Vol. 189, No. 2, 1992 December 15, 1992
BIOCHEMICAL
A COMPARISON MISPAIRING
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1202-1206
OF SINGLE NUCLEOTIDE PRIMER EXTENSION PCR-RFLP IN DETECTING A POINT MUTATION
WITH
Fu-Hai Lin* and Ruth Lin New York State Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314 Received
November
5,
1992
SUMMARY: A recent report by Petruzzella et al. (BBRC 186, 491-497, 1992) raised a question as to whether a point mutation in the mitochondrial ND2 gene (BBRC 182, 238246, 1992) is relevant to Alzheimer’s disease. The argument was based on their inability to detect the point mutation at position 5460 in codon 331 in the DNAs extracted from 15 patients with Alzheimer’s disease using mispairing PCR-RFLP. To clarify the discrepancy, we tested the DNAs reported by Petruzzella et al. for the mutation by single-nucleotide primer extension. The present work confirms our previous report and extends our finding of the point mutation in 8 of the 15 AD DNAs. o 1992Academic PESS, IX.
After the report of a point mutation in codon 331 of the ND2 gene in Alzheimer brain mitochondrial
DNA (mtDNA, l), a number of laboratories have shown an interest in
the subject. Petruzzella et al. (2) used the mispairing PCR (3) in conjunction with restiction fragment length polymorphism (RFLP) to examine brain DNAs from 15 Alzheimer patients and were unable to detect the mutation. To evaluate the validity of our report (l), samples from 3 patients were tested for the mutation by the mispairing PCR-RFLP
method (2).
Cases #90 and #28095 were found to contain normal and mutant mtDNA in homoplasmic form respectively (1). This finding was confirmed by the mispairing PCR-RFLP method (2). However, there was disagreement in case #3925 regarding the presence of the mutation (2). Because of the absence of the mutation in the 15 AD patients in their study, these workers concluded that this mutation is not AD specific (2). mtDNA
in patient 3925 was heteroplasmic and it contained about equal amount of
normal and mutant forms (1). detect the point mutation
The inability of the mispairing PCR-RFLP
technique to
in heteroplasmic mtDNAs seems to indicate an allele-specific
* To whom corresponce should be addressed. Abbreviations: AD, Alzheimer’s disease; ND2, subunit 2 of rnitochondrial NADH dehydrogenase-ubiquinone oxidoreductaxe (EC 1.6.99.3); PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SNuPE, single nucleotide primer extension. 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
1202
Vol.
189, No. 2, 1992
amplification
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of the normal form in the mispairing PCR (4). To test whether this was the
case, we amplified the target region by using the mispairing (2) and the pairing oligomers (1) as the primers in PCR. The resultant products were tested for the point mutation by single nucleotide primer extention (SNuPE) as described previously (1). The results of the present study confirm our previous finding (1). In addition, we detected a G to T base substitution at nt 5460 (5) in 8 of the 15 AD samples (2) and found that the mtDNA of the 8 patients was heteroplasmic.
This finding explains the difference in the data reported by
the two laboratories (1,2).
MATERIALS
AND METHODS
Total DNA extracted from 15 AD patients, 5 normal individuals and 2 mtDNAs, 2122, as well as the mispairing primers (2) were kindly provided by Dr. Eric A. Schon of Columbia University.
Chemical reagents were obtained from the same sources reported
previously (1). The primers for PCR amplification
and for SNuPE were synthesized in this
Institute as reported (1). Amplification
of the target region by PCR was done as described (1). The pairing
primers ND2-001 and ND2-003 generated a fragment of 350 bp (1) whereas the mispairing primers produced
a fragment
of 214 bp (2).
The PCR products were purified
by
electrophoresis in a 1.2% agarose gel and were used as the template for SNuPE as described (1).
