Vol.
170,
August
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1990
994-997
RAPID SHIFT IN GENOTYPE OF HUMAN MITOCHONDRIAL DNA IN A FAMILY WITH LEBER'S HEREDITARY OPTIC NEUROPATHY E.M. Bleeker-Wagemakers2, N.J. Ponnel, M.J. Van A. Westerveld3, C. Van den Bogert4 and H.F. Tabakl
P.A. Bolhuisl", Schooneveld2, 'Academic
Medical
2The Netherlands 3Academic 4E.C.
Center, Department of Neurology, Amsterdam, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands
1105 AZ 1100 AZ
Medical Center, Department of Anthropogenetics, 1105 AZ Amsterdam, The Netherlands
Slater
Institute for Biochemical Research, Amsterdam, The Netherlands
1105 AZ
Received June 26, 1990 Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis. 0 1990Academic Press,Inc.
Leber's hereditary optic neuropathy (LHON, McKusick No. 30890) is a maternally inherited disorder. Most patients suffer from acute, bilateral and severe loss of vision. The peak age of onset is at about 20 years. Peripapillary microangiopathy is often present as a characteristic finding. mutation in mitochondrial
Wallace et al (5) discovered a point DNA (mtDNA) from several families
affected by LHON. The mutation results in the substitution highly conserved arginine into histidine in subunit ND4 of complex I (NADH CoQ dehydrogenase) of the mitochondrial *To whom correspondence Abbreviations: mitochondrial.
should
LHON, Leber's
be addressed. hereditary
0006-291X/90$1.50 Copyright All rights
0 1990 by Academic Press, Inc. in any form reserved.
of reproduction
of a
994
optic
neuropathy;
mt,
Vol.
170,
No.
respiratory families
3, 1990
with
found i.e. Demonstration atrophy
of
discriminate investigation yield
AND
chain. The mutation is LHON (S-7). In a few
both
can
BIOCHEMICAL
be important
in
present families,
mutated and normal mtDNA the mutation in a patient for
the
LHON from multiple of the mutation
insight
BIOPHYSICAL
the
genotypes
of
MATERIALS
AND METHODS
differential
sclerosis. in families
prevalence
and in
RESEARCH
in
some but heteroplasmy
were present with optic
segregation
not
all was
(7). nerve
diagnosis Moreover, affected
the
COMMUNICATIONS
e.g.
to
by LHON can of
mutated
mtDNA.
12 Dutch families affected by LHON were investigated for the presence of the mutation and the occurrence of heteroplasmy. DNA was isolated from white blood cells as described (8) and samples of 1 ug were used for amplification of the mtDNA segment 11501 Primers corresponding to sequences 11501 to 11520 and to 11900. 11881 to 11900 were obtained from Eurogentec (Liege, Belgium). The polymerase chain reaction was carried out in 30 cycles (1 min 95', 2 min 55', 3 min 72'). Samples of 250 ng amplified DNA were digested with Sfa Nl as recommended by the supplier (Biolabsll. Agarose gel electrophoresis and ethidium bromide staining resulted in patterns as shown in Fig. 1A for a control The presence of the and two patients from different families. mutation was confirmed by direct sequencing of the fragment 1150 to 11900 from two patients and by Southern blots of total DNA digested with SfaNl from two other patients. Percentages of mutated and normal mtDNA were determined in case of heteroplasmy from calibration curves containing samples of intact and SfaNl digested normal mtDNA, diluted to 80, 60, 40, 20 and 10 per cent The yield of segment 11501 to of the initial concentration, 11900 in the polymerase chain reaction was reproducible, and similar for normal and mutated mtDNA.
A
z;r,
6
C
;;a;;ectrophoresis
DEFGHI
of mtDNA fragments. Top: +, digested B, LHON patient with digested; A, control; mtDNA muta;ed'at nucleotide position 11778 resulting in loss of a SfaNl site; C, LHON patient without this mutation; D, markers; E, blank (polymerase chain reaction); F, grandmother; G, mother; H, son 1; I, son 2. Left: length of fragments (base pairs). not
995
Vol.
170,
No.
