American Journal of Medical Genetics 42:173-179 (1992)
Mitochondrial DNA Mutation and Heteroplasmy in Type I Leber Hereditary Optic Neuropathy Danping Zhu, Effrosini P. Economou, Stylianos E. Antonarakis, and Irene H. Maumenee The Johns Hopkins Center for Hereditary Eye Diseases of the Wilmer Ophthalmological Institute (D.Z.,I.H.M.) and the Center for Medical Genetics, Department of Pediatrics (E.P.E., S.E.A.), The Johns Hopkins Medical Institutions, Baltimore, Maryland able for energy production and provides approximately 90% of the total adenosine triphosphate in humans [Wallace, 19861.The central nervous system is the most dependent tissue. Leber hereditary optic neuropathy fLHONf is a maternally inherited disorder characterized by acute or subacute bilateral loss of central vision, primarily in young males. The age-of-onset ranges from adolescence to late adulthood, with an average of 20-24 years [Nikoskelainen, 1984; McKusick, 1987; Wallace et al., 1988;Newman and Wallace, 19901. The maternal inheritance pattern suggests mitochondrial inheritance [Erickson, 1972; Giles et al., 1980; Egge and Wilson, 1983; Nikoskelainen et al., 19871. Recent studies have shown a G-A nucleotide substitution at 11778nt of the mitochondrial genome identified by Wallace et al., [1988], which converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene [Chomyn et al., 1985, 19861 and eliminated a SfaNI site [Wallace et al., 19881or created a Mae 111site [Coppinger et al., 1990; Stone et al., 19901. This point mutation was found in more than 60%of the families studied. [Wallaceet al., 1988;Holt et al., 1989;Hotta et al., 1989; Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989; Stone et al., 1990;Lott et al., 1990.1 All previously reported families except those reported by Holt et al. [19891 and Lott et al. [19901were homoplasmic. Heteroplasmy could theoretically arise during meiotic andlor mitotic divisions. We studied members from 4 families with LHON KEY WORDS: maternally inherited disorder, using SfaNI and Mae I11 digestion of a 201 base pair (bp) polymerase chain reaction, mtDNA fragment amplified by polymerase chain reacloss of central vision tion (PCR) encompassing the 11778nt mutation to verify the G+A 11778nt mutation and correlation of mtDNA heteroplasmy with the disease. All of the proINTRODUCTION bands had the G+A mutation and all individuals with Mitochondrial DNA (mtDNA) is an extranuclear this mutation showed heteroplasmy in their mtDNA; DNA exclusivelyinherited from the mother [Gileset al., i.e., they had both normal and mutant mitochondria at 19801. It encodes a set of proteins which are indispens- 11778nt. The severity of the disease appeared to correlate with the proportion of mutant DNA from platelets as judged by densitometry.
Leber hereditary optic neuropathy (LHON)is a maternally inherited disorder characterized by bilateral acute or subacute loss of central vision, primarily in young males. A &A single base mutation at 11778nt of the mitochondrial genome which eliminates a SfaNI restriction site [Wallace et al., 1988;Holt et al., 1989;Hotta et al., 1989;Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989;Stone et al., 1990; Lott et al., 1990.1 has been found in more than 60% of the families with LHON studied. We studied 25 persons from 4 families with LHON using SfaNI and Mae I11 digestion of a 201 base pair polymerase chain reaction (PCR)product encompassing the 11778ntmutation. The loss of the SfaNI site and the acquisition of a Mae I11 site at 11778nt were identified in all maternal relatives of the LHON families studied. The mutation was heteroplasmic in all affected individuals, female carriers, and males at-risk. The heteroplasmy of mitochondrial DNA (mtDNA)was also identified by direct DNA sequencing of PCR amplified mtDNA digested by SfaNI or Mae 111. It appears that the proportion of the mutant mtDNA correlates with the severity of the disease.
Received for publication November 16, 1990; revision received April 26, 1991. Address reprint requests to Danping Zhu, M.D., The Johns Hopkins Center for Hereditary Eye Diseases, Maumenee Building Room 321, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Md 21205.
0 1992 Wiley-Liss, Inc.
SUBJECTS AND METHODS Subjects Blood samples were drawn from 25 members of 4 multi-generation families with LHON, including 6 af-
174
Zhu et al.
Pedigree:
B
A
C
D
's w
J f 1
66 i
111
1
2
2
Jf3
3
IV 1
2
3Jf4
5
6
7
11
12
13
Fig. 1. Pedigrees of 4 multigenerational families with LHON. The ethic origins are A: Eastern Europe; B USA, NC; C: USA, WVA; D Canada. Roman numerals I, 11, 111, and IV number the generations. Numbers below the symbols are case numbers. Circles represent females; squares represent males. 0,O= normal subjects; m,0 = affected, 0 = normal carrier female; /1 = proband; M = DNA size marker.
fected persons (5 males and 1 female), 15 carriers, 2 males at-risk, and 2 normal control individuals (Fig. 1). All affected and obligatory carriers were examined at the Wilmar Eye Institute at the Johns Hopkins Hospital. The manifestations are shown in Table I. The onset ranged from 5-24 years. The follow-up period was from 2-10 years. Two of the 6 affected regained some vision. Affected male B-111-3 regained useful vision in the left eye from 41200 to 20130 20 months after onset. Affected male D-IV-4 regained vision in both eyes from 20/100 and count finger (CF) to 20125 at one year after onset. Other patients had persistent visual loss for periods of 9-11 years after onset.