RESULTS Since the apparent difference in SNuPE (1) and the mispairing PCR-RFLP methods (2) lies in the production of DNA by using different primers in PCR, it seems possible that the latter method preferentially population.
amplified the normal form of mtDNA in a heteroplasmic
To test whether this was the case, the 350 bp and 214 bp DNA preparations
(Fig 1A) were assayed for the presence of the point mutation in codon 331 of the ND2 gene by SNuPE. The results are shown in Figure 1. In panel B, it is seen that DNA generated from sample 22 (2) which was extracted from case #3925 (I), with the pairing primers ND2001 and ND2-003 (1) consisted of 2 forms of mtDNA.
One form contained the normal
codon CCC and the other TCC (22-P) confirming the previous report (1). In contrast to sample 22-P, only GCC, the normal codon, was detected in the DNA produced with the mispairing primers (22-M).
The 214 bp DNA samples #16 through #20 in panel B were
produced by the mispairing primers and no mutation was detected in these samples. On the 1203
Vol.
189, No. 2, 1992
A
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
P F) Pairing
P (Rev)
f-35o
bpf-------------)l
I k------,214 5160 Mispairing
bp--Jb4Q6 5498 MP (Rev)
5262 MP F) B
22-P --/I---II
22-M
16
17
16
19
,ssl"ma &,a &@_
--_----_--__-_.-_~~~--_ GATGATGATGATGATGATGAT
C
r---r
16
17
20
18
19
20
I
___-_----------
GATGATGATGATGAT
Figure 1. Detection of a point mutation by SNuPE. The target region in mtDNA was amplified by PCR as described (1) by using the mispairing (B) and pairing (C) oligomers as the primers. For detail regarding the primers, see references 1 and 2. For SNuPE, 3 sets of reaction were set up. Each reaction mixture contained 20 k 1incubation buffer and 1 unit Taq DNA polymerase (Boehringer Mannheim), 50 ng DNA template, 1 pmole extension primer and 1 p Ci of a [nP]dNTP (3000 Ci/mmole, Amersham). The reaction was performed in an automatic thermocycler (Coy) for 2 min each at 96oC, 55oC and 72oC. Equal volumes of the reaction mixture and a stop solution (95% formamide, 5 mM EDTA, 0.05% bromophenol blue and xylene cyanol) were then mixed and heated at 96oC for 2 min; 5 @1 of the mixture was loaded onto a gel (11 x 14 cm) containing 3% NuSiev (FMC) and 1% agarose. Electrophoresis was run at 200 V at room temperature until bromophenol blue reached l/4 from the bottom of the gel. Autoradiography was performed on Kodak AR Xray film at -7ooC for 4 h. A. A schematic diagram shows the locations of the primers and the sixes of PCR products. P (F) and P (Rev ) are pairing primers forward and reverse,repectively. MP = mispairing primer. The numbers indicate the position of nucleotides where the primers locate. B. DNA template was prepared in PCR with the mispairing primers except sample 22-P which was produced with the pairing primers. The spots show the incorporation of the indicated nucleotide onto the 3’-end of the extension primer. The numbers on the top of the Figure are sample numbers corresponding to those in Table 1. C. DNA template was produced in PCR with the pairing primers.
other hand, when the 350 bp sample #16 generated by the pairing primers was tested for the mutation, a G to T change was detected as shown in panel C. The G to T transversion was also detected in 7 other AD samples as summarized in Table 1. It is noted that the mtDNA
in the 8 AD patients is heteroplasmic and the TCC mutant represents a minor
fraction. 1204
Vol.
189,
No.
