Copyright 0 1992 by the Genetics Society of America

Mitochondrial DNA Complex I and I11 Mutations AssociatedWith Leber’s Hereditary Optic Neuropathy Michael D. Brown,* AlexanderS. Voljavec,*’t Marie T. Lott,* Antonio Torroni,* Chi-Chuan Yang* and Douglas C. Wallace* “Center for Genetics and Molecular Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, and +Department of Nephrology, Emory University School of Medicine, Atlanta, Georgia 30322 Manuscript received April 10, 1991 Accepted for publication September 6, 1991 ABSTRACT Four new missense mutations have been identified through restriction analysis and sequencing of the mitochondrial DNAs (mtDNA) from Leber’s hereditary optic neuropathy (LHON) patients who lacked the previously identified 11778 mutation. Each altered a conserved amino acid and correiated with the LHON phenotype in population and phylogenetic analyses. The nucleotide pair (np) 13708 mutation (G to A, ND5 gene) changedan alanine to a threonine andwas found in 6/25 (24%)of non1 1778 LHONpedigrees and in 5.0% of controls, the np 15257mutation (G to A, cytochrome b gene) changed an aspartate to an asparagine and was found in 4 of the 13708-positive pedigrees and 0.3% of controls, the np 15812 mutation (G to A, cytochrome b gene) changed a valine to a methionine and was detected in two of the 15257-positive pedigrees and 0.1% of controls and the np 5244 mutation (G to A, ND2 gene) changed a glycine to a serine and was found in one of the 15812positive patients and none of 2103 controls. The 15257 mutation altered a highly conserved amino acid in an extramembrane domain of cytochrome b that is associated with the ligation of the low potential bsciciheme and the 5244mutation altered a strongly evolutionarily conserved region of the ND2 polypeptide. The 13708 and 15812 mutations changed moderately conserved amino acids. Haplotype and phylogenetic analysis of the four np 15257 mtDNAs revealed that all harbored the same rare Caucasian haplotype and that the np 13708, np 15257, np 15812 and np 5244 mutations were added sequentially along this mtDNA lineage. Since the percentage of sighted controls decreases as these mutationsaccumulate, it appears that they interact synergistically, each increasing the probability of blindness. The involvement of both mitochondrial complex I (np 5244, 1 1778, 13708) and complex 111 (np 15257, 15812) mutations in LHON indicates that the clinical manifestations of this disease are the productof an overall decrease in mitochondrial energy production rather than a defect in a specific mitochondrial enzyme.

L

EBER’S hereditary optic neuropathy (LHON) is a maternally inherited disease of young adults which results in rapid bilateral loss of central vision due to opticnerve death (NEWMAN and WALLACE 1990). Penetrance is variable along homoplasmic maternal lineages and the disease exhibits a male bias with an overall ratio of greater than 2 affected males to 1 affected female (NEWMAN and WALLACE 1990). T h e maternal transmission of LHON suggested that this disease might bedue toa mitochondrialDNA (mtDNA) mutation (GILESet al. 1980). Th’1s was confirmed by the discovery of a G to A transition mutation at nucleotide pair (np) 11778 in the mtDNAs of 9 of 1 1 LHONpedigrees (WALLACE et al. 1988). This mutation replaces an arginine with a histidine at position 340 in the ND4 gene of the mitochondrial NADHxbiquinone oxidoreductase (respiratory complex I) and eliminates a SfaNI restriction enzyme recognition site. Several lines of evidence indicate that the 11778 mutation is the cause of most LHON cases. T h e mutation alters an evolutionary conserved amino Genetics 130: 163-173 (January, 1992)

acid, is found in 50% to 80% of LHON patients but not controls, correlates with the symptoms regardless of mtDNA haplotype,and can be heteroplasmic (WALLACE et al. 1988; SINGH, LOTT and WALLACE 1989; LOTT, VOLJAVEC and WALLACE 1990; HOLT,MILLER and HARDINC 1989). While the majority of LHON cases can be attributed to the 11778 mutation, other LHON patients do not have this mutation. Wenow describe the mtDNA sequence of a nom11778 patient and provide evidence that the primary cause of the disease in this patient and three others is a mutation in a highly conserved amino acid of the mtDNAcytochrome b gene of respiratory complex 111. Three additional mutations, in the ND2, ND5 and cytochrome b genes, were also found which appear to augment the deleterious effects of this mutation. MATERIALS AND METHODS LHON patients and controls: Twenty-five non-11778 LHON families, 24 11778 LHONfamilies and 292 controls

M. D. Brown et al.

164 I AOE 2 1

II I A 0 1 24

111

IV

ME17

1

2

3

FIGURE1.-Pedigree of 11778-negative LHON family. Blackened symbols indicate individualswith LHON. mtDNA was isolated from the proband (IV-1) and subjected to DNA sequence analysis. The age of LHON onset is indicated.

