Vol.
187,
No.
September
3, 1992
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1992
1551-1557
AN ND-6 MITOCHONDRIAL DNA MUTATION ASSOCIATED WITH LEBER HEREDITARY OPTIC NEUROPATHY Donald R. Johns*, Michael J. Neufeld,
and Raymond D. Park
Department of Neurology, Meyer 6-119, Johns Hopkins School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-7619 Received
August
13,
1992
SUMMARY: A mitochondrial DNA mutation at nucleotide position 14,484 was found in 14 independent probands with Leber hereditary optic neuropathy and in O/250 controls. The 14,484 mutation, which changes methionine-64 to valine in a conserved domain of the ND-6 gene, occurred in association with a mitochondrial DNA haplotype that includes the 13,708 secondary mutation in lo/14 probands. An associated mutation at nucleotide position 3,394, which changes conserved tyrosine-30 to histidine in the ND-1 gene, was observed in 5/14 probands positive for the 14,484 mutation, all of whom harbored the same mitochondrial DNA haplotype. Multiple mitochondrial DNA mutations may interact in the pathogenesis of Leber hereditary optic neuropathy and the 13,708 secondary mutation appears to play a central role in this process. 0 1992RcademlcPress. Inc.
Leber hereditary optic neuropathy (LHON) visual loss which predominantly
been identified
in multiple mutation
LHON
mutations
in mitochondrial
families to date, at nucleotide
gene) (3,4), and 15,257 (apocytochrome
DNA (mtDNA)
(2).
positions 11,778 (ND-4 b gene) (5,6). Another
was found at nucleotide position 4,160 (ND-l
large family with a maternally other mtDNA
mutation
mutations which appear to be proximate causative mutations have
gene) (2), 3,460 (ND-l primary mtDNA
inherited form of acute
affects young men (1). LHON was the first human disease
to be linked conclusively to an inherited Three primary mtDNA
is a maternally
inherited
gene) in a single
disease that included optic atrophy (7). Several
have been found that appear to play secondary roles in LHON,
including those at nucleotide positions 4,216 (ND-l gene) (8), 4,917 (ND-2 gene) (8), 5,244 (ND-2 gene) (6), 13,708 (ND-5 gene) (5,6,8), and 15,812 (apocytochrome
b gene) (5,6). We
*To whom correspondence should be addressed. The abbreviations used are: LHON = Leber hereditary optic neuropathy; mtDNA = mitochondrial DNA; Complex I = mitochondrial NADH:ubiquinone oxidoreductase (EC 1.6.99.3); Complex III = ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2); ND-l, ND-2, ND-3, ND-4, ND4L, ND-5, ND-6 = subunits of NADH dehydrogenase.
1551
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0006-29 I x/92 $4.00 by Academic Press. Inc. in any form resrvvrd.
Vol.
187,
No.
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
proposed that some of these mutations might be synergistic in their action with the primary mutations or that they were linked with another primary pathogenetic The present study was designed to discover pathogenetically mutations
in LHON
mutations.
probands who did not harbor any of the three known primary
We found an mtDNA
position 14,484 in 14 independent tightly linked with the LHON 13,708 secondary mtDNA
missense mutation LHON
probands.
mutation,
simultaneous
mtDNA
of the 13,708 mutation
The 14,484 mutation
and was not found in controls.
appears to be
in association with the The 14,484 mutation
haplotype that included five m&sense mutations in Five families also harbored a missense
Complex I and III genes in 10 of 14 families. in the ND-l
in the ND-6 gene at nucleotide
phenotype, was found predominantly
occurred as a component of an mtDNA mutation
mutation (5,8). significant mtDNA
gene at nucleotide
position
3,394.