2,
1992
BIOCHEMICAL
TABLE 1. NUCLEOTIDE *Sample
Age
1
AND
BIOPHYSICAL
RESEARCH
COMMUNICA9ONS
COMPOSITION OF CODON 331 OF THE ND2 GENE Diagnosis
Codon 331
AD
CCC/KC
aa AIafSer
2
76
AD
ND
3
90
AD
GCC/TCC
Ala/.%x
4
64
Normal
GCC
Ala
5
78
AD
GCC
Ala
6
63
AD
GCC
Ala
7
63
AD
GCC/TCC
AIa/Ser
8
68
Normal
GCC
Ala
9
81
AD
GCC/TCC
AIa/Ser
10
64
Normal
GCC
Ala
11
88
AD
GCC
Ala
12
70
AD
GCC/TCC
AlaJSer
13
59
AD
GCCflCC
AIa/Ser
14
61
Normal
GCC
Ala
15
87
AD
GCC/TCC
AIa/Ser
16
93
AD
GCC/TCC
AIa/Ser
17
68
AD
GCC
Ala Ala
18
78
AD
GCC
19
88
AD
GCC
Ala
20
75
Normal
GCC
Ala
21
79
AD
GCC
Ala
CCC/l-CC 22 76 AD *r I he sample was numbered accordmg to reference 2. ND= Not done.
AIa/Ser
DISCUSSION The present study confirms our previous report (1) that mtDNA
from patient 3925 consists of normal and mutant forms, and shows that little, if any, of the mutant mtDNA was produced in PCR by using the mispairing oligomers as the primers (2). The mechanism is not understood.
It is possible, however, that the failure of Taq DNA polymerase to amplify
the mutant molecule is due to allele-specific PCR (4) in which a base-pair mismatch at the 3’-end of a primer can reduce the thermal stability of the primer-template
complex. From
the present observation, we conclude that the SNuPE method (1) is more reliable than the mispairing PCR-RFLP
(2) in detecting a point mutation in heteroplasmic mtDNAs. 1205
avot. 489,
No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The G to T transversion was detected at the first position of codon 331 of the ND2 gene in 8 of 15 AD DNAs (Table 1). mtDNA in these patients was heteroplasmic and the ratio of normal to mutant form was about 91 (data not shown). Whether the level of this change in coding sequence significantly affects the catalytic activity of complex I remains to be elucidated.
The study detected the mutation in about 50% of AD patients and the
change was a G to T transversion.
This pattern is similar to that reported previously (1).
The data suggest that mutations may occur in other mitochondrial genes, one of which could be the gene coding for cytochrome oxidase (6).
This is consistent with the finding of
multigene mutations in other degenerative diseases (7). It is believed that several factors are involved in Alzheimer’s disease (7). Thus, a defect in mitochondrial to be an important
function is likely
contributing cause of the disease.
ACKNOWLEDGMENTS R.L was supported by an NIH grant PO1 AGO 4220 to Dr. Henry M. Wisniewski to whom we give thanks for his support This work was partially supported by New York State Office of Mental Retardation and Developmental Disabilities. We thank Dr. Eric A. Schon of Columbia University for providing us with the DNAs and the mispairing primers; Drs. David L. Miller, Richard I. Carp and Richard Rubenstein for reviewing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Lin, F.H., Lin, R., Wisniewski, H-M., Hwang, Y.W., Grundke-Iqbal, I., HealyLouie, G. and Iqbal, K (1992) Biochem. Biophys. Res. Comm 182, 238246. Petruzzella, V., Chen, X. and Schon, E.A (1992) B&hem. Biophys. Res. Comm 186, 491-497. Seibel, P., Degoul, F., Romero, N., Marsac, C. and Kadenbach, B. (1990) B&hem. Biophys. Res. Comm. 173,561-565. Amheim, N. and Erlich, H. (1992). Annu. Rev. B&hem. 61,131-156. Anderson, S., Bankier, AT., Barrell, B.S., deBruijn, M.H.L., Co&on, AR., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B-k, Sanger, F., Schreier, P.H., Smith, AJ.H., Staden, R. and Young, LG. (1981) Nature 290, 457-465. Parker, Jr., W.D., Filley, C.M. and Parks, J.K. (1990) Neurology, 40, 1302- 1303. Wallace, D.C. (1992) Annu. Rev. Biochem. 61, 1175-1212.