Amplification of mtDNA One hundred nanograms of mitochondria1rich DNA isolated from platelets was amplified using the PCR in a 100 pl reaction mixture with 30 nM of each primer, 200 p M of each dNTP, and 2 u of Amplitaq (Taq I) DNA polymerase [Saiki et al., 19881. The premix of PCR reagents, primers, and dNTPs was treated under 254 nm short wave ultraviolet and 365 nM medium wave UV light for 20 minutes to prevent DNA contaminations [Kwok and Higuchi, 19891 and then the DNA template was added. The mixture was denatured for 3 minutes a t 94°C followedby 30 cycles of amplification (denatured at 94°C for one minute, annealing a t 59°C for one minute, extension at 72°C for 3 minutes) using a DNA thermal DNA Extraction cycler (Perkin-Elmer-Cetus, Norwalk, CT). The amplimtDNA was extracted from peripheral blood platelets fied DNA was purified using Centricon 30 (Amicon, [Giles et al., 19801 within 48 hours after the blood was Danvers, MA). drawn. Primers Nineteen and 23 base oligonucleotide primers [Wallace et al., 19881 were prepared by an Applied Biosystems DNA Synthesizer, and were used to amplify the 201 bp mtDNA fragment. These primers were: Primer A: 11673-5' CCCCC TGAAG CTTCA CCGG 3'-11691 Primer B: 11851-5' TGGGG GGTAA GGCGA GGTTA GCG 3'-11873
Restriction Fragment Length Polymorphism (RFLP) and Southern Blot Analysis Two hundred nanograms of amplified mtDNA was digested with 2 u of restriction enzyme SfaNI (New England Biolabs) or Mae I11 (Boehringer) overnight according to the manufacturer's recommendation. Plasmid pBR322 DNA was used as a test DNA for completion of digestion with restriction enzymes. The digests were separated on a 4% agarose gel (BRL) or 3%Nusieve and
TABLE I. Clinical Findines in 6 Patients With LHON and the &A ~~
Pea Case A-11-1 B-11-1 B-11-2 B-111-3 c-11-1 D-IV-4
11778nt Mutation in the Mitochondria1 Genome
~
Age-ofonset 16 15 16 6 15 24
"Follow-up. bFamily history. 'Not personally examined.
FIU" (yrs) 9 10 10 10 11
7
First visual acuity OD 21200 81200
-
101200 10180 20/100
Last visual acuity OD 21200 51125
f
-E
Last visual acuity 0s 21200 51100
41200 11200 CF
201100 CF 20125
20130 CF 20125
First visual acuity 0s 21200 51200
-
-c
FH~
+ + + ++
Visual recovery -
+t
mtDNA Mutation in Leber Optic Neuropathy 1%agarose gel with Hae III-cut QFX174RF DNA as DNA size marker. After SfaNI digestion, the 201 bp amplified mtDNA fragment was cleaved into 115 bp and 86 bp fragments in normal controls. The mutant mtDNA in LHON lacking the SfaNI site showed a 201 bp fragment. In Mae I11 digestion the 11778nt mutation generated an extra Mae I11 site and showed 105 bp and 96 bp fragments; in contrast, no cleavage was observed in amplified DNA from normal control individuals. The mtDNA Mae I11 digests of all members studied were transferred by Southern blotting to a nitrocellulose membrane (Schleicher & Schuell) and hybridized with a 201 bp normal mtDNA fragment labeled with d2PdCTPusing a random priming method [Feinberg and Vogelstein, 19841. The relative proportions of the mutant vs. normal mtDNA were assessed from autoradiographs using the Pharmicia LKBXL laser Ultroscan densitometer except for cases C-1-2 and C-11-2, which were scanned from photographs of the ethidium bromide stained gel.