(147 Caucasians, 53 Asians, 41 Africans, 48 Amerindians and 3 Black Americans) were studied. All LHON probands experienced painless, precipitous and progressive bilateral central vision loss after childhood. Cases of blindness showing a paternal transmission were excluded from the study. The pedigree of the sequenced proband (a male)contained four affected individuals (3 male, 1 female) ranging over four generations (Figure 1). The otherthree probands containing the np 13708 and 15257 mutations included two familial cases and one singleton case. Preparation of mtDNAsamples: mtDNA for analysis was extracted from whole blood (200 pl), fractionated lymphocytes, or Epstein-Barr virus-transformed lymphoblastoid cell lines(WALLACEetal. 1986, 1988). Following extraction, mtDNA was isolated by anion-exchange affinitycolumn (Qiagen, Inc., Studio City, California) or by proteinase K/ phenol-chloroform extraction (WALLACE et al. 1988; LOTT, VOLJAVEC and WALLACE 1990). Polymerase chain reaction (PCR)amplification of double-stranded DNA: DNA was amplified using 35 cycles of denaturation (45 sec at 94"), annealing (30 sec at 5" below the Tm of the most A T - rich primer pair) and extension (60 sec at 72"). No oil overlay was used. The 100-rl PCR reactions contained 200 p~ of each dNTP (dATP, dCTP, dGTP and dTTP), 50 mM KCI, 10 mM Tris-C1 (pH 8.3), 1.5 mM MgC12, 0.01% gelatin (wt/vol),30pmolof each primer (Microchemical Facility, Emory University),and 2.5 units of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, Connecticut). Double-stranded PCR products were visualized by ethidium bromide staining of agarose gels. Restriction enzyme analysis:The mutations at np 5244, 13708, 15812and15257eliminateHpaII/MspI,BstNI,RsaI and AccI restriction endonuclease sites, respectively.T o test for the5244 mutation, a 1086-np fragment was PCRamplified between np 4831 and 59 17 which included HpaII/ MspI recognition sites at nps 4846, 5242, 5742, and 5776. Following HpaII digestion, normal mtDNAs were cleaved into 500-, 396-, 151-, 24- and 15-np fragments, while in the heteroplasmic patient an additional 896-np fragment was found resulting from the fusion of the 500-np and the396np digestion products. Alternatively, a 330-np PCR fragment (np 5150-5480) was amplified and digested with HpaII to screen for this mutation. T o test for the 13708 mutation, a 754-np fragment was PCR-amplified between np 13 196 and 13950 and subjected to BstNI digestion. Normal mtDNAs were cleaved into 5 12 242-np fragments while mutant fragments remained uncut. T o test for the 15257 mutation, a 532-np PCR fragment was generated between np 14829 and 15360 and digested with AccI. Normal rntDNAs were cleaved into 426 + 106-

+

+

np fragments while mutant fragments remain uncut. To createa positivetest for this mutation, a forward PCR primer (15257Mse) was prepared whose3'-OHis at np 15256, immediately adjacent to the mutant nucleotide, and whose 5' end is at np 15234. The sequence of the primer differs from the normal mtDNA at np 15254 such that if the mutation is present, a MseI restriction site is created (Figure 2). This primer is opposed by a reverse primer from 15340 to 15360 (3' to 5') generating a PCR fragment of 127 np. If the patient has the mutation, digestion withMseI cuts the fragment into 21- and 106-np fragments. If the sequence is wild type, it remains uncut. T o test for the np 15812 mutation, a 161-np fragment was PCR-amplified between np 15704 and 15874 and digested with RsaI. Normal mtDNAs were cleaved into 108 53-np fragments while mutant fragments remain uncut. For haplotype analysis, a series of nine overlapping mtDNA fragments were generated by PCR which encompassed theentire mtDNA genome: np 16453-1696; np 1561-3717; np 3007-5917; np 5317-7608; np 7392-8921; np 8282-10107; np 9911-1 1873; np 11673-13950; and np 13914-16547. Each fragment was digested with 14 restriction endonucleases: AluI, AvaII, BamHI, DdeI, HaeII, HaeIII, HhaI, HincII, Hinfl,HpaI, HpaII/MsPI, MboI, RsaI, and TaqI. All restriction endonuclease digestions were performed according to manufacturer's conditions using approximately 100-200 ng of PCR-generated DNA digested with 5 units ofenzyme and visualized by ethidium bromide staining. Restriction fragments of less than 200 bp were separated on 2.5% NuSieveplus 0.9% SeaKem (FMC Bioproducts) while allother digests wereseparated on 1.O-1.5% SeaKem agarose gels. Sequencing of asymmetrically amplified patient mtDNAs: DNA sequence analysis of the majority of the sequenced proband's mtDNA as well as analysis ofmutations causing restriction endonuclease site losses in other patients was accomplished by direct sequencing of asymmetrically PCR-amplifiedmtDNA. Single-stranded DNA was produced by reamplifying 2 pl of a 100-pl double-stranded PCR reaction using primers at a 20 pmol:0.2 pmolratio (INNIS et al. 1988). dNTPS were added at 20 PM and gelatin was deleted from the reaction. The single-stranded DNAwas precipitated with ammonium acetate/isopropanol, washed with 500 pl of 70% ethanol, dried under vacuum, resuspended in 7 pl of sterile water and sequenced. Primers included either 0.5 pmol of the limiting asymmetric PCR primer or an internal primer. The chain extension used dideoxyribonucleotides, Taq polymerase (Amplitaq, PerkinElmer/Cetus), and 35S-labeleddATP (SHOFFNER et al. 1989). Sequencing reactions were resolved on either a 6% polyacrylamide/8M urea or 60%Hydrolink (AT Biochem, Malvern, Pennsylvania) sequencing gel and visualized by autoradiography. The ND1, ND2, ND5 and ND6 genes of the sequenced proband were cloned into the M13mp19 vector and singlestranded templates were sequenced using the M 13 universal primers and internal slip primers. The ND1 and ND2 genes were cloned byPCR amplification of the genesusing primers with 5'HindIII extensions followed by HindIII restriction endonuclease digestion using a 100-fold excess of the enzyme. The ND5 gene insert was generated by PCR amplification of a 2877-np (np 1171 1-14588) fragment followed by BclI and BamHI double digestion at internal restriction endonuclease sites. The ND6 insert was generated by PCR amplification of a 2668 np (np 13197-15865) fragment followed by BamHI and XhoI double digestion at

