Neuroseience Letters, 133 (1991) 1-2 1 © 1991 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/91/$ 03.50

NSL 08175

Sequencing of exons 16 and 17 of the fl-amyloid precursor protein gene in 14 families with early onset Alzheimer's disease fails to reveal

mutations in the fl-amyloid sequence

Fiona Crawford , John Hardy , Mike Mul lan , Alison Goate , David Hughes, L iana Fidani , Pene lope Roques , Mar t in Rossor and Marie-Chris t ine Char t ie r -Har l in

Alzheimer's Disease Research Group, Departments of Biochemistry and Neurology, St. Mary's Hospital Medical School, London ( U.K.)

(Received 25 July 1991; Accepted 4 August 1991)

Key words: Alzheimer's disease; Chromosome 21; Genetics, fl-Amyloid precursor protein

A mutation within exon 17 at codon 717 of the fl-amyloid protein precursor (APP) gene is one cause of early onset familial Alzheimer's disease. Direct sequencing of exons 16 and 17 of the fl-amyloid precursor protein gene in 14 families with familial early onset Alzheimer's disease without the known pathogenic mutation (APP717) failed to reveal other mutations within the fl-amyloid sequence in this form of the disorder.

The valine to isoleucine mutation at residue 717 of the fl-amyloid precursor protein (APP) gene [2] has been found in several families with early onset familial Alz- heimer's disease (AD) [2, 3]. These data, together with the fact that this variant has not been found in the general population [2, 3] strongly suggests that this mu- tation is a rare cause of early onset familial AD in Caucasian population [2, 3], though it may be a rela- tively common cause of this form of AD in Japanese populations [3, 4]. While there is strong genetic evidence that not all cases of the early onset familial AD are encoded within the APP locus [7, 10, 11], mutations within the fl-amyloid section of the APP gene (exons 16 and 17 [13]) other than APP717 are likely causes of some the other cases. For this reason we decided to sequence these exons in affected individuals from 11 other families in our collection with familial AD (Table I) 1, and three families (FAD1, FAD2, FAD4) from the Coriell cell repository [9].

PCR direct sequencing was carried out exactly as we have previously described [1, 7]. PCR amplification of exon 17 was carried out using primers of sequence: (A) 5'-CCT CAT CCA AAT GTC CCC GTC ATT-3' and (B) 5'-GCC TAA TTC TCT CAT AGT CTT AAT TCC CAC-3'. to amplify the exon. PCR conditions were 94°C,

Correspondence." M.C. Chartier-Harlin, Alzheimer's Disease Research Group, Departments of Biochemistry and Neurology, St. Mary's Hos- pital Medical School, London W2 IPG, U.K.

10 min to denature; then 35 cycles of 60°C, 1 min; 72°C, 3 min; 94°C, 1.5 min and a single cycle of 72°C, 10 min. The reaction volume used was 25 pl with a final concen- tration of primers at 50 pmol, dNTPs of 200/~M and a magnesium chloride concentration of 1.5 mM. A second PCR reaction as then performed with a final concentra- tion of 50 pmol of primer A and 0.5 pmol of primer B. The PCR product was purified on a Centricon 100 mi- croconcentrator (Amicon) and used directly for sequenc- ing with the Sequenase kit (version 2.0) (USB) following the manufacturer's protocol. PCR amplification of exon 16 was initially carried out using primers of sequence: - G G G TAG GCT TTG TCT TAC AG and GGC AAG ACA AAC AGT AGT GG. Direct sequencing of this exon used an internal primer of sequence: ACA AAC AGT AGT GGA AAG AGG TAA ATT AT. The con- ditions used for the amplification and sequencing reac- tions were initially the same as for exon 17. Sequencing exon 16 by this method, while it generated some data, proved troublesome. We, therefore, switched to using biotinylated primers with Dynabeads (M280 Streptavi- din, Dynal, Merseyside, U.K.) (sequence G G G TAG GCT TTG TCT TAC AG) to produce single stranded DNA for sequencing of this exon. Single strand confor- mation analysis [5, 6] of these exons using these PCR primers failed to reveal any mutations in either exon 16 or exon 17 although it does show band shifts in exon 17 with families with both APP693 and APP717 mutations (unpublished data).

TABLE I

Brief clinical information from the FAD series of families have been previously reported (see ref. 9). We have previously reported the se- quence data for FAD4 [1]. Many of these families are also discussed in ref. 12.

Families Age of onset No. of affected No. path. (range) confirmed

F53 52 (43 60) 5 1 F74 43 (39-50) 7 2 F75 51 (44-58) 11 0 F84 61 (59-64) 3 0 F97 62 (62-63) 2 0 FI05 38 (36-39) 14 0 FI21 37 (35-39) 6 0 F 126 54 (48-63) 4 0 F134 45 (41-50) 6 2 F 148 44 (42-46) 5 1 FI60 37 (36-38) 4 2

Examina t i on o f the sequence da t a f rom these families

identif ied no o ther mu ta t ions within the f l -amyloid con-

ta ining exons. There are two possible exp lana t ions for

these f inding which are not mutua l ly exclusive. The first

is that there are mu ta t ions elsewhere in the A P P gene

(either within the open reading frame, or within cont ro l -

ling elements) which are pathogenic . The second is tha t

these families have mu ta t ions in genes o ther than the

A P P gene. Whi le the precedence o f o ther diseases sug-

gests tha t o ther mu ta t ions within the A P P locus are like-

ly causes o f A D (for example , there are m a n y muta t ions

within the pr ion gene which are pathogenic) , it is no tab le

that the l inkage da t a suggesting a locus on c h r o m o s o m e

21 are largely con t r ibu ted by those families with the

APP717 m u t a t i o n (these pedigrees are F23 (pedigree 26

[8]) and F372 [8]). Thus, one does no t need to pos tu la te

the occurrence o f o ther mu ta t ions elsewhere on ch romo-

some 21 to expla in the d a t a relat ing to c h r o m o s o m e 21

l inkage for A D .

This work was suppor t ed by grants f rom Research

into Ageing, IPSEN, the Men ta l Hea l th F o u n d a t i o n , the

Roya l Society, and the Medica l Research Council . The

samples f rom the F A D series o f pedigrees which were

used in this s tudy were AG06849 and AG07647 (FAD 1) ,

AG08554 and AG08526 ( F A D 2 ) and AG08554 and

AG08109A ( F A D 4 ) f rom the Coriel l Cell reposi tory .

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I0

11

12

13

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