RESULTS Normal was
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION digestion
present
families
in
patterns
were
found
the
5 other
families
heteroplasmy
was
detected.
in
7 families.
and In
in
2 out
one
family
of
The
mutation
these
5
with
heteroplasmy, grandmother, shown in Fig.
the percentage of mutated mtDNA was 60 in the 55 in the mother and more than 95 in two sons, as 1B. The grandmother and mother had no signs of the two sons suffered from severe loss neuropathy, however,
optic of vision
(visual
The with
presence earlier
out
of
acuity
0.125/0.02
of the mutation data. Wallace 11 families, Vilkki
and Holt
et
Heteroplasmy (6). et al.
al.
(7)
was Holt
in
and
in et et
4 out
not reported et al. (7)
5 out
al al.
0.02/0.02).
of
12 families
mtDNA
affected Holt et in
females
the disorder, reflect the
sons to al. (7) could
of
provided populations
Our data, however, show human mtDNA are possible: increased Fig. 1B. This white
from
observation blood
55 in
the
implies cells
from
68 in a cousin suggested that the of
to
(7). the
estimate
data obtained mtDNA in ova.
that very rapid the percentage mother
that females
line 9
8 families.
by Wallace et al. observed heteroplasmy
be used
in
(5) observed the mutation in (6) in 10 out of 19 families,
families with mutated mtDNA. The percentage nucleotide position 11778 in the mtDNA from was higher than 95 in 2 families and varied >95 in finding
is
to
the has
in
(5)
4
of DNA mutated at white blood cells in one family from Based on this percentage of
mutated
the
transmit
chance
to
peripheral
changes in of mutated
> 95 in
and Vilkki in 3 of the
her
sons,
percentage
of
limited
predicting
blood
genotype DNA as
of
shown
in
mtDNA
in
mutated
value.
The rapid shift of genotype of mtDNA demonstrated here in humans is in line with studies on bovine mitochondria (9-11). Analysis of mtDNA in Holstein cows showed that rapid segregation of populations of mtDNA may occur. This finding is surprising in view of the large number of mitochondria normally present in mamm>lian cells. However, the population passes a developmental bottleneck during
of mtDNA molecules bovine oogenesis
(9,
10)
and therefore a highly asymmetric distribution of mtDNA can be transmitted to the progeny. Segregation to homoplasmic genotypes of bovine mtDNA could occur within 2 or 3 generations (11). The
Vol.
170,
present mtDNA.
No.
BIOCHEMICAL
3, 1990
data
show
that
a
similar
AND
BIOPHYSICAL
phenomenon
RESEARCH
applies
COMMUNICATIONS
to
human
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Albrecht v. Graefes. Arch. Ophthalmol. 17, Leber, T. (1871). 249-291. Van Senus, A.H.C. (1963). Dot. Ophthalmol. 17, l-62. Went, L.N. (1972). In: Vinken, P.J., Bruyn, G.W., eds. Handbook of Clinical Neurology, Vol. 13. Amsterdam: North Holland Publishing. pp. 94-110. Nikoskelainen, E.K., Savontaus, M.L., Wanne, O.P., Katila, M. J. and Nummelin, K.U. (1987). Arch. Ophthalmol. 105, 665671. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T. G. , Lezza, A.M.S., Elsas, L.J. and Nikoskelainen, E.K. (1988). Science 242, 1427-1430. Vilkki, S., Savontaus, M. L. and Nikoskilainen, E.K. (1989) Am. J. Hum. Genet. 45, 206-211. Holt, I.J., Miller, D.H. and Harding, E.A. (1989) J. Med. Genet. 26, 739-743. Bowtell, D.D.L. (1987) Anal. Biochem. 162, 463-465. Hauswirth, W.W. and Laipis, P.J. (1985) In: Achievements and perspectives of mitochondrial research, E. Quagliarello, ed. Vol. II, Elsevier Science Publishers pp. 49-59. Amsterdam, Laipis, P.J., Van de Walle, M.J. and Hauswirth, W.W. (1988) Proc. Natl. Acad. Sci. USA 85, 8107-8110. Ashley, M.V., Laipis, P.J. and Hauswirth, W.W. (1989) Nucl. Acid. Res. 17, 7325-7331.
997
170,
August
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1990
994-997
RAPID SHIFT IN GENOTYPE OF HUMAN MITOCHONDRIAL DNA IN A FAMILY WITH LEBER'S HEREDITARY OPTIC NEUROPATHY E.M. Bleeker-Wagemakers2, N.J. Ponnel, M.J. Van A. Westerveld3, C. Van den Bogert4 and H.F. Tabakl
P.A. Bolhuisl", Schooneveld2, 'Academic
Medical
2The Netherlands 3Academic 4E.C.