The concept of multiple,
mutations interacting in LHON is strengthened, and the pivotal role in LHON
is given further credence. METHODS
A. Patient Selection. Prototypical LHON probands who harbored the 13,708 secondary mutation, but who did not have any of the three primary mtDNA mutations, were chosen for the initial detailed sequence analysis. After the 14,484 mutation was discovered, specific sequence analysis of this region was carried out on the remaining LHON probands and on neurological disease and normal controls. B. mtDNA Sequence Analysis. Extensive regions of multiple Complex I and III genes (ND1, ND-2, ND-3, ND-4, ND4L, ND-5, apocytochrome b) had previously been sequenced in the prototypical probands. A detailed sequence analysis of the ND-6 gene was then undertaken, by using the amplification and sequencing primers for PCR given in Table 1. The PCR products were purified in an ultrafiltration microconcentrator (Amicon, Beverly, MA) and sequenced directly with 32P-end labelled primers as described previously (9). The 14,484 mutation was assessed after PCR amplification with forward primer 14,200-218 and reverse primer 14,538-519 and sequenced with forward primer 14,389-410 (Table 1). C. mtDNA HaulotvDe Analvsis. The 14,484 mutation was found predominantly in association with an mtDNA haplotype which consisted of four missense mutations and two
Table
1
PCR PRIMERS FOR ND-6 SEQUENCING Forward 14,200-218 14,389-410
Primers Reverse /3'-5') Primers TCAACCAGTAACTACTACT 14,205-186 GGTTGACCATTGTTTGTTGG CCCCACTAAMCACTCACCAAG 14,470-452 ATATACTACAGCGATGGCT 14,538-519 TTTGGGGGAGGTTATATGGG 14.686-663 CCGTGCGAGAATAATGATGTATGC
C5 I -3 ’ )
The forward primers are complementary to the H-strand and the reverse primers are complementary to the L-strand of human mtDNA. All primer sequences are given in the 5'-to-3' direction and are numbered according to the Cambridge human mtDNA sequence (10). 7552
Vol.
187,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
silent mutations. The six components of the mtDNA haplotype were assessed as follows: nucleotide positions 4,216 (ND-l gene) (Nla III site gain (8)); 10,398 (ND-3 gene) (Dde I site gain); 13,708 (ND-5 gene) (BstN I and Fnu4H I site losses (8)); 15,452 (cytochrome b gene) (direct sequence analysis); 16,065 (D-loop) (Hinf I site loss); and 16,517 (D-loop) (Hae III site loss). An associated mtDNA mutation at position 3,394 (ND-l gene) was assessed by the gain of an Hae III site.
A. ND-6 Mutations.
A missense T-to-C transition at nucleotide position 14,484, which
changes the 64th amino acid residue from methionine independent
to valine (M64V), was found in 14
LHON probands. A silent T-to-C transition at nucleotide position 14,485 was
found in multiple
samples. Three sequence differences were noted consistently at nucleotide
positions 14,199 (C-to-A), 14,272 (G-to-C), and 14,368 (G-to-C), which probably represent errors in the original Cambridge B. mtDNA mtDNA
HaDIotvne.
sequence (10).
The 14,484 mutation occurred in association with a six-component
haplotype in 10 of 14 families:
ND-l
gene, 4,216 (Y304H);
ND-3 gene, 10,398
(T114A); ND-5 gene, 13,708 (A458T); cytochrome b gene, 15,452 (L2361); D-loop, 16,065; and D-loop, 16,517. An additional
missense mutation
at nucleotide position 3,394 (ND-l
gene, Y30H) was found in 5 of 14 probands, all of whom harbored the core mtDNA haplotype. C. Occurrence with known nrimarv LHON 2 of 13 LHON
mutations. The 14,484 mutation
probands with the 15.257 mutation,
Table ASSOCIATION POPULATION
OF THE 13,708
FREOUENCY 13.708+ 24/480
Controls
was found in
but was not found in association with
2 MUTATION
WITH
RELATIVE
ENRICHMENT
(5%)“
LHON p-VALUE
--
--
LHON:
11,778+
14/77
(18%)
3.6x
0.0002
LHON :
3,460+
3/13
(23%)
4.6x
0.029
LHON:
15,257+
12/13
(92%)
18.4x
10.0001
LHON:
14,484+
10/14
(71%)
14.2x
187,
No.