+

165

mtDNA Mutations and Leber's Disease

I

MUTANT

I

PCR primer with introduced mismatch 1

0

I.. T C A t T A > /

1. . T

15257

...

T C A G T A ~ A C

A;g"T

15257

C A t T A>/ T C A G T A ~ A C

...

_AIGIA / A T C Al't T A m A C A A G T A A T T T G A

A G T A A T C T G A

No M s e I site created with wild type "G" at 15257.

M s e I site (TTAA) created by a combination of the transition

at 15257 and the introduced mismatch at15254.

15234

15380

I

127 bp

I

106 bp

wr

I

21 bp

MUTANT

15254

FIGURE2.-Schematic showing MseI restriction enzyme digestion assay for the 15257 mutation using a mutant PCR primer. Shaded box highlights mismatched thymine in the forward primer, GAATCTGAGGAGGCTACTCAtTA, and in the resulting double strand PCR product. The thymine (t) in the primer replaces the wild type guanine (G) normally found at np 15254. Restriction map shows position of the MseI site created at np 15254 only when the G to A mutation is present at np 15257. internal restriction sites. Mutations were verified by sequencing at least two independent clones. Phylogeneticanalysis: The evolutionary relationship among 32 Caucasian controls and six non-11778 LHON with the patientswereinferredusingparsimonyanalysis computer programPAUP (SWOFFORD 1990).A hypothetical African ancestor (ancestor "a" from CANN,STONEKING and WIWN 1987) was used to root the resulting phylogenetic tree.

RESULTS

Proband sequencing: To identify additional mtDNApointmutations which can cause LHON, 97% of the mtDNA protein coding and tRNA genes were sequenced from the proband of a maternally inherited LHON pedigree which lacked the 11778 mutation (Figure 1). Twenty-seven nucleotide substitutions were found relative to the normal Cambridge et al. 1981). Ten of the mtDNA sequence (ANDERSON nucleotide substitutions did not change an aminoacid and five others are probable errors in the Cambridge sequence (Table 1). Two mutations altered bases in tRNAs (np 5633, tRNA"'" and np 7476, tRNA&) which were not evolutionarily conserved (Table 1). The np7476 mutation also altered anAluI restriction and site known to be polymorphic (CANN,STONEKING WIUON1987). Hence these mutationsare unlikely to be the primary cause of LHON. Ten mutations altered aminoacids (Table 1). Seven ofthese(np4216,8860,10398,14199,14484,15326

and 15452) changed nonconserved amino acids and thus were unlikely to be functionally significant. The np 8860, 10398 and 14199 mutations also altered restriction endonuclease recognition sites known to be polymorphic in the general population at frequencies of >go%, 26% and >go%, respectively, confirming that they are not related to thedisease phenotype (BALLINGER et al. 1992; TORRONI et al. 1992). The eighth amino acid substitution mutation at np 13708 is a G to A transition which changes the 458th amino acid in the ND5 gene from a nonpolar alanine to a polar threonine.This change could be significant since all vertebrates have an alanine or leucine in this position (Table 1). This mutation eliminates a BstNI site, a polymorphism which has been reported in up to 7% of individuals in various normal human popuet al. 1988; VILKKIet al. 1989; HOLT, lations (GELINAS HARDING and MORGAN-HUGHES 1988). We tested 27 additional Caucasian controls, none of which had the BstNI site loss, giving an overall frequency of 5% (14 of 278) (Table 2). By contrast, 6 of 25 (24%) LHON patients without the 11778 mutation including the sequenced proband had lost this site (Table 2). This difference was significant at the0.001 level. However, 5 of 23 (22%) patients with the 11778 mutation had also lost this site (Table 2). Thus, this mutation was found at a higher frequency in LHON patients than normal individuals, but it didnot discriminate be-