Nucleotide Sequencing of PCR Amplified mtDNA To check for the presence of adenine or guanine at 11778nt, direct nucleotide sequencing of PCR products was performed using the single primer method, with T7 DNA polymerase (Sequenase, USB Cleveland, OH) [Wrischnik et al., 1987; Saiki et al., 1988;Higuchi et al., 19901. Primer A was end labeled with y3'P-ATP (3000 ci/ mmol; New England Nuclear) using T4 DNA polynucleotide kinase (BRL).Sixt nanograms of PCR product was annealed with 20 ng &P radiolabeled primer A in a volume of 11p,I and heated to 95°Cfor 5 minutes. 2.5 pl of the annealed sample was added to each of four A or C or G or T tubes with 3 p1 of sequencing mixture (62 FM dNTP, 6.2 pM ddNTP, and 2 u of T7 DNA polymerase in buffer 25 mM Tris-HC1, pH 7.5,lO mM MgC12,70 mM NaCl, and 7 mM dithiothreitol). The mixture was incubated a t 40-42°C for 10 minutes and 3 p1 of stop buffer was added (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). Three microliter of samples were boiled for 3 minutes and loaded onto 6% polyacrylamide/BMurea gel. After electrophoresis for 2.5 hours at 58 watts, gels were dried and exposed to Kodak XAR-5 film for 16-48 hours. Nucleotide Sequencing of Heteroplasmic Mitochondria1 DNA Individuals C-1-2 and C-11-2 are clinically normal women who showed heteroplasmy for the 11778nt mutation in the SfaNI or Mae I11 digestion (Fig. 2). They had both the normal and mutant alleles of the mtDNA. Two hundred nanograms of the 201-bp PCR product was digested with SfaNI or Mae I11 and purified by gel electrophoresis. Sixty nanograms of the digested mtDNA was subjected to a second PCR DNA amplification, using Primer A, Primer B, and 20 pM of dNTP. In SfaNI digests, only the 201 bp mutant allele which was not cut by the SfaNI amplified, and in Mae I11 digestion, only the 201 bp normal allele which did not gain an extra Mae I11site amplified. If the digestion is incomplete, both the
175
mutant and normal alleles will be amplified. The second PCR products were purified using the Centricon 30 and subjected to direct DNA sequencing as previously described.
RESULTS We compared the SfaNI restriction digestion patterns of the 201 bp PCR amplified mtDNA in members of 4 families with LHON. The SfaNI or Mae I11 DNA fragments specific for the presence of the 11778nt mutation in the mtDNA were found to be associated with all maternal lineage members of the 4 famiIies. All lost a SfaNI site and gained an extra Mae I11 site because of the &A mutation. All were heteroplasmic with different proportions (37-92%) of mutant mtDNA; i.e., they contained mtDNA molecules with both G and A at 11778nt. The typical restriction enzyme digestion patterns are shown in Figure 2. In SfaNI digestion (Fig. 2A) the normal control (lane 1)had the 115 bp and 86 bp fragments. The affected male (lane 2) lost the SfaNI site, generating mainly a 201 bp fragment. The small portion of normal mtDNA was only visible on autoradiography after 4-7 days of exposure (data not shown). Two carriers (lanes 3 and 4) had both normal and mutant mtDNA, the 115 bp, 86 bp, and 201 bp fragments. In Mae I11 digestion (Fig. 2B) the &A mutation generated an extra Mae I11 site in mutant mtDNA. The 1 fragment; an afnormal control (lane 1)had ~ 2 0 bp fected male (lane 2) had the 105bp and 96 bp fragments, and a faint band at 201 bp represented the small proportion of the normal allele; female carriers (lanes 3 and 4:l had both mutant and normal mtDNA. In Mae I11 digestion and Southern blot analysis, the 105bp, 96 bp, and201bp bands representing the mutant and normal mtDNA were present in all members from maternal linkage in LHON families. All affected individuals, carriers, and males at-risk showed different proportions of mutant and normal mtDNA, as shown in Figure 3A, B. The 105bp and 96 bp fragments represent the mutant mtDNA. The 201 bp fragments represent the normal mtDNA. A normal control (lane 12)showed a 201 bp fragment. Affected (lanes 1-41, female carriers (lanes 5-11), and males at-risk (data not shown) had both the 105 bp and 96 bp mutant allele and the 201 bp normal allele. In some cases the small proportion of normal allele was only clearly shown in audioradiograms. The proportions of mutant mtDNA are given in Table 11. In the 6 affected persons the proportions of mutant mtDNA ranged from 62-92%. Four had no visual recovery (Table I). Two affected males (cases B-111-3 and D-IV-4) who had 90 and 62% of mutant mtDNA, respectively, regained vision to 20130 in the left eye (case B-11-3) and 20/25 in both eyes (case D-IV-4)at 20 and 12 months after onset, respectively. The patient with 90% mutant DNA was, a t age 5 years, the youngest affected in our series and had severe visual field loss in both eyes. Female carriers had 32-92% mutant mtDNA. Two males at-risk in one sibship (Cases D-IV-2 and D-IV-5) had 37% and 55% of mutant mtDNA, respectively. Two normal controls had 0% mutant mtDNA. Nucleotide sequence analysis was performed in sev-
176
Zhuet al. Pedigree C
PCR of amplified Mae I11 digested mtDNA showed a G at 11778nt of the normal allele (lane 3).
