaskell, P., Yamaoka, L., Hung, W.-Y., Alberts, M., Walker, A., Bartlett, R., Haynes, C., Welsh, K., Earl, N., Heyman, A., Clark, C. and Roses, A., Linkage studies in familial Alzheimer disease: evidence for chromosome 19 linkage, Am. J. Hum. Genet., 48 (1991) 1034-1050. 18 PreUi, F., Castano, E.M,, Van Duinen, S.G., Bots, G.T.A.M., Luyendijk, W. and Frangione, B., Different processing of Alzheimer's fl-protein precursor in the vessel wall of patients with hereditary cerebral hemorrhage with amyloidosis-Dutch type, Biochem. Biophys. Res. Commun., 151 (1988) 1150 1155. 19 Schellenberg, G., Bird, T., Wijsman, E., Moore, D., Boehnke, M., Bryant, E., Lampe, T., Nochlin, D., Sumi, S., Deeb, S., Beyreuther, K. and Martin, G., Absence of linkage of chromosome 21q21 markers to familial Alzheimer's disease, Science, 241 (1988) 1507-1510. 20 Selkoe, D., The molecular pathology of Alzheimer's disease, Neuron, 6 (1991) 487-498. 21 St. George-Hyslop, P.H., Haines, J.L., Farrer, L.A., Polinsky, R., Van Broeckhoven, C., Goate, A.M. and Crapper-McLachlan, D.R., Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder, Nature, 347 (1990) 194-197. 22 Tanzi, R.E., Vaula, G., Romano, D.M., Mortilla, M., Huang, T.L., Tupler, R.G., Wasco, W., Hyman, B.T., Haines, J.L. and Jenkins, B.J., Assessment of amyloid fl-protein precursor gene mutations in a large set of familial and sporadic Alzheimer's disease cases, Am. J. Hum. Genet., 51 (1992) 273-282. 23 Tanzi, R.E., St. George-Hyslop, P.H., Haines, J., Polinsky, R., Nee, L., Foncin, J.-F., Neve, R., McClatchey, A., Conneally, P. and Gusella, J., The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene, Nature, 329 (1987) 15(~157. 24 Van Broeckhoven, C., Genthe, A., Vandenberghe, A., Horsthemke, B., Backhovens, H., Raeymaekers, P., Van Hul, W., Wehnert, A., Gheuens, J., Cras, P., Bruyland, M., Martin, J., Salbaum, M., Multhaup, G., Masters, C., Beyreuther, K., Gurling, H., Mullan, M., Holland, A., Barton, A., Irving, N., Williamson, R., Richards, S. and Hardy, J., Failure of familial Alzheimer's disease to segregate with the A4-amyloid gene in several European families, Nature, 329 (1987) 153-155. 25 Van Duijn, C.M., Stijnen, T. and Hofman, A., Risk factors for Alzheimer's disease: overview of the EURODEM collaborative reanalysis of case-control studies, Int. J. Epidemiol., 20 (Suppl. 2) (1991) $4-S12. 26 Vinters, H.V., Nishimura, G.S., Secor, D.L. and Pardridge, W.M., Immunoreactive A4 and gamma-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer's disease, Am. J. Pathol, 137 (1990) 233-240.