166

M. D. Brown et al. TABLE 1 mtDNA mutations in tRNA and protein-coding genes tRNA genes Nucleotide conservation (H/C/M/X)

NucleotideNucleotide tRNA posttlon region tRNA change

tRNA""' t

~

~

A

UCN

$

e

~

5633 7476 C

C +T +T

Unpaired base C/C/T/C Anti codon stem C/T/T/T

Protein coding genes

Gene

NDl ATP6 ND3 ND5 ND6 Cytb

Nucleotide Nucleotide Amino positionchange change

4216 8860 10398 13708 14199 14484 15257 15326 15452 15812

T+C A +G A+G G+A C +A A .--,G G +A A +G C +A G+A

acid

Tyr+His Thr +Ala T h r + Ala Ala+Thr Pro + Thr Met + Val Asp+ Asn Thr- Ala Leu + Ile Val+Met

Amino acid conservation (H/C/M/X)

Y/H/H/H T/A/A/T T/T/T/A A/L/A/A P/S/S/D M/M/L/L D/D/D/D T/M/I/L L/I/F/L V/V/I/V

Ten synonymous base substitutions were found within the sequenced region: 3423 (G to T), 4985 (G to A), 10172 (G to A), 10966(TtoC),11251(AtoG),11335(TtoC),11719(GtoA), 12441 (T to C), 12612 (A to G ) and 14365 (G to C).Based on DNA sequence analysis and restriction enzyme polymorphism studies, fiveother mutations were detected which are likely to be errors in the human Cambridge mtDNA sequence: 4769 (A to C), 7028 (C to T), 13702 (G to C), 14272 (G to C) and 14368 (G to C) (unpublished data from this laboratory and BALLINGERet al. 1992; TORRONI el al. 1992; WALLACE et al. 1988). Mutations are reported as L-strand base changes. All tRNA genes were sequenced except for proline. Ribosomal RNA genes were not sequenced. Restriction endonuclease polymorphisms have been reported for thefollowing STONEKING and mutations: 7476 results in an AluI site loss (CANN, WILSON 1987), 8860 results in a Hhal site gain (BALLINGER et at. 1992; CANN,STONEKING and WILSON 1987; HORAIand MATSUNAGA 1986), 10398 results in a DdeI site gain (BALLINGER et al. 1992; et al. 1992), 13708 CANN,STONEKING and WILSON1987; TORRONI et al. 1989; HOLT,HARDINC results in a BstNI site loss (GELINAS and MORGAN-HUGHES 1988; VILKKIet al. 1989), 14199 results in et al. 1992: TORRONI et at. 1992), and a Hincll site loss (BALLINGER 15812 results in a RsaI site loss (HORAI and MATSUNAGA1986). et al. 1981), H = human Cambridge mtDNA sequence (ANDERSON et al. 1982), M = mouse C = cow mtDNA sequence (ANDERSON mtDNA sequence (HOWELL 1989), X = Xenopus mtDNA sequence (ROEet al. 1985).

tween LHON patients with and without the 11778 mutation. Hence it is unlikely to be the primary cause of LHON in the proband. T h e ninth amino acid substitution mutation at np 15257 is a G to A transition in the cytochrome b gene which converts amino acid 171 from an acidic aspartate to anuncharged asparagine.An aspartate is found at this position in all vertebrate mtDNAs as well as in Drosophila, sea urchin and plant(wheat) mtDNAs. Further, it is contained within a highly conserved region of cytochrome b (Table 3). Hence, this mutation probably reduces the efficiency of this key electron carrier. T h e mutation eliminates an AccI restriction enzyme site at 15254-15259. Previously, 70 Cau-