f
1
3
2
1
2
3
4
4
u
M
Fig. 2. SfaNI and Mae I11 digestion patterns of a 201 bp PCR amplified mtDNA encompassing the 11778nt mutation of LHON in pedigree C on an ethidium bromide stained gel. A SfaNI digestion. Lane 1: Father shows 115bp and 86bp. Lane 2 An affected male lost the SfaNI site and shows a 201 bp fragment; the trace of 115 bp and 86 bp fragments were only shown after hybridization to 32Plabeled 201 bp mtDNA. (Data not shown.)Lanes 3 and 4: Obligate carrier female and possible carrier female, who have both the mutant and normal mtDNA (the 201 bp, 115 bp, and 86 bp fragments). M is Hae 111-cut QX174 RF DNA marker. B: Mae I11 Digestion. Lane 1: Father shows a 201 bp fragment with no extra Mae I11 sites. Lane 2 Affected male has an extra Mae I11 site and shows the 107 bp and 94 bp fragments. A very faint 201 bp fragment represents the small proportion of normal mtDNA. Lanes 3 and 4 Obligate carrier and possible carrier have both the mutant and normal mtDNA, the 105 bp, 96 bp, and 201 bp fragments. The heteroplasmy of mtDNA of the affected male in lane 2 who had >90% mutant mtDNA can only be identified in Mae III digestion. but not in SfaNI digestion.
era1 PCR products. The direct sequencing of the 201 bp PCR products showed (Fig. 3A) a G at 11778nt in the normal control individual (lane 1). Lanes 2-4 contain DNA from 2 carrier females and one affected male who had >84% of mutant mtDNA. A &A mutation was shown. Due to small proportions of normal mtDNA the normal G at 11778nt was not clearly visible. The direct sequencing of the heteroplasmic mtDNA of a carrier female (case C-1-21is shown in Figure 3B, lane 1. Both G and A were shown a t 11778nt. The second PCR product of amplified SfaNI digested mtDNA showed an A a t 11778nt of the mutant allele (lane 2) and a second
DISCUSSION The single nucleotide change a t position 11778 in the mitochondrial genome converts the evolutionary highly conserved codon 340 of the NADH dehydrogenase subunit 4 gene from an arginine to a histidine [Wallace et al., 19881.This mutation is present in over 60% of families with LHON as judged by RFLP and Southern blot analysis of mtDNA amplified by PCR. The results of this study demonstrate the 11778nt mutation in platelet mtDNA from all members in maternal lineage of the 4 families with LHON studied. There is heterogeneity in LHON. The molecular basis for disease in cases lacking the 11778nt mutation is unclear. Sequencing of the complete NADH CoQ reductase (ND4) gene of an affected male showed absence of the 11778nt mutation and suggests that a mutation in a mitochondrial gene other than the ND4 gene may lead to the disease [Coppinger et al., 19901. It has been suggested that the 11778nt mutation is associated with a poor prognosis for visual recovery in LHON [Holt et al., 19891. Two of our affected males (cases B-111-3and D-IV-4)regained vision to 20/30 in the left eye and 20125 in both eyes, respectively, 12 and 20 months after onset with follow-up periods of 7 and 10 years. These observations refute the previously proposed poor visual prognosis in patients with the 11778nt mutation. mtDNA from platelets from all maternal lineages of all families was heteroplasmic. Small proportions of normal mtDNA (115 bp and 86 bp fragments in SfaNI digestion and 201 bp fragment in Mae I11 digestion) are difficult to detect in 4% ethidium bromide stained agarose gel (Fig. 2A, lane 2, and Fig. 4A), but can be demonstrated on autoradiograms using Southern blot analysis (Fig. 4B). Hence, it is easy to misinterpret results as indicating homoplasmy in affected males or carrier females. We have identified mtDNA heteroplasmy in all obligate carriers in the 4 families. Proportions of mutant mtDNA in affected and in carriers were found to vary from 62-90% and 67-92%, respectively. The different proportion of mtDNA heteroplasmy in LHON may explain the fact that not all sons or daughters of carrier females are affected or carriers, unless the mutation was close to homoplasmy [Holt et al., 19893. The results from densitometry (Table 111)demonstrates that the proportion of mutant mtDNA may vary significantly between family members. A shift towards normal or mutant mtDNA homoplasmy must have occurred. Two males at-risk (cases D-IV-2 and D-IV-5)had 37 and 55% of mutant mtDNA, respectively. Two females atrisk (cases D-IV-1 and D-IV-7) in the same sibship has 90-91% of mutant mtDNA. A shift towards normal reaching homoplasmy would lead to a healthy individual, and a shift towards mutant reaching homoplasmy would lead to a high risk individual. Different tissues of a heteroplasmic individual may have different proportions of mutant mtDNA molecules [Lott et al., 19901. mtDNA in platelets do not necessarily represent the proportions in the optic nerve. However,
mtDNA Mutation in Leber Optic Neuropathy A C G T
A C G T
A C G T
177
A C G T
A C
c -A
I1778nt G-,
C
A 2
I
A C G T
A C G T
-:....i..
11773
4
3
A C G T
~.
