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Improved direct sequencing of Alzheimer's amyloid precursor protein (APP) exons 16 and 17
Neuroscience Letters, 141 (1992)6%71
69
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08728
Improved direct sequencing of Alzheimer's amyloid precursor protein (APP) exons 16 and 17 R o s a A d r o e r a, 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 b, F i o n a C r a w f o r d b a n d R a f a e l Oliva a aMolecular Genetics Research Group, Faculty of Medicine, University of Barcelona, Barcelona (Spain) and bDepartment of Biochemistry and Molecular Genetics, Alzheimer's Disease Research Group, St Mary's Hospital Medical School, London ( UK)
(Received 17 January 1992;Revised version received30 March 1992;Accepted 3 April 1992) Key words: Alzheimer'sdisease; Chromosome21; Genetics;fl-Amyloidprecursor protein; Polymerasechain reaction; Direct sequencing;amyloid
precursor protein mutation Direct sequencing of exon 17 of the amyloid precursor protein (APP) gene led to the identificationof 3 different types of APP717 pathogenic mutations associatedwith familialAlzheimer'sdisease(FAD). The low frequencyof these mutations results in havingto screenmany samples in order to identify new families affectedby them, which is laborious and time consuming. Thus, in order to help the identificationof these mutations in additional countriesand to search for new mutations in APP, perhaps in other exons also causing FAD, we have optimizedthe procedure and reduced the time necessaryfor sample preparation from 11 h to 31/2 h.
Direct sequencing of amyloid precursor protein (APP) exon 17 led to the identification of the pathogenic mutation APP717 Val---)Ile causing early onset Alzheimer's disease [ 1 4 , 6, 7, 9-11]. At present, 5 families have been reported having this mutation : 1 in England [4], 1 in USA [4] and 3 in Japan [7, 11]. Two other single aminoacid substitutions have also been described in the APP 717 codon : a Val--+Gly substitution [2], and a Val--~Phe [6]. All of these cases are associated with the Alzheimer's phenotype, although they represent a low fraction of the patients whose APP exon 717 has been screened. Thus, because of the large number of samples that must be sequenced in order to identify these mutations in the different populations, we decided to optimize the procedure and reduce the time necessary for sample processing before sequencing. The procedure described below requires merely 31/2 h for sample processing (instead of 11 h which were required when using the standard methods), is simpler to use and gives consistently better sequences. This marked reduction in time has been possible by: (1) performing a single PCR amplification instead of two PCRs (the first one to amplify the DNA, and the second one to obtain ssDNA), and (2) standardizing the single Correspondence: R. Oliva, Molecular Genetics Research Group, Faculty of Medicine, Universityof Barcelona,Diagonal 643, 08028 Barcelona, Spain. Fax: (34) (3) 4909346.
strand purification method based on affinity with streptavidin coated magnetic beads [5] and the subsequent direct sequencing for its use with the specific oligonucleotides described below. The amplification reactions contained 20 ng of DNA, 10 pmol of each primer (To amplify exon 16: Primer 1, b i o t i n - G G G T A G G C T T T G T C T T A C A G - 3 ' and primer 2, 5'-ACAAACAGTAGTGGAAAGAGGTAAATTAT3"; To amplify exon 17: Primer 3: biotin-GCCTAATTCTCTCATAGTCTTAATTCCCAC-3' and primer 4: 5'G T T G G G C A G A G A A T A T A C T G A - 3 ' ) , dNTPs at 200 pM, 1.5 m M MgCI2, 10 m M Tris-HCl, pH 8.3, 50 m M KC1 and 1 unit Taq polymerase in a final volume of 25 /tl. PCR conditions were as follows: an initial denaturation at 94°C for 5 min, 35 cycles consisting of 1 min at 60°C, 1 rain at 72°C and 1 min at 94°C, and a final elongation of 5 min at 72°C. To purify ssDNA using streptavidin coated magnetic beads the manufacturer's protocols were followed with modifications: 20/A of the PCR reaction was mixed with prewashed Dynabeads (10 /tl of beads prewashed 3 times with 200/.tl ofTWS: 0.17% Triton X-100, 500 m M NaC1, 10 m M Tris, p H 7.5, 1 m M EDTA pH 8.0 and resuspended in 80/11 of TWS) at room temperature on a rotary mixer for 30 min. The Dynabeads are then separated with the aid of a Magnetic Particle Concentrator (MPC) and washed twice with 20/al of TWS as follows: the microcentrifuge tubes containing
70 T
C
G
A
j
c T A
C T
O,11,
C A
G T G b
T G A
A A
G,4o A
C G 5'