Center, Department of Neurology, Amsterdam, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands
1105 AZ 1100 AZ
Medical Center, Department of Anthropogenetics, 1105 AZ Amsterdam, The Netherlands
Slater
Institute for Biochemical Research, Amsterdam, The Netherlands
1105 AZ
Received June 26, 1990 Mitochondrial DNA isolated from white blood cells was investigated in families suffering from Leber's hereditary optic neuropathy. A recently described mutation at nucleotide position 11778 was present in 5 out of 12 families and heteroplasmic mitochondrial DNA was observed in 2 of these 5 families. A rapid shift in genotype was found in one of the families with heteroplasmy: the grandmother had 60 percent mitochondrial DNA mutated at nucleotide position 11778, the mother 55 percent, and the two sons at least 95 percent. These data indicate that the number of mitochondrial DNA molecules transmitted to the progeny passes a developmental bottleneck, as previously proposed to occur in bovine oogenesis. 0 1990Academic Press,Inc.
Leber's hereditary optic neuropathy (LHON, McKusick No. 30890) is a maternally inherited disorder. Most patients suffer from acute, bilateral and severe loss of vision. The peak age of onset is at about 20 years. Peripapillary microangiopathy is often present as a characteristic finding. mutation in mitochondrial
Wallace et al (5) discovered a point DNA (mtDNA) from several families
affected by LHON. The mutation results in the substitution highly conserved arginine into histidine in subunit ND4 of complex I (NADH CoQ dehydrogenase) of the mitochondrial *To whom correspondence Abbreviations: mitochondrial.
should
LHON, Leber's
be addressed. hereditary
0006-291X/90$1.50 Copyright All rights
0 1990 by Academic Press, Inc. in any form reserved.
of reproduction
of a
994
optic
neuropathy;
mt,
Vol.
170,
No.
respiratory families
3, 1990
with
found i.e. Demonstration atrophy
of
discriminate investigation yield
AND
chain. The mutation is LHON (S-7). In a few
both
can
BIOCHEMICAL
be important
in
present families,
mutated and normal mtDNA the mutation in a patient for
the
LHON from multiple of the mutation
insight
BIOPHYSICAL
the
genotypes
of
MATERIALS
AND METHODS
differential
sclerosis. in families
prevalence
and in
RESEARCH
in
some but heteroplasmy
were present with optic
segregation
not
all was
(7). nerve
diagnosis Moreover, affected
the
COMMUNICATIONS
e.g.
to
by LHON can of
mutated
mtDNA.
12 Dutch families affected by LHON were investigated for the presence of the mutation and the occurrence of heteroplasmy. DNA was isolated from white blood cells as described (8) and samples of 1 ug were used for amplification of the mtDNA segment 11501 Primers corresponding to sequences 11501 to 11520 and to 11900. 11881 to 11900 were obtained from Eurogentec (Liege, Belgium). The polymerase chain reaction was carried out in 30 cycles (1 min 95', 2 min 55', 3 min 72'). Samples of 250 ng amplified DNA were digested with Sfa Nl as recommended by the supplier (Biolabsll. Agarose gel electrophoresis and ethidium bromide staining resulted in patterns as shown in Fig. 1A for a control The presence of the and two patients from different families. mutation was confirmed by direct sequencing of the fragment 1150 to 11900 from two patients and by Southern blots of total DNA digested with SfaNl from two other patients. Percentages of mutated and normal mtDNA were determined in case of heteroplasmy from calibration curves containing samples of intact and SfaNl digested normal mtDNA, diluted to 80, 60, 40, 20 and 10 per cent The yield of segment 11501 to of the initial concentration, 11900 in the polymerase chain reaction was reproducible, and similar for normal and mutated mtDNA.
A
z;r,
6
C
;;a;;ectrophoresis
DEFGHI
of mtDNA fragments. Top: +, digested B, LHON patient with digested; A, control; mtDNA muta;ed'at nucleotide position 11778 resulting in loss of a SfaNl site; C, LHON patient without this mutation; D, markers; E, blank (polymerase chain reaction); F, grandmother; G, mother; H, son 1; I, son 2. Left: length of fragments (base pairs). not
995
Vol.