casians from Finland had been screened with AccI, but none had lost this site (VILKKIet al. 1989). We extended this analysis by screening 291 additional controls and foundonly one Caucasian which had lost the site (Table 2). Hence, only 1 in 217 Caucasians (0.5%) and 1 in 362 total controls (0.3%) had lost the AccI site. To determine if this mutation was associated with LHON, we tested 23 non-11778 LHON patients. Four (including theproband) of thenon-11778 LHON patients (1 7%) lacked the AccI recognition site (Figure 3a and Table 2). This frequency difference is significant at the 0.001 level.We also screened 24 11778 LHON patients, but none had lost the AccI restriction enzyme site (Table 2). T o more precisely test for thenp 15257 mutation,we developed a second restriction endonuclease assay in which mtDNAs containingthe np 15257 G to Amutation would be cleaved by MseI, while all other mtDNAs would not be cut (Figure 2). The four non-11778 LHON patients which lacked the AccI restriction site (np 1525415259) had the MseI site (Figure 3b). Hence, all four have the15257mutation. Coincidently, all four of these patients were also individuals which harbor the np 13708 mutation. The positive Caucasian control also had the MseI site (data not shown). Three family members of the sequenced proband had the MseI site and lacked the AccI site, confirming the maternal inheritance of the np 15257 mutation (Figure 3, and a b). Hence, this mutation is likely tocontributeto LHON since it changes a functionally important amino acid, is a very rare mutation in the general population, and is maternally inherited. T h e tenth amino acid substitution mutation at np 158 12is a G to A transition in the cytochrome b gene which converts amino acid 356 from a valine to a methionine. Since all vertebrates have either a valine, leucine or isoleucine in this position, this substitution could also be functionally significant (Table 4) (DESJARDINS and MORAIS 1990). This mutation also eliminates a RsaI restriction endonuclease recognition site atnp 15812-15815. RsaI is commonly used in mtDNApopulation surveys, hencea total of 759 unaffected individuals have been screenedfor this mutation (Table 2). Only one Japanese (0.13%) had this site loss (HORAIand MATSUNAGA1986). Restriction endonuclease and sequence analysis of our 23 non-11778 LHON patients revealed one additional patient which harbored this mutation giving a total of 2 or 9% (Table 2). This difference is significant at the 0.001 level. None of the np 11778 patients had this mutation. Surprisingly, bothpatients with thenp 158 12mutation also had the np 13708 and np 15257 mutations. Three family members of the sequenced proband also lacked this RsaI site, confirming its maternal inheritance. Thus, the np 1581 2 mutation alters a moderately conserved amino acid, correlates

DiseaseLeber'smtDNA and Mutations

167

TABLE 2 Frequency of the mutations in LHON patients and controls 15257 Source

LHON (non-11,778) LHON (1 1,778) Controls

13708

w

N

%

N

46

N

%

N

24.0 21.7 5.0

6/25 5/23 141278

17.4 0 0.3

4/23 0124 11.362

8.7 0 0.1

2/23 0124 11759

4.5

1/22 0124 012 103

0 0

The x' value calculated from these data indicate a highly significant (P< 0.001, 1 d.f.) association between the mutations and non-11778 LHON: np 13708 = 13.380 (total number of controls used in calculation), np 15257 = 49.200/19.595 (total controls/Caucasian controls only), np 15812 = 42.84111 1.367 (total controls/Caucasian controls only), np 5244 = 95.636 (total number of controls used in calculation). The non-11778 LHON pedigree with the np 5244 mutation also contains the 15812, 15257, and 13708 mutations; the two pedigrees with the 15812 mutation contain the 15257 and 13708 mutations; and the four pedigrees with the 15257 mutation also contain the 13708 mutation. The frequency of the 13708 mutation was calculated from GELINAS et al. (1989), HOLT,HARDING and MORGAN-HUGH= (1988), and VILKKIet al. (1989). The frequency of the 15257 mutation was calculated from VILKKIet al. (1989) and the control group from this study which consisted of 147 Caucasians, 101 Mongoloids (48 Amerindians, 19 Taiwanese, 1 Chinese, 10 Malay, 13 Borneons, and 10 et al. (1992), Koreans), 4 1Africans (Senegalese), 3 Black Americans. The frequency of the 158 12 mutation was calculated from BALLINGER et al. (1990). TORRONI et al. (1992), and unpublished data from this laboratory. The frequency HORAIand MATSUNACA(1986), STONEKING et al. (1990), SCHURR et al. (1990), CANN,STONEKING and WILSON (1987), BALLINGER of the 5244 mutation was calculated from STONEKING et al. (1992), TORRONI et al. (1992),JOHNsoN et al. (1983), SCOZZARI et a/. (1988), SEMINO et al. (1989), DEBENEDICTIS et al. (1989). TORRONI et al. (1990), HORAIand MATSUNAGA(1986), BREGA et al. (1986a), BONNE-TAMIR et al. (1986), SARTORIS et al. (1 988). BREGA et al. (1986b), and VILKKIet al. (1989). TABLE 3 Amino acid conservationof cytochrome b aspartate 171 Cytochrome Source

Human, LHON Human, Cambridge Bovine Mouse Rat Chicken Xenopus h v i s Sea Urchin Drosophila yakdm Wheat Paramecium

References sequence b amino acid GGYSV GGYSV GGFSV GGFSV GGFSV GGFSV GGFSV GGFSV GGFAV GGFSV

N D D D D D D D D D

SPTL SPTL NATL KATL KATL NPTL

NATL NATL NATL NATL FTDQK N TDTL

This paper ANDERSON et aL (1981) ANDERSONet al. (1982) BIBBet ol. (1981) GADALETA et al. (1989) DESJARDINS and MORAIS(1990) ROEet al. (1985) JACOBS et al. (1988) CLARY and WOLSTENHOLME (1985) BOERet al. (1985) PRITCHARD et al. (1989)

Boxed residues correspond to the human mitochondrial cytochrome b amino acid 171 or the residue found in the equivalent position in other organisms.

strongly with non-11778 LHON, and is maternally inherited. Consequently, it could also contribute to the disease. Haplotype analysis: T h e striking association between the np 13708, 15257 and 15812mutations led us tofurther investigate the relationship between these mutations and the LHON phenotype through mtDNA haplotype analysis and phylogenetic investigation. T h e mtDNA haplotypes were determined for the four np 15257-positive patients, two np 15257 negative patients, the one np 15257 positive control and 32 np 15257-negative controls. All patients were also 11778 negative. Each individual's mtDNA was PCR amplified and screened for restriction site variants using the 14restriction endonucleasesmost commonly employed in population surveys. Their mtDNAs were also tested for the three LHON associated mutations at nps 13708, 15812 and 15257.