the P C R p r o d u c t - D y n a b e a d s mix are placed on the magnet to pull the PCR p r o d u c t - D y n a b e a d s complexes to the bottom of the tube. The supernatant is then removed with the aid of a micropipette, 20/11 of TWS are added to the PCR p r o d u c t - D y n a b e a d s complex, the tube removed from the MPC, mixed for 15 s and again introduced into the MPC-Magnet, the supernatant being removed as above. This procedure is repeated once more and the PCR product Dynabeads mix is then resuspended in 32/~1 of TE and 8/11 of 1 M N a O H , 4 m M E D T A are added to denature the two strands of the PCR product. At this point the D N A strand containing biotin at the 5' end remains strongly bound to the streptavidin coated magnetic beads whereas the other strand without biotin remains free in solution. The tube is then placed in the MPC to pull the complex formed by the magnetic particle-streptavidin 5' biotin-single stranded D N A to the bottom of the tube and the supernatant removed as above. The beads are finally washed twice with 200/A of TWS and suspended in 7 HI of H20. Exon 16 was sequenced with primer 2 and exon 17 was sequenced with primer 4, in both cases using Sequenase (USB), [35S]dATP~S and following manufacturer's instructions. The method described here should be of great value to rapidly screen for the APPTI7 mutation in a large number of samples and to search for new mutations of A P E perhaps in other loci, linked to familial Alzheimer's disease. This approach is faster and more efficient than alternative methods such as sub-cloning the P C R product, sequencing double stranded D N A or doing asymmetric PCR followed by template purification. BclI digestion of the exon 17 PCR product produces two extra bands if the mutation APP717 Val~Ile is present, but fails to detect the other mutations. Another alternative to D N A sequencing is SSCP analysis [8] but this technique is not quicker than direct sequencing and fails to identify all nucleotide changes. With the increasing introduction of automatic sequencers, the sample preparation method described here should also be very valuable because it can be easily automated and adapted to the use of fluorescent primers or terminators. This work has been supported by G r a n t SAL90-0334 from the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT) from Spain to R.O. and by the Ipsen foundation and MRC. Chartier-Harlin, M.-C., Crawlord, F., Hamandi, K., Mullan, M., Goate, A., Hardy, J., Backhovens, H., Martin, 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. 2 Chartier-Harlin, M.-C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, R, Hardy, 1
Fig. 1. DNA sequence of APP exon 17 obtained by direct sequencing using streptavidin coated magnetic beads from a FAD patient lacking the pathogenic APP717 mutation. ~, site of the described APP717 mutation linked to some types of FAD. (), site of the Hereditary Cerebral Hemorrhage With Amyloidosisof Dutch Type (HCHWA-D).
71
3
4
5
6
J. and Mullah, M., Early-onset Alzheimer's disease caused by mutations at codon 717 of the fl-amyloid precursor protein gene, Nature, 353 (1991) 844-846. 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 14 families with early onset Alzheimer's disease fails to reveal mutations in the fl-amyloid sequence, Neurosci. Lett., 133 (1991) 1-2. 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., Owen, M. and Hardy, J., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nature, 349 (1991) 704-706. Hultman, T., St~ihl, S., Hornes, E. and Uhl6n, M., Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support, Nucl. Acids Res., 17 (1989) 4937- 4946. Murrell, J., Farlow, M., Ghetti, B. and Benson, M.D., A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease, Science, 254 (1991) 97-99.
7 Naruse, S., Igarashi, S., Kobayashi, H., Aoki, K., Inuzuka, T., Kaneko, K., Shimizu, T., Iihara, K., Kojima, T., Miyatake, T. and Tsuji, S., Mis-sense mutation Val---)Ilein exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer's disease, Lancet, 337 (1991) 978-979. 8 Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K., Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction, Genomics, 5 (1989) 874- 879. 9 Schellenberg, G.D., Anderson, L., O'dahl, S., Wisjman, E.M., Sadovnick, A.D., Ball, M.J., Larson, E.B., Kukull, W.A., Martin, G.M., Roses, A.D. and Bird, T.D., APP717, APP693, and PRIP gene mutations are rare in Alzheimer Disease, Am. J. Hum. Genet. 49 (1991) 511-517. 10 Van Duijn, C.M., Hendriks, L., Cruts, M., Hardy, J., Hofman, A. and Van Broeckhoven, C., Amyloid precursor protein gene mutation in early-onset Alzheimer's disease, Lancet, 337 (1991) 978. 11 Yoshioka, K., Miki, T., Katsuya, T., Ogihara, T. and Sakaki, Y., The 717Val~Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups, Biochem. Biophys. Res. Commun., 178 (1991) 1141-1146.