-G
I I778 nt G and A-
B 2
I
3
Fig. 3. A Direct DNA sequencing of 11778nt mutation in LHON. Lane 1: Normal control (case A-1-1) shows a G at 11778nt. Lane 2 Carrier female (case A-1-2). Lanes 3 , 4 Affected males (cases A-11-1 and (2-11-1). DNA in lanes 2-4 show heteroplasmy with
Mitochondrial DNA Mutation and Heteroplasmy in Type I Leber Hereditary Optic Neuropathy Danping Zhu, Effrosini P. Economou, Stylianos E. Antonarakis, and Irene H. Maumenee The Johns Hopkins Center for Hereditary Eye Diseases of the Wilmer Ophthalmological Institute (D.Z.,I.H.M.) and the Center for Medical Genetics, Department of Pediatrics (E.P.E., S.E.A.), The Johns Hopkins Medical Institutions, Baltimore, Maryland able for energy production and provides approximately 90% of the total adenosine triphosphate in humans [Wallace, 19861.The central nervous system is the most dependent tissue. Leber hereditary optic neuropathy fLHONf is a maternally inherited disorder characterized by acute or subacute bilateral loss of central vision, primarily in young males. The age-of-onset ranges from adolescence to late adulthood, with an average of 20-24 years [Nikoskelainen, 1984; McKusick, 1987; Wallace et al., 1988;Newman and Wallace, 19901. The maternal inheritance pattern suggests mitochondrial inheritance [Erickson, 1972; Giles et al., 1980; Egge and Wilson, 1983; Nikoskelainen et al., 19871. Recent studies have shown a G-A nucleotide substitution at 11778nt of the mitochondrial genome identified by Wallace et al., [1988], which converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene [Chomyn et al., 1985, 19861 and eliminated a SfaNI site [Wallace et al., 19881or created a Mae 111site [Coppinger et al., 1990; Stone et al., 19901. This point mutation was found in more than 60%of the families studied. [Wallaceet al., 1988;Holt et al., 1989;Hotta et al., 1989; Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989; Stone et al., 1990;Lott et al., 1990.1 All previously reported families except those reported by Holt et al. [19891 and Lott et al. [19901were homoplasmic. Heteroplasmy could theoretically arise during meiotic andlor mitotic divisions. We studied members from 4 families with LHON KEY WORDS: maternally inherited disorder, using SfaNI and Mae I11 digestion of a 201 base pair (bp) polymerase chain reaction, mtDNA fragment amplified by polymerase chain reacloss of central vision tion (PCR) encompassing the 11778nt mutation to verify the G+A 11778nt mutation and correlation of mtDNA heteroplasmy with the disease. All of the proINTRODUCTION bands had the G+A mutation and all individuals with Mitochondrial DNA (mtDNA) is an extranuclear this mutation showed heteroplasmy in their mtDNA; DNA exclusivelyinherited from the mother [Gileset al., i.e., they had both normal and mutant mitochondria at 19801. It encodes a set of proteins which are indispens- 11778nt. The severity of the disease appeared to correlate with the proportion of mutant DNA from platelets as judged by densitometry.
Leber hereditary optic neuropathy (LHON)is a maternally inherited disorder characterized by bilateral acute or subacute loss of central vision, primarily in young males. A &A single base mutation at 11778nt of the mitochondrial genome which eliminates a SfaNI restriction site [Wallace et al., 1988;Holt et al., 1989;Hotta et al., 1989;Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989;Stone et al., 1990; Lott et al., 1990.1 has been found in more than 60% of the families with LHON studied. We studied 25 persons from 4 families with LHON using SfaNI and Mae I11 digestion of a 201 base pair polymerase chain reaction (PCR)product encompassing the 11778ntmutation. The loss of the SfaNI site and the acquisition of a Mae I11 site at 11778nt were identified in all maternal relatives of the LHON families studied. The mutation was heteroplasmic in all affected individuals, female carriers, and males at-risk. The heteroplasmy of mitochondrial DNA (mtDNA)was also identified by direct DNA sequencing of PCR amplified mtDNA digested by SfaNI or Mae 111. It appears that the proportion of the mutant mtDNA correlates with the severity of the disease.
Received for publication November 16, 1990; revision received April 26, 1991. Address reprint requests to Danping Zhu, M.D., The Johns Hopkins Center for Hereditary Eye Diseases, Maumenee Building Room 321, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Md 21205.
0 1992 Wiley-Liss, Inc.
SUBJECTS AND METHODS Subjects Blood samples were drawn from 25 members of 4 multi-generation families with LHON, including 6 af-
174
Zhu et al.
Pedigree:
B
A
C
D
's w
J f 1
66 i
111
1
2
2
Jf3
3
IV 1
2
3Jf4
5
6
7
11
12
13
Fig. 1. Pedigrees of 4 multigenerational families with LHON. The ethic origins are A: Eastern Europe; B USA, NC; C: USA, WVA; D Canada. Roman numerals I, 11, 111, and IV number the generations. Numbers below the symbols are case numbers. Circles represent females; squares represent males. 0,O= normal subjects; m,0 = affected, 0 = normal carrier female; /1 = proband; M = DNA size marker.
fected persons (5 males and 1 female), 15 carriers, 2 males at-risk, and 2 normal control individuals (Fig. 1). All affected and obligatory carriers were examined at the Wilmar Eye Institute at the Johns Hopkins Hospital. The manifestations are shown in Table I. The onset ranged from 5-24 years. The follow-up period was from 2-10 years. Two of the 6 affected regained some vision. Affected male B-111-3 regained useful vision in the left eye from 41200 to 20130 20 months after onset. Affected male D-IV-4 regained vision in both eyes from 20/100 and count finger (CF) to 20125 at one year after onset. Other patients had persistent visual loss for periods of 9-11 years after onset.