The 32random Caucasian controls showed the extensive restriction site variation previously reported in population surveys (JOHNSON et al. 1983; TORRONI et al. 1990; BRECAet al. 1986b; SANTACHIARA-BENERECETTI et al. 1988; CANN, STONEKINC and WILSON 1987). Similarly, the non-15257 and non-11778 patients (PF and PS, Figure 4) had standard Caucasian haplotypes. By contrast, all five individuals with the np 15257 mutation hadvery similar haplotypes which included a mutation at 13708 andrestriction enzyme site losses at nps 7474 ( A M ) , 16065 (Hinfl)and 165 17 (HaeIII). Previous population surveys had identified two Caucasians with the np 16065 site loss, one of which also had the Ah1 site loss at np 7474 (CANN, STONEKINC and WIWN 1987). Hence, the mutations at np 1651 7, 7474, 13708, and 16065 define a rare Caucasian haplotype on which the np 15257 mutation occurred. The fact that four randomly chosen non-

M. D. Brown et al.

168

I

2

3

P1

P2 P3 P4 Cl c2 c3

c4

1 2 3 P1

p2

P3 P4

c1

c2 CJ c4

Ef3

127 bp 106 bp

;. 106 bp F

a) Accl digestion

b) Msel digestion

FIGURE3.-Restriction enzyme analysis of the 15257 mutation. (a) Accl restriction enzyme digestion of non-11778 patients and controls. (b) MseI restriction enzyme digestion (Figure 1) of non-11778 LHON patients and controls. P = patient, C = control (C1 and C2 = Caucasian, C3 = African, C4 = Amerindian); 1, 2, 3 = maternal relatives of sequenced proband P1 (2 = IV-2. 3 = IV-3 and proband P1 = IV-I of Figure 1); circles = females, squares = males, filled symbols = LHON phenotype; bp = base pairs. TABLE 4 Amino acid conservation of cytochrome b valine 356 Cytochrome Source

Human, LHON Human, Cambridge Bovine Mouse Rat Chicken Xenopus laevis Sea urchin Drosophila yakuba Wheat Paramecium

b amino acid sequence

GQVAS GQVAS GQLAS GQLAS GQLAS GQMAS

GQLAS GQI'AS GQILT GQIPS FFIML

M LYFT

V LYFT V LYFL I SYFS I SYFS L SYFT V IYFS V LYFS I IYFL V FFFL C LYTP

References

This paper et al. (1981) ANDERSON ANDERSON et al. (1982) BIBBet al. (1981) GADALETA et al. (1989) DESJARDINS and MORAIS (1990) ROEet af. (1985) JACOBS et al. (1988) CLARY and WOLSTENHOLME (1985) BOERet al. (1985) PRITCHARD et al. (1989)

Boxed residues correspond to the human mitochondrial cytochrome b amino acid 356 or the residue found in the equivalent position in other organisms.

1 1778 LHON patients collected from Ohio,Michigan and California in the United States and from western Germany all have this same rare Caucasian mtDNA haplotype demonstrates that LHON is linked to this mtDNA. Phylogenetic analysis confirmed that the mtDNAs of all four np15257 LHON patients were very closely related (Figure 4). T h e Caucasian controls and the two non-15257/non-11778patients (PS and PF) were dispersed across multiple branches of the Caucasian and mtDNA tree, while all of the np 15257 (Pl-4 CC) and np 15812 individuals were associated in one and cluster defined by the np 16517, 7474, 16065, 13708 polymorphisms (Figure 4).This phylogeny also and 15812 demonstrates that the np 13708, 15257 mutations accumulated sequentially on this maternal lineage and that the np 15257 and np 15812 mutations each arose once. Additional mutations on this lineage separate patient P2 (np 5242)and patient P3 (np 7607). These must have also arisen once, after the np 13708 and np 15257 mutations. T h e sequential accumulation of the np 13708, 2 supports the importance 15257 and 1581 mutations

of the np 15257 mutation in generating the LHON phenotype and of the np 15812 mutation in exacerbating its expression. The mutations responsible for and 13708 site losses all the np 16517, 7474, 16065, occur in the generalpopulation and thus predated the np 15257 mutation and, with the possible exception of np 13708, are not involved in LHON. The np 16065 and 1651 7 mutations occur in the non-coding D-loop region of the mtDNA and are unlikely to be functionally significant. The 7476 mutation in the tRNA?& gene alters anon-conserved nucleotide and is alsounlikely to bethe cause of the disease. By contrast, the appearance of the np 15257 mutation correlates with the appearance of the LHON phenotype, with four out of five individuals with this mutation being blind. This demonstrates that thenp 15257 mutation predisposes individuals to blindness, though its presence in one control (CC) indicates that it does not necessitate blindness. This implies that additional genetic and/or environmental factors are required for expression of LHON in np 15257 individuals. The 2 arose phylogeny shows that the np 1581 mutation after the np 15257 mutation, being present intwo

169

mtDNA Mutations and Leber’sDisease

9052-,

I

6Sl7.