BIOCHEMICAL
A COMPARISON MISPAIRING
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1202-1206
OF SINGLE NUCLEOTIDE PRIMER EXTENSION PCR-RFLP IN DETECTING A POINT MUTATION
WITH
Fu-Hai Lin* and Ruth Lin New York State Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314 Received
November
5,
1992
SUMMARY: A recent report by Petruzzella et al. (BBRC 186, 491-497, 1992) raised a question as to whether a point mutation in the mitochondrial ND2 gene (BBRC 182, 238246, 1992) is relevant to Alzheimer’s disease. The argument was based on their inability to detect the point mutation at position 5460 in codon 331 in the DNAs extracted from 15 patients with Alzheimer’s disease using mispairing PCR-RFLP. To clarify the discrepancy, we tested the DNAs reported by Petruzzella et al. for the mutation by single-nucleotide primer extension. The present work confirms our previous report and extends our finding of the point mutation in 8 of the 15 AD DNAs. o 1992Academic PESS, IX.
After the report of a point mutation in codon 331 of the ND2 gene in Alzheimer brain mitochondrial
DNA (mtDNA, l), a number of laboratories have shown an interest in
the subject. Petruzzella et al. (2) used the mispairing PCR (3) in conjunction with restiction fragment length polymorphism (RFLP) to examine brain DNAs from 15 Alzheimer patients and were unable to detect the mutation. To evaluate the validity of our report (l), samples from 3 patients were tested for the mutation by the mispairing PCR-RFLP
method (2).
Cases #90 and #28095 were found to contain normal and mutant mtDNA in homoplasmic form respectively (1). This finding was confirmed by the mispairing PCR-RFLP method (2). However, there was disagreement in case #3925 regarding the presence of the mutation (2). Because of the absence of the mutation in the 15 AD patients in their study, these workers concluded that this mutation is not AD specific (2). mtDNA
in patient 3925 was heteroplasmic and it contained about equal amount of
normal and mutant forms (1). detect the point mutation
The inability of the mispairing PCR-RFLP
technique to
in heteroplasmic mtDNAs seems to indicate an allele-specific
* To whom corresponce should be addressed. Abbreviations: AD, Alzheimer’s disease; ND2, subunit 2 of rnitochondrial NADH dehydrogenase-ubiquinone oxidoreductaxe (EC 1.6.99.3); PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SNuPE, single nucleotide primer extension. 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
1202
Vol.
189, No. 2, 1992
amplification
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of the normal form in the mispairing PCR (4). To test whether this was the
case, we amplified the target region by using the mispairing (2) and the pairing oligomers (1) as the primers in PCR. The resultant products were tested for the point mutation by single nucleotide primer extention (SNuPE) as described previously (1). The results of the present study confirm our previous finding (1). In addition, we detected a G to T base substitution at nt 5460 (5) in 8 of the 15 AD samples (2) and found that the mtDNA of the 8 patients was heteroplasmic.
This finding explains the difference in the data reported by
the two laboratories (1,2).
MATERIALS
AND METHODS
Total DNA extracted from 15 AD patients, 5 normal individuals and 2 mtDNAs, 2122, as well as the mispairing primers (2) were kindly provided by Dr. Eric A. Schon of Columbia University.
Chemical reagents were obtained from the same sources reported
previously (1). The primers for PCR amplification
and for SNuPE were synthesized in this
Institute as reported (1). Amplification
of the target region by PCR was done as described (1). The pairing
primers ND2-001 and ND2-003 generated a fragment of 350 bp (1) whereas the mispairing primers produced
a fragment
of 214 bp (2).
The PCR products were purified
by
electrophoresis in a 1.2% agarose gel and were used as the template for SNuPE as described (1).