Amplification of mtDNA One hundred nanograms of mitochondria1rich DNA isolated from platelets was amplified using the PCR in a 100 pl reaction mixture with 30 nM of each primer, 200 p M of each dNTP, and 2 u of Amplitaq (Taq I) DNA polymerase [Saiki et al., 19881. The premix of PCR reagents, primers, and dNTPs was treated under 254 nm short wave ultraviolet and 365 nM medium wave UV light for 20 minutes to prevent DNA contaminations [Kwok and Higuchi, 19891 and then the DNA template was added. The mixture was denatured for 3 minutes a t 94°C followedby 30 cycles of amplification (denatured at 94°C for one minute, annealing a t 59°C for one minute, extension at 72°C for 3 minutes) using a DNA thermal DNA Extraction cycler (Perkin-Elmer-Cetus, Norwalk, CT). The amplimtDNA was extracted from peripheral blood platelets fied DNA was purified using Centricon 30 (Amicon, [Giles et al., 19801 within 48 hours after the blood was Danvers, MA). drawn. Primers Nineteen and 23 base oligonucleotide primers [Wallace et al., 19881 were prepared by an Applied Biosystems DNA Synthesizer, and were used to amplify the 201 bp mtDNA fragment. These primers were: Primer A: 11673-5' CCCCC TGAAG CTTCA CCGG 3'-11691 Primer B: 11851-5' TGGGG GGTAA GGCGA GGTTA GCG 3'-11873
Restriction Fragment Length Polymorphism (RFLP) and Southern Blot Analysis Two hundred nanograms of amplified mtDNA was digested with 2 u of restriction enzyme SfaNI (New England Biolabs) or Mae I11 (Boehringer) overnight according to the manufacturer's recommendation. Plasmid pBR322 DNA was used as a test DNA for completion of digestion with restriction enzymes. The digests were separated on a 4% agarose gel (BRL) or 3%Nusieve and
TABLE I. Clinical Findines in 6 Patients With LHON and the &A ~~
Pea Case A-11-1 B-11-1 B-11-2 B-111-3 c-11-1 D-IV-4
11778nt Mutation in the Mitochondria1 Genome
~
Age-ofonset 16 15 16 6 15 24
"Follow-up. bFamily history. 'Not personally examined.
FIU" (yrs) 9 10 10 10 11
7
First visual acuity OD 21200 81200
-
101200 10180 20/100
Last visual acuity OD 21200 51125
f
-E
Last visual acuity 0s 21200 51100
41200 11200 CF
201100 CF 20125
20130 CF 20125
First visual acuity 0s 21200 51200
-
-c
FH~
+ + + ++
Visual recovery -
+t
mtDNA Mutation in Leber Optic Neuropathy 1%agarose gel with Hae III-cut QFX174RF DNA as DNA size marker. After SfaNI digestion, the 201 bp amplified mtDNA fragment was cleaved into 115 bp and 86 bp fragments in normal controls. The mutant mtDNA in LHON lacking the SfaNI site showed a 201 bp fragment. In Mae I11 digestion the 11778nt mutation generated an extra Mae I11 site and showed 105 bp and 96 bp fragments; in contrast, no cleavage was observed in amplified DNA from normal control individuals. The mtDNA Mae I11 digests of all members studied were transferred by Southern blotting to a nitrocellulose membrane (Schleicher & Schuell) and hybridized with a 201 bp normal mtDNA fragment labeled with d2PdCTPusing a random priming method [Feinberg and Vogelstein, 19841. The relative proportions of the mutant vs. normal mtDNA were assessed from autoradiographs using the Pharmicia LKBXL laser Ultroscan densitometer except for cases C-1-2 and C-11-2, which were scanned from photographs of the ethidium bromide stained gel.