7697+, 12946+,

13325-

9714+,

16517+

14304- I

-

16436+,

13325.

E

10394. -

-,

13957-,

16389+

FIGURE4.-Phylogenetic tree generated by parsimony analysis showing evolutionary relationships among Caucasian non-11778 LHON patients and unaffected Caucasian controls. Circles indicate mtDNA haplotypes of individuals tested. P1-4 = LHON patients 14 containing the np 13708and 15257 mutations and P1 is the sequenced proband, CC = control containing the np 13708 and 15257 mutations, PS = singleton non-11778 and non-15257 patient (without other family history of disease), PF = non-11778 and non15257 patient with family history; HYP = hypothetical African ancestral mitochondrial DNA; numbers 1-32 = independent Caucasian controls; CA = published “Cambridge” mitochondrial DNA sequence. Horizontal branch lengths are proportional to the number of steps between nodes. Numbers on top of branches indicate nucleotide position of restriction endonuclease site loss (-) or gain (+).

go52-p 5584-,

1715,

4529-, 8249+, 1W28+,

16389+

15754+

v

~

255+,

6260-

7474,

p 5 E 16065- q i) 1 ,J ,

27 16517-

individuals, both blind. Hence the np 158 12 mutation appears to be one genetic factor that can augment the expression of the np 15257 mutation. T h e mutation which resulted in the HpaII/MspI restriction endonuclease siteloss at np 5242 in patient P2 (Figure 4) appears to be a second mutation which increases the probability of blindness in this lineage. This restrictionsitepolymorphism was unique because it was heteroplasmic (Figure 5). Direct sequencing confirmed the heteroplasmy (Figure 6 ) and revealed that the mutationwas a G to A transition at np 5244 of the ND2 gene. T h e mutation was found in

one of 22 (4.5%) non-11778 LHON patients and in none of 2103 unaffectedcontrols(Table 2). This frequency difference is significant at the 0.001 level. T h e mutationsubstitutedaserine for a glycine at amino acid 259 in the ND2 polypeptide. A glycine is found in the mtDNAs of this position in all species including animals, fungi, plants and protozoa and the region chloroplast DNA of tobacco. Further the entire is evolutionarily conserved (Table 5). Hence this mutation is likely to be functionally significant. T h e mutation creating a HaeIII restriction endonuclease site at np7607 in patient P3 (Figure 4) is not

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np 5244

FIGURE5.--HpaII digest showing loss of HpaII recognition site at np 5242 in one 1 1778-negative LHON patient. Heteroplasmy is indicated by presence of the 500 and 396 np bands as well as the fusion band of 896 np. P2 corresponds to P2 of Figure 3; P+ = 11778-positiveLHON patient; P- = 11778-negativeLHON patient not harboring np 5244 mutation; C = unaffected Caucasian controls.

relevant to blindness. This T to C transition at np 7609 does not change an aminoacid. DISCUSSION

Previously, we identified a mtDNA mutation which accounts for between 50% and 80% of LHON cases et al. 1988). We now demonstrate that the (WALLACE symptoms of at least some of the remaining LHON Patients are also the product of mtDNA mutations. Four seemingly unrelatednon-11778 LHONprobandsrepresentingone singleton case andthree LHON families fromNorth America andEurope werefoundto all have the same uniquemtDNA haplotype defined by the three rare restriction site polymorphisms at nps 16065, 13708, 7476 and the more common np 1651 7 polymorphism. Since the probability of this haplotype occurring by chance in four randomly selected LHON patients is essentially zero, the LHON phenotype is linked to this mtDNA haplotype. Sequence analysis of 97% of the protein and tRNA gene coding regions from one of these patients revealed three potentially significant mutations, one in the ND5 gene (np 13708) andtwo in the cytochrome b gene (nps 15257 and 15812). Subsequent haplotype analysis of three otherpatients with the np 13708 and 15257mutations revealed a fourth mutation(np 5244, ND2gene) likely tobe causally related to LHON. The np 13708 mutation was associated withall mtDNAs in the LHON lineage and is known to be widely distributed among Caucasians. Hence, this mutation probably predatedthe np 15257mutation. While this raises the possibility that the np 13708 mutation is merely a linked polymorphism, the mutation does substitute a polar threonine for anon-polar alanine in a postulated hydrophobic region of ND5 and thus might impede electron transport through and SIMPSON complex I (DE LA CRUZ, NECKELMANN 1984;CHOMYNet al. 1988). Further, the mutation was found in both 11778and non-11778LHON