RESULTS Since the apparent difference in SNuPE (1) and the mispairing PCR-RFLP methods (2) lies in the production of DNA by using different primers in PCR, it seems possible that the latter method preferentially population.
amplified the normal form of mtDNA in a heteroplasmic
To test whether this was the case, the 350 bp and 214 bp DNA preparations
(Fig 1A) were assayed for the presence of the point mutation in codon 331 of the ND2 gene by SNuPE. The results are shown in Figure 1. In panel B, it is seen that DNA generated from sample 22 (2) which was extracted from case #3925 (I), with the pairing primers ND2001 and ND2-003 (1) consisted of 2 forms of mtDNA.
One form contained the normal
codon CCC and the other TCC (22-P) confirming the previous report (1). In contrast to sample 22-P, only GCC, the normal codon, was detected in the DNA produced with the mispairing primers (22-M).
The 214 bp DNA samples #16 through #20 in panel B were
produced by the mispairing primers and no mutation was detected in these samples. On the 1203
Vol.
189, No. 2, 1992
A
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
P F) Pairing
P (Rev)
f-35o
bpf-------------)l
I k------,214 5160 Mispairing
bp--Jb4Q6 5498 MP (Rev)
5262 MP F) B
22-P --/I---II
22-M
16
17
16
19
,ssl"ma &,a &@_
--_----_--__-_.-_~~~--_ GATGATGATGATGATGATGAT
C
r---r
16
17
20
18
19
20
I
___-_----------
GATGATGATGATGAT
Figure 1. Detection of a point mutation by SNuPE. The target region in mtDNA was amplified by PCR as described (1) by using the mispairing (B) and pairing (C) oligomers as the primers. For detail regarding the primers, see references 1 and 2. For SNuPE, 3 sets of reaction were set up. Each reaction mixture contained 20 k 1incubation buffer and 1 unit Taq DNA polymerase (Boehringer Mannheim), 50 ng DNA template, 1 pmole extension primer and 1 p Ci of a [nP]dNTP (3000 Ci/mmole, Amersham). The reaction was performed in an automatic thermocycler (Coy) for 2 min each at 96oC, 55oC and 72oC. Equal volumes of the reaction mixture and a stop solution (95% formamide, 5 mM EDTA, 0.05% bromophenol blue and xylene cyanol) were then mixed and heated at 96oC for 2 min; 5 @1 of the mixture was loaded onto a gel (11 x 14 cm) containing 3% NuSiev (FMC) and 1% agarose. Electrophoresis was run at 200 V at room temperature until bromophenol blue reached l/4 from the bottom of the gel. Autoradiography was performed on Kodak AR Xray film at -7ooC for 4 h. A. A schematic diagram shows the locations of the primers and the sixes of PCR products. P (F) and P (Rev ) are pairing primers forward and reverse,repectively. MP = mispairing primer. The numbers indicate the position of nucleotides where the primers locate. B. DNA template was prepared in PCR with the mispairing primers except sample 22-P which was produced with the pairing primers. The spots show the incorporation of the indicated nucleotide onto the 3’-end of the extension primer. The numbers on the top of the Figure are sample numbers corresponding to those in Table 1. C. DNA template was produced in PCR with the pairing primers.
other hand, when the 350 bp sample #16 generated by the pairing primers was tested for the mutation, a G to T change was detected as shown in panel C. The G to T transversion was also detected in 7 other AD samples as summarized in Table 1. It is noted that the mtDNA
in the 8 AD patients is heteroplasmic and the TCC mutant represents a minor
fraction. 1204
Vol.
189,
No.