Nucleotide Sequencing of PCR Amplified mtDNA To check for the presence of adenine or guanine at 11778nt, direct nucleotide sequencing of PCR products was performed using the single primer method, with T7 DNA polymerase (Sequenase, USB Cleveland, OH) [Wrischnik et al., 1987; Saiki et al., 1988;Higuchi et al., 19901. Primer A was end labeled with y3'P-ATP (3000 ci/ mmol; New England Nuclear) using T4 DNA polynucleotide kinase (BRL).Sixt nanograms of PCR product was annealed with 20 ng &P radiolabeled primer A in a volume of 11p,I and heated to 95°Cfor 5 minutes. 2.5 pl of the annealed sample was added to each of four A or C or G or T tubes with 3 p1 of sequencing mixture (62 FM dNTP, 6.2 pM ddNTP, and 2 u of T7 DNA polymerase in buffer 25 mM Tris-HC1, pH 7.5,lO mM MgC12,70 mM NaCl, and 7 mM dithiothreitol). The mixture was incubated a t 40-42°C for 10 minutes and 3 p1 of stop buffer was added (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). Three microliter of samples were boiled for 3 minutes and loaded onto 6% polyacrylamide/BMurea gel. After electrophoresis for 2.5 hours at 58 watts, gels were dried and exposed to Kodak XAR-5 film for 16-48 hours. Nucleotide Sequencing of Heteroplasmic Mitochondria1 DNA Individuals C-1-2 and C-11-2 are clinically normal women who showed heteroplasmy for the 11778nt mutation in the SfaNI or Mae I11 digestion (Fig. 2). They had both the normal and mutant alleles of the mtDNA. Two hundred nanograms of the 201-bp PCR product was digested with SfaNI or Mae I11 and purified by gel electrophoresis. Sixty nanograms of the digested mtDNA was subjected to a second PCR DNA amplification, using Primer A, Primer B, and 20 pM of dNTP. In SfaNI digests, only the 201 bp mutant allele which was not cut by the SfaNI amplified, and in Mae I11 digestion, only the 201 bp normal allele which did not gain an extra Mae I11site amplified. If the digestion is incomplete, both the
175
mutant and normal alleles will be amplified. The second PCR products were purified using the Centricon 30 and subjected to direct DNA sequencing as previously described.
RESULTS We compared the SfaNI restriction digestion patterns of the 201 bp PCR amplified mtDNA in members of 4 families with LHON. The SfaNI or Mae I11 DNA fragments specific for the presence of the 11778nt mutation in the mtDNA were found to be associated with all maternal lineage members of the 4 famiIies. All lost a SfaNI site and gained an extra Mae I11 site because of the &A mutation. All were heteroplasmic with different proportions (37-92%) of mutant mtDNA; i.e., they contained mtDNA molecules with both G and A at 11778nt. The typical restriction enzyme digestion patterns are shown in Figure 2. In SfaNI digestion (Fig. 2A) the normal control (lane 1)had the 115 bp and 86 bp fragments. The affected male (lane 2) lost the SfaNI site, generating mainly a 201 bp fragment. The small portion of normal mtDNA was only visible on autoradiography after 4-7 days of exposure (data not shown). Two carriers (lanes 3 and 4) had both normal and mutant mtDNA, the 115 bp, 86 bp, and 201 bp fragments. In Mae I11 digestion (Fig. 2B) the &A mutation generated an extra Mae I11 site in mutant mtDNA. The 1 fragment; an afnormal control (lane 1)had ~ 2 0 bp fected male (lane 2) had the 105bp and 96 bp fragments, and a faint band at 201 bp represented the small proportion of the normal allele; female carriers (lanes 3 and 4:l had both mutant and normal mtDNA. In Mae I11 digestion and Southern blot analysis, the 105bp, 96 bp, and201bp bands representing the mutant and normal mtDNA were present in all members from maternal linkage in LHON families. All affected individuals, carriers, and males at-risk showed different proportions of mutant and normal mtDNA, as shown in Figure 3A, B. The 105bp and 96 bp fragments represent the mutant mtDNA. The 201 bp fragments represent the normal mtDNA. A normal control (lane 12)showed a 201 bp fragment. Affected (lanes 1-41, female carriers (lanes 5-11), and males at-risk (data not shown) had both the 105 bp and 96 bp mutant allele and the 201 bp normal allele. In some cases the small proportion of normal allele was only clearly shown in audioradiograms. The proportions of mutant mtDNA are given in Table 11. In the 6 affected persons the proportions of mutant mtDNA ranged from 62-92%. Four had no visual recovery (Table I). Two affected males (cases B-111-3 and D-IV-4) who had 90 and 62% of mutant mtDNA, respectively, regained vision to 20130 in the left eye (case B-11-3) and 20/25 in both eyes (case D-IV-4)at 20 and 12 months after onset, respectively. The patient with 90% mutant DNA was, a t age 5 years, the youngest affected in our series and had severe visual field loss in both eyes. Female carriers had 32-92% mutant mtDNA. Two males at-risk in one sibship (Cases D-IV-2 and D-IV-5) had 37% and 55% of mutant mtDNA, respectively. Two normal controls had 0% mutant mtDNA. Nucleotide sequence analysis was performed in sev-
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Zhuet al. Pedigree C
PCR of amplified Mae I11 digested mtDNA showed a G at 11778nt of the normal allele (lane 3).