69
© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00 NSL 08728
Improved direct sequencing of Alzheimer's amyloid precursor protein (APP) exons 16 and 17 R o s a A d r o e r a, 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 b, F i o n a C r a w f o r d b a n d R a f a e l Oliva a aMolecular Genetics Research Group, Faculty of Medicine, University of Barcelona, Barcelona (Spain) and bDepartment of Biochemistry and Molecular Genetics, Alzheimer's Disease Research Group, St Mary's Hospital Medical School, London ( UK)
(Received 17 January 1992;Revised version received30 March 1992;Accepted 3 April 1992) Key words: Alzheimer'sdisease; Chromosome21; Genetics;fl-Amyloidprecursor protein; Polymerasechain reaction; Direct sequencing;amyloid
precursor protein mutation Direct sequencing of exon 17 of the amyloid precursor protein (APP) gene led to the identificationof 3 different types of APP717 pathogenic mutations associatedwith familialAlzheimer'sdisease(FAD). The low frequencyof these mutations results in havingto screenmany samples in order to identify new families affectedby them, which is laborious and time consuming. Thus, in order to help the identificationof these mutations in additional countriesand to search for new mutations in APP, perhaps in other exons also causing FAD, we have optimizedthe procedure and reduced the time necessaryfor sample preparation from 11 h to 31/2 h.
Direct sequencing of amyloid precursor protein (APP) exon 17 led to the identification of the pathogenic mutation APP717 Val---)Ile causing early onset Alzheimer's disease [ 1 4 , 6, 7, 9-11]. At present, 5 families have been reported having this mutation : 1 in England [4], 1 in USA [4] and 3 in Japan [7, 11]. Two other single aminoacid substitutions have also been described in the APP 717 codon : a Val--+Gly substitution [2], and a Val--~Phe [6]. All of these cases are associated with the Alzheimer's phenotype, although they represent a low fraction of the patients whose APP exon 717 has been screened. Thus, because of the large number of samples that must be sequenced in order to identify these mutations in the different populations, we decided to optimize the procedure and reduce the time necessary for sample processing before sequencing. The procedure described below requires merely 31/2 h for sample processing (instead of 11 h which were required when using the standard methods), is simpler to use and gives consistently better sequences. This marked reduction in time has been possible by: (1) performing a single PCR amplification instead of two PCRs (the first one to amplify the DNA, and the second one to obtain ssDNA), and (2) standardizing the single Correspondence: R. Oliva, Molecular Genetics Research Group, Faculty of Medicine, Universityof Barcelona,Diagonal 643, 08028 Barcelona, Spain. Fax: (34) (3) 4909346.
strand purification method based on affinity with streptavidin coated magnetic beads [5] and the subsequent direct sequencing for its use with the specific oligonucleotides described below. The amplification reactions contained 20 ng of DNA, 10 pmol of each primer (To amplify exon 16: Primer 1, b i o t i n - G G G T A G G C T T T G T C T T A C A G - 3 ' and primer 2, 5'-ACAAACAGTAGTGGAAAGAGGTAAATTAT3"; To amplify exon 17: Primer 3: biotin-GCCTAATTCTCTCATAGTCTTAATTCCCAC-3' and primer 4: 5'G T T G G G C A G A G A A T A T A C T G A - 3 ' ) , dNTPs at 200 pM, 1.5 m M MgCI2, 10 m M Tris-HCl, pH 8.3, 50 m M KC1 and 1 unit Taq polymerase in a final volume of 25 /tl. PCR conditions were as follows: an initial denaturation at 94°C for 5 min, 35 cycles consisting of 1 min at 60°C, 1 rain at 72°C and 1 min at 94°C, and a final elongation of 5 min at 72°C. To purify ssDNA using streptavidin coated magnetic beads the manufacturer's protocols were followed with modifications: 20/A of the PCR reaction was mixed with prewashed Dynabeads (10 /tl of beads prewashed 3 times with 200/.tl ofTWS: 0.17% Triton X-100, 500 m M NaC1, 10 m M Tris, p H 7.5, 1 m M EDTA pH 8.0 and resuspended in 80/11 of TWS) at room temperature on a rotary mixer for 30 min. The Dynabeads are then separated with the aid of a Magnetic Particle Concentrator (MPC) and washed twice with 20/al of TWS as follows: the microcentrifuge tubes containing
70 T
C
G
A
j
c T A
C T
O,11,
C A
G T G b
T G A
A A
G,4o A
C G 5'