September
3, 1992
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1992
1551-1557
AN ND-6 MITOCHONDRIAL DNA MUTATION ASSOCIATED WITH LEBER HEREDITARY OPTIC NEUROPATHY Donald R. Johns*, Michael J. Neufeld,
and Raymond D. Park
Department of Neurology, Meyer 6-119, Johns Hopkins School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-7619 Received
August
13,
1992
SUMMARY: A mitochondrial DNA mutation at nucleotide position 14,484 was found in 14 independent probands with Leber hereditary optic neuropathy and in O/250 controls. The 14,484 mutation, which changes methionine-64 to valine in a conserved domain of the ND-6 gene, occurred in association with a mitochondrial DNA haplotype that includes the 13,708 secondary mutation in lo/14 probands. An associated mutation at nucleotide position 3,394, which changes conserved tyrosine-30 to histidine in the ND-1 gene, was observed in 5/14 probands positive for the 14,484 mutation, all of whom harbored the same mitochondrial DNA haplotype. Multiple mitochondrial DNA mutations may interact in the pathogenesis of Leber hereditary optic neuropathy and the 13,708 secondary mutation appears to play a central role in this process. 0 1992RcademlcPress. Inc.
Leber hereditary optic neuropathy (LHON) visual loss which predominantly
been identified
in multiple mutation
LHON
mutations
in mitochondrial
families to date, at nucleotide
gene) (3,4), and 15,257 (apocytochrome
DNA (mtDNA)
(2).
positions 11,778 (ND-4 b gene) (5,6). Another
was found at nucleotide position 4,160 (ND-l
large family with a maternally other mtDNA
mutation
mutations which appear to be proximate causative mutations have
gene) (2), 3,460 (ND-l primary mtDNA
inherited form of acute
affects young men (1). LHON was the first human disease
to be linked conclusively to an inherited Three primary mtDNA
is a maternally
inherited
gene) in a single
disease that included optic atrophy (7). Several
have been found that appear to play secondary roles in LHON,
including those at nucleotide positions 4,216 (ND-l gene) (8), 4,917 (ND-2 gene) (8), 5,244 (ND-2 gene) (6), 13,708 (ND-5 gene) (5,6,8), and 15,812 (apocytochrome
b gene) (5,6). We
*To whom correspondence should be addressed. The abbreviations used are: LHON = Leber hereditary optic neuropathy; mtDNA = mitochondrial DNA; Complex I = mitochondrial NADH:ubiquinone oxidoreductase (EC 1.6.99.3); Complex III = ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2); ND-l, ND-2, ND-3, ND-4, ND4L, ND-5, ND-6 = subunits of NADH dehydrogenase.
1551
Copyright 0 1992 A// rights oj. reproducrion
0006-29 I x/92 $4.00 by Academic Press. Inc. in any form resrvvrd.
Vol.
187,
No.
BIOCHEMICAL
3, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
proposed that some of these mutations might be synergistic in their action with the primary mutations or that they were linked with another primary pathogenetic The present study was designed to discover pathogenetically mutations
in LHON
mutations.
probands who did not harbor any of the three known primary
We found an mtDNA
position 14,484 in 14 independent tightly linked with the LHON 13,708 secondary mtDNA
missense mutation LHON
probands.
mutation,
simultaneous
mtDNA
of the 13,708 mutation
The 14,484 mutation
and was not found in controls.
appears to be
in association with the The 14,484 mutation
haplotype that included five m&sense mutations in Five families also harbored a missense
Complex I and III genes in 10 of 14 families. in the ND-l
in the ND-6 gene at nucleotide
phenotype, was found predominantly
occurred as a component of an mtDNA mutation
mutation (5,8). significant mtDNA
gene at nucleotide
position
3,394.