f
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3
2
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u
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Fig. 2. SfaNI and Mae I11 digestion patterns of a 201 bp PCR amplified mtDNA encompassing the 11778nt mutation of LHON in pedigree C on an ethidium bromide stained gel. A SfaNI digestion. Lane 1: Father shows 115bp and 86bp. Lane 2 An affected male lost the SfaNI site and shows a 201 bp fragment; the trace of 115 bp and 86 bp fragments were only shown after hybridization to 32Plabeled 201 bp mtDNA. (Data not shown.)Lanes 3 and 4: Obligate carrier female and possible carrier female, who have both the mutant and normal mtDNA (the 201 bp, 115 bp, and 86 bp fragments). M is Hae 111-cut QX174 RF DNA marker. B: Mae I11 Digestion. Lane 1: Father shows a 201 bp fragment with no extra Mae I11 sites. Lane 2 Affected male has an extra Mae I11 site and shows the 107 bp and 94 bp fragments. A very faint 201 bp fragment represents the small proportion of normal mtDNA. Lanes 3 and 4 Obligate carrier and possible carrier have both the mutant and normal mtDNA, the 105 bp, 96 bp, and 201 bp fragments. The heteroplasmy of mtDNA of the affected male in lane 2 who had >90% mutant mtDNA can only be identified in Mae III digestion. but not in SfaNI digestion.
era1 PCR products. The direct sequencing of the 201 bp PCR products showed (Fig. 3A) a G at 11778nt in the normal control individual (lane 1). Lanes 2-4 contain DNA from 2 carrier females and one affected male who had >84% of mutant mtDNA. A &A mutation was shown. Due to small proportions of normal mtDNA the normal G at 11778nt was not clearly visible. The direct sequencing of the heteroplasmic mtDNA of a carrier female (case C-1-21is shown in Figure 3B, lane 1. Both G and A were shown a t 11778nt. The second PCR product of amplified SfaNI digested mtDNA showed an A a t 11778nt of the mutant allele (lane 2) and a second
DISCUSSION The single nucleotide change a t position 11778 in the mitochondrial genome converts the evolutionary highly conserved codon 340 of the NADH dehydrogenase subunit 4 gene from an arginine to a histidine [Wallace et al., 19881.This mutation is present in over 60% of families with LHON as judged by RFLP and Southern blot analysis of mtDNA amplified by PCR. The results of this study demonstrate the 11778nt mutation in platelet mtDNA from all members in maternal lineage of the 4 families with LHON studied. There is heterogeneity in LHON. The molecular basis for disease in cases lacking the 11778nt mutation is unclear. Sequencing of the complete NADH CoQ reductase (ND4) gene of an affected male showed absence of the 11778nt mutation and suggests that a mutation in a mitochondrial gene other than the ND4 gene may lead to the disease [Coppinger et al., 19901. It has been suggested that the 11778nt mutation is associated with a poor prognosis for visual recovery in LHON [Holt et al., 19891. Two of our affected males (cases B-111-3and D-IV-4)regained vision to 20/30 in the left eye and 20125 in both eyes, respectively, 12 and 20 months after onset with follow-up periods of 7 and 10 years. These observations refute the previously proposed poor visual prognosis in patients with the 11778nt mutation. mtDNA from platelets from all maternal lineages of all families was heteroplasmic. Small proportions of normal mtDNA (115 bp and 86 bp fragments in SfaNI digestion and 201 bp fragment in Mae I11 digestion) are difficult to detect in 4% ethidium bromide stained agarose gel (Fig. 2A, lane 2, and Fig. 4A), but can be demonstrated on autoradiograms using Southern blot analysis (Fig. 4B). Hence, it is easy to misinterpret results as indicating homoplasmy in affected males or carrier females. We have identified mtDNA heteroplasmy in all obligate carriers in the 4 families. Proportions of mutant mtDNA in affected and in carriers were found to vary from 62-90% and 67-92%, respectively. The different proportion of mtDNA heteroplasmy in LHON may explain the fact that not all sons or daughters of carrier females are affected or carriers, unless the mutation was close to homoplasmy [Holt et al., 19893. The results from densitometry (Table 111)demonstrates that the proportion of mutant mtDNA may vary significantly between family members. A shift towards normal or mutant mtDNA homoplasmy must have occurred. Two males at-risk (cases D-IV-2 and D-IV-5)had 37 and 55% of mutant mtDNA, respectively. Two females atrisk (cases D-IV-1 and D-IV-7) in the same sibship has 90-91% of mutant mtDNA. A shift towards normal reaching homoplasmy would lead to a healthy individual, and a shift towards mutant reaching homoplasmy would lead to a high risk individual. Different tissues of a heteroplasmic individual may have different proportions of mutant mtDNA molecules [Lott et al., 19901. mtDNA in platelets do not necessarily represent the proportions in the optic nerve. However,
mtDNA Mutation in Leber Optic Neuropathy A C G T
A C G T
A C G T
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A C G T
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Fig. 3. A Direct DNA sequencing of 11778nt mutation in LHON. Lane 1: Normal control (case A-1-1) shows a G at 11778nt. Lane 2 Carrier female (case A-1-2). Lanes 3 , 4 Affected males (cases A-11-1 and (2-11-1). DNA in lanes 2-4 show heteroplasmy with