the P C R p r o d u c t - D y n a b e a d s mix are placed on the magnet to pull the PCR p r o d u c t - D y n a b e a d s complexes to the bottom of the tube. The supernatant is then removed with the aid of a micropipette, 20/11 of TWS are added to the PCR p r o d u c t - D y n a b e a d s complex, the tube removed from the MPC, mixed for 15 s and again introduced into the MPC-Magnet, the supernatant being removed as above. This procedure is repeated once more and the PCR product Dynabeads mix is then resuspended in 32/~1 of TE and 8/11 of 1 M N a O H , 4 m M E D T A are added to denature the two strands of the PCR product. At this point the D N A strand containing biotin at the 5' end remains strongly bound to the streptavidin coated magnetic beads whereas the other strand without biotin remains free in solution. The tube is then placed in the MPC to pull the complex formed by the magnetic particle-streptavidin 5' biotin-single stranded D N A to the bottom of the tube and the supernatant removed as above. The beads are finally washed twice with 200/A of TWS and suspended in 7 HI of H20. Exon 16 was sequenced with primer 2 and exon 17 was sequenced with primer 4, in both cases using Sequenase (USB), [35S]dATP~S and following manufacturer's instructions. The method described here should be of great value to rapidly screen for the APPTI7 mutation in a large number of samples and to search for new mutations of A P E perhaps in other loci, linked to familial Alzheimer's disease. This approach is faster and more efficient than alternative methods such as sub-cloning the P C R product, sequencing double stranded D N A or doing asymmetric PCR followed by template purification. BclI digestion of the exon 17 PCR product produces two extra bands if the mutation APP717 Val~Ile is present, but fails to detect the other mutations. Another alternative to D N A sequencing is SSCP analysis [8] but this technique is not quicker than direct sequencing and fails to identify all nucleotide changes. With the increasing introduction of automatic sequencers, the sample preparation method described here should also be very valuable because it can be easily automated and adapted to the use of fluorescent primers or terminators. This work has been supported by G r a n t SAL90-0334 from the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT) from Spain to R.O. and by the Ipsen foundation and MRC. Chartier-Harlin, M.-C., Crawlord, F., Hamandi, K., Mullan, M., Goate, A., Hardy, J., Backhovens, H., Martin, 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. 2 Chartier-Harlin, M.-C., Crawford, F., Houlden, H., Warren, A., Hughes, D., Fidani, L., Goate, A., Rossor, M., Roques, R, Hardy, 1
Fig. 1. DNA sequence of APP exon 17 obtained by direct sequencing using streptavidin coated magnetic beads from a FAD patient lacking the pathogenic APP717 mutation. ~, site of the described APP717 mutation linked to some types of FAD. (), site of the Hereditary Cerebral Hemorrhage With Amyloidosisof Dutch Type (HCHWA-D).
71
3
4
5
6
J. and Mullah, M., Early-onset Alzheimer's disease caused by mutations at codon 717 of the fl-amyloid precursor protein gene, Nature, 353 (1991) 844-846. 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 14 families with early onset Alzheimer's disease fails to reveal mutations in the fl-amyloid sequence, Neurosci. Lett., 133 (1991) 1-2. 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., Owen, M. and Hardy, J., Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease, Nature, 349 (1991) 704-706. Hultman, T., St~ihl, S., Hornes, E. and Uhl6n, M., Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support, Nucl. Acids Res., 17 (1989) 4937- 4946. Murrell, J., Farlow, M., Ghetti, B. and Benson, M.D., A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease, Science, 254 (1991) 97-99.
7 Naruse, S., Igarashi, S., Kobayashi, H., Aoki, K., Inuzuka, T., Kaneko, K., Shimizu, T., Iihara, K., Kojima, T., Miyatake, T. and Tsuji, S., Mis-sense mutation Val---)Ilein exon 17 of amyloid precursor protein gene in Japanese familial Alzheimer's disease, Lancet, 337 (1991) 978-979. 8 Orita, M., Suzuki, Y., Sekiya, T. and Hayashi, K., Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction, Genomics, 5 (1989) 874- 879. 9 Schellenberg, G.D., Anderson, L., O'dahl, S., Wisjman, E.M., Sadovnick, A.D., Ball, M.J., Larson, E.B., Kukull, W.A., Martin, G.M., Roses, A.D. and Bird, T.D., APP717, APP693, and PRIP gene mutations are rare in Alzheimer Disease, Am. J. Hum. Genet. 49 (1991) 511-517. 10 Van Duijn, C.M., Hendriks, L., Cruts, M., Hardy, J., Hofman, A. and Van Broeckhoven, C., Amyloid precursor protein gene mutation in early-onset Alzheimer's disease, Lancet, 337 (1991) 978. 11 Yoshioka, K., Miki, T., Katsuya, T., Ogihara, T. and Sakaki, Y., The 717Val~Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups, Biochem. Biophys. Res. Commun., 178 (1991) 1141-1146.