2,
1992
BIOCHEMICAL
TABLE 1. NUCLEOTIDE *Sample
Age
1
AND
BIOPHYSICAL
RESEARCH
COMMUNICA9ONS
COMPOSITION OF CODON 331 OF THE ND2 GENE Diagnosis
Codon 331
AD
CCC/KC
aa AIafSer
2
76
AD
ND
3
90
AD
GCC/TCC
Ala/.%x
4
64
Normal
GCC
Ala
5
78
AD
GCC
Ala
6
63
AD
GCC
Ala
7
63
AD
GCC/TCC
AIa/Ser
8
68
Normal
GCC
Ala
9
81
AD
GCC/TCC
AIa/Ser
10
64
Normal
GCC
Ala
11
88
AD
GCC
Ala
12
70
AD
GCC/TCC
AlaJSer
13
59
AD
GCCflCC
AIa/Ser
14
61
Normal
GCC
Ala
15
87
AD
GCC/TCC
AIa/Ser
16
93
AD
GCC/TCC
AIa/Ser
17
68
AD
GCC
Ala Ala
18
78
AD
GCC
19
88
AD
GCC
Ala
20
75
Normal
GCC
Ala
21
79
AD
GCC
Ala
CCC/l-CC 22 76 AD *r I he sample was numbered accordmg to reference 2. ND= Not done.
AIa/Ser
DISCUSSION The present study confirms our previous report (1) that mtDNA
from patient 3925 consists of normal and mutant forms, and shows that little, if any, of the mutant mtDNA was produced in PCR by using the mispairing oligomers as the primers (2). The mechanism is not understood.
It is possible, however, that the failure of Taq DNA polymerase to amplify
the mutant molecule is due to allele-specific PCR (4) in which a base-pair mismatch at the 3’-end of a primer can reduce the thermal stability of the primer-template
complex. From
the present observation, we conclude that the SNuPE method (1) is more reliable than the mispairing PCR-RFLP
(2) in detecting a point mutation in heteroplasmic mtDNAs. 1205
avot. 489,
No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The G to T transversion was detected at the first position of codon 331 of the ND2 gene in 8 of 15 AD DNAs (Table 1). mtDNA in these patients was heteroplasmic and the ratio of normal to mutant form was about 91 (data not shown). Whether the level of this change in coding sequence significantly affects the catalytic activity of complex I remains to be elucidated.
The study detected the mutation in about 50% of AD patients and the
change was a G to T transversion.
This pattern is similar to that reported previously (1).
The data suggest that mutations may occur in other mitochondrial genes, one of which could be the gene coding for cytochrome oxidase (6).
This is consistent with the finding of
multigene mutations in other degenerative diseases (7). It is believed that several factors are involved in Alzheimer’s disease (7). Thus, a defect in mitochondrial to be an important
function is likely
contributing cause of the disease.
ACKNOWLEDGMENTS R.L was supported by an NIH grant PO1 AGO 4220 to Dr. Henry M. Wisniewski to whom we give thanks for his support This work was partially supported by New York State Office of Mental Retardation and Developmental Disabilities. We thank Dr. Eric A. Schon of Columbia University for providing us with the DNAs and the mispairing primers; Drs. David L. Miller, Richard I. Carp and Richard Rubenstein for reviewing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Lin, F.H., Lin, R., Wisniewski, H-M., Hwang, Y.W., Grundke-Iqbal, I., HealyLouie, G. and Iqbal, K (1992) Biochem. Biophys. Res. Comm 182, 238246. Petruzzella, V., Chen, X. and Schon, E.A (1992) B&hem. Biophys. Res. Comm 186, 491-497. Seibel, P., Degoul, F., Romero, N., Marsac, C. and Kadenbach, B. (1990) B&hem. Biophys. Res. Comm. 173,561-565. Amheim, N. and Erlich, H. (1992). Annu. Rev. B&hem. 61,131-156. Anderson, S., Bankier, AT., Barrell, B.S., deBruijn, M.H.L., Co&on, AR., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B-k, Sanger, F., Schreier, P.H., Smith, AJ.H., Staden, R. and Young, LG. (1981) Nature 290, 457-465. Parker, Jr., W.D., Filley, C.M. and Parks, J.K. (1990) Neurology, 40, 1302- 1303. Wallace, D.C. (1992) Annu. Rev. Biochem. 61, 1175-1212.