The concept of multiple,
mutations interacting in LHON is strengthened, and the pivotal role in LHON
is given further credence. METHODS
A. Patient Selection. Prototypical LHON probands who harbored the 13,708 secondary mutation, but who did not have any of the three primary mtDNA mutations, were chosen for the initial detailed sequence analysis. After the 14,484 mutation was discovered, specific sequence analysis of this region was carried out on the remaining LHON probands and on neurological disease and normal controls. B. mtDNA Sequence Analysis. Extensive regions of multiple Complex I and III genes (ND1, ND-2, ND-3, ND-4, ND4L, ND-5, apocytochrome b) had previously been sequenced in the prototypical probands. A detailed sequence analysis of the ND-6 gene was then undertaken, by using the amplification and sequencing primers for PCR given in Table 1. The PCR products were purified in an ultrafiltration microconcentrator (Amicon, Beverly, MA) and sequenced directly with 32P-end labelled primers as described previously (9). The 14,484 mutation was assessed after PCR amplification with forward primer 14,200-218 and reverse primer 14,538-519 and sequenced with forward primer 14,389-410 (Table 1). C. mtDNA HaulotvDe Analvsis. The 14,484 mutation was found predominantly in association with an mtDNA haplotype which consisted of four missense mutations and two
Table
1
PCR PRIMERS FOR ND-6 SEQUENCING Forward 14,200-218 14,389-410
Primers Reverse /3'-5') Primers TCAACCAGTAACTACTACT 14,205-186 GGTTGACCATTGTTTGTTGG CCCCACTAAMCACTCACCAAG 14,470-452 ATATACTACAGCGATGGCT 14,538-519 TTTGGGGGAGGTTATATGGG 14.686-663 CCGTGCGAGAATAATGATGTATGC
C5 I -3 ’ )
The forward primers are complementary to the H-strand and the reverse primers are complementary to the L-strand of human mtDNA. All primer sequences are given in the 5'-to-3' direction and are numbered according to the Cambridge human mtDNA sequence (10). 7552
Vol.
187,
No.
3, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
silent mutations. The six components of the mtDNA haplotype were assessed as follows: nucleotide positions 4,216 (ND-l gene) (Nla III site gain (8)); 10,398 (ND-3 gene) (Dde I site gain); 13,708 (ND-5 gene) (BstN I and Fnu4H I site losses (8)); 15,452 (cytochrome b gene) (direct sequence analysis); 16,065 (D-loop) (Hinf I site loss); and 16,517 (D-loop) (Hae III site loss). An associated mtDNA mutation at position 3,394 (ND-l gene) was assessed by the gain of an Hae III site.
A. ND-6 Mutations.
A missense T-to-C transition at nucleotide position 14,484, which
changes the 64th amino acid residue from methionine independent
to valine (M64V), was found in 14
LHON probands. A silent T-to-C transition at nucleotide position 14,485 was
found in multiple
samples. Three sequence differences were noted consistently at nucleotide
positions 14,199 (C-to-A), 14,272 (G-to-C), and 14,368 (G-to-C), which probably represent errors in the original Cambridge B. mtDNA mtDNA
HaDIotvne.
sequence (10).
The 14,484 mutation occurred in association with a six-component
haplotype in 10 of 14 families:
ND-l
gene, 4,216 (Y304H);
ND-3 gene, 10,398
(T114A); ND-5 gene, 13,708 (A458T); cytochrome b gene, 15,452 (L2361); D-loop, 16,065; and D-loop, 16,517. An additional
missense mutation
at nucleotide position 3,394 (ND-l
gene, Y30H) was found in 5 of 14 probands, all of whom harbored the core mtDNA haplotype. C. Occurrence with known nrimarv LHON 2 of 13 LHON
mutations. The 14,484 mutation
probands with the 15.257 mutation,
Table ASSOCIATION POPULATION
OF THE 13,708
FREOUENCY 13.708+ 24/480
Controls
was found in
but was not found in association with
2 MUTATION
WITH
RELATIVE
ENRICHMENT
(5%)“
LHON p-VALUE
--
--
LHON:
11,778+
14/77
(18%)
3.6x
0.0002
LHON :
3,460+
3/13
(23%)
4.6x
0.029
LHON:
15,257+
12/13
(92%)
18.4x
10.0001
LHON:
14,484+
10/14
(71%)
14.2x