170,
No.
RESULTS Normal was
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION digestion
present
families
in
patterns
were
found
the
5 other
families
heteroplasmy
was
detected.
in
7 families.
and In
in
2 out
one
family
of
The
mutation
these
5
with
heteroplasmy, grandmother, shown in Fig.
the percentage of mutated mtDNA was 60 in the 55 in the mother and more than 95 in two sons, as 1B. The grandmother and mother had no signs of the two sons suffered from severe loss neuropathy, however,
optic of vision
(visual
The with
presence earlier
out
of
acuity
0.125/0.02
of the mutation data. Wallace 11 families, Vilkki
and Holt
et
Heteroplasmy (6). et al.
al.
(7)
was Holt
in
and
in et et
4 out
not reported et al. (7)
5 out
al al.
0.02/0.02).
of
12 families
mtDNA
affected Holt et in
females
the disorder, reflect the
sons to al. (7) could
of
provided populations
Our data, however, show human mtDNA are possible: increased Fig. 1B. This white
from
observation blood
55 in
the
implies cells
from
68 in a cousin suggested that the of
to
(7). the
estimate
data obtained mtDNA in ova.
that very rapid the percentage mother
that females
line 9
8 families.
by Wallace et al. observed heteroplasmy
be used
in
(5) observed the mutation in (6) in 10 out of 19 families,
families with mutated mtDNA. The percentage nucleotide position 11778 in the mtDNA from was higher than 95 in 2 families and varied >95 in finding
is
to
the has
in
(5)
4
of DNA mutated at white blood cells in one family from Based on this percentage of
mutated
the
transmit
chance
to
peripheral
changes in of mutated
> 95 in
and Vilkki in 3 of the
her
sons,
percentage
of
limited
predicting
blood
genotype DNA as
of
shown
in
mtDNA
in
mutated
value.
The rapid shift of genotype of mtDNA demonstrated here in humans is in line with studies on bovine mitochondria (9-11). Analysis of mtDNA in Holstein cows showed that rapid segregation of populations of mtDNA may occur. This finding is surprising in view of the large number of mitochondria normally present in mamm>lian cells. However, the population passes a developmental bottleneck during
of mtDNA molecules bovine oogenesis
(9,
10)
and therefore a highly asymmetric distribution of mtDNA can be transmitted to the progeny. Segregation to homoplasmic genotypes of bovine mtDNA could occur within 2 or 3 generations (11). The
Vol.
170,
present mtDNA.
No.
BIOCHEMICAL
3, 1990
data
show
that
a
similar
AND
BIOPHYSICAL
phenomenon
RESEARCH
applies
COMMUNICATIONS
to
human
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Albrecht v. Graefes. Arch. Ophthalmol. 17, Leber, T. (1871). 249-291. Van Senus, A.H.C. (1963). Dot. Ophthalmol. 17, l-62. Went, L.N. (1972). In: Vinken, P.J., Bruyn, G.W., eds. Handbook of Clinical Neurology, Vol. 13. Amsterdam: North Holland Publishing. pp. 94-110. Nikoskelainen, E.K., Savontaus, M.L., Wanne, O.P., Katila, M. J. and Nummelin, K.U. (1987). Arch. Ophthalmol. 105, 665671. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T. G. , Lezza, A.M.S., Elsas, L.J. and Nikoskelainen, E.K. (1988). Science 242, 1427-1430. Vilkki, S., Savontaus, M. L. and Nikoskilainen, E.K. (1989) Am. J. Hum. Genet. 45, 206-211. Holt, I.J., Miller, D.H. and Harding, E.A. (1989) J. Med. Genet. 26, 739-743. Bowtell, D.D.L. (1987) Anal. Biochem. 162, 463-465. Hauswirth, W.W. and Laipis, P.J. (1985) In: Achievements and perspectives of mitochondrial research, E. Quagliarello, ed. Vol. II, Elsevier Science Publishers pp. 49-59. Amsterdam, Laipis, P.J., Van de Walle, M.J. and Hauswirth, W.W. (1988) Proc. Natl. Acad. Sci. USA 85, 8107-8110. Ashley, M.V., Laipis, P.J. and Hauswirth, W.W. (1989) Nucl. Acid. Res. 17, 7325-7331.
997