-

-

G G C+T C G A

1; FIGURE 6.-Direct DNA sequencing autoradiogram showing nucleotide sequencefrom np 5239 to np 5249 in patient with np 5244 mutation. Sequence shown is H-strand sequence. Heteroplasmy at np 5244 is confirmed by the presence of both C and T signal (Hstrand) at np 5244 (corresponding to G and A of L-strand).

patients in oursandother studies. The associated BstNI site loss was reported in three of 17 (18%) Finnish LHON families (VILKKIet al. 1989), in 43% non-11778 and 17% 11778 LHON families in a Baland in 24% timore study (JOHNS and BERMAN 1991) non-11778 and 22% 11778 LHON familiesin our study. It has been proposed that this mutation might be contributory to the LHON phenotype, perhaps in a synergistic manner(JOHNS andBERMAN1991), which would be consistent with our data. The np 15257 mutation is mostlikely to be the primary cause of the disease. Phylogenetic analysis indicates that its occurrence correlates with the appearance of LHON. This mutationalso substitutes an asparagine for a highly conserved aspartate that is located in a conserved region of the cytochrome b gene. This region (approximately 20amino acids) comprises part of an extramembrane domain located adjacent to an invariant histidine residue (His-183) which is involved in ligating the low potential 6566 heme (HOWELL1989). The association between the np 15257 mutation and LHON is further strengthened by the observation that two of 17 (12%) Finnish LHON families had lost the AccI restriction site at np 15254-15259 (VILKKIet al. 1989). It is not known which of these families harbor the 11778 mutation. Thus, six independent LHONpedigrees from Europe and the United States likely harbor this mutation. While the np 15257 mutation is likely the primary factor in causing LHON in these patients, the presence of an unaffected control with this mutation indicates that other factors must contribute to expression of the disease phenotype. This phenomenon is characteristic of LHON since LHON family members which are homoplasmic for the 1 1778 mutation have been identified which are not blind. The np 158 12 mutation was clearly associated with LHON, occurring at the end of the LHON lineage and, in that context, foundonly in affected pedigrees. Since it substitutes amethionine for a moderately conserved valine in cytochrome 6 , this mutation prob-

mtDNA and Mutations

Leber’s Disease

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TABLE 6 Amino acid conservation of ND2 glycine 269 Source Human, LHON Human, Cambridge Bovine Mouse Rat Chicken Xenopus laeuU Sea urchin Drosophila yakuba Paramecium

Neurospora crassa Tobacco (chloroplast)

ND2 amino acid sequence GLPPLT GLPPLT GLPPLS GLPPLT GLPPLT GLPPLT GLPPLS GLPPLT GLPPFL GIPPLL GIPPLV GLPPLA

S FLPKW G FLPKW G FMPKW G FLPKW G FLPKW G FMPKW G FVPKW G FILKF G FLPKW GFFLKF G FFAKQ G FFGK-

References This paper ANDERSON et al. (1981) ANDERSON et al. (1982) BIBBet al. (1981) GADALETA et al. (1989) DESJARDINS and MORAIS(1990) ROEet al. (1985) JACOBS et 01. (1988) CLARY and WOLSTENHOLME (1985) PRITCHARD et al. (1989) DEVRIES et al. (1986) SHINOZAKI et al. (1986)

Boxed residues correspond to the human mitochondrial ND2 amino acid 259 or the residue found in the equivalent position in other organisms. A dash indicates a gap in amino acid sequence alignment.

ably contributes to the non-11778 LHON phenotype. The np 15812 mutation and possibly the np 13708 mutation are likely examples of additional, but not primary, genetic factors which contribute to LHON. Yet the combined presence of the np 13708, 15257 and15812 mutationsdoes not necessarily ensure blindness as the two female siblings of the sequenced proband are visually asymptomatic (Figure 3). The np 5244 mutation detectedby haplotype analysis also appears to be a major factor in blindness. It is found only in an LHONpatient and not in over 2000 controls. It changes a very highly conserved amino acid 259 of the ND2 polypeptide from a glycine to a serine in aregion of ND2(amino acids 248-263) identified by FUJIIet al. (1 988)as one of six evolutionarilly conserved regions within the ND2 protein (Table 5) (FUJII et al. 1988).Hence, this mutation probably disrupts electron flow through respiratory complex I. Finally, the np 5244 mutation is heteroplasmic. This indicates that the mutation arose quite recently (LOTT,VOLJAVEC and WALLACE 1990) andis thus an important factor in the blindness of this proband. Even though the np 5244mutation meets all of the criteria of an LHON mutation, phylogenetic analysis raises the possibility that in its heteroplasmic state it is not sufficient to cause blindness. This mutation is the most recent in a series of potentially deleterious mtDNAmutations (np13708,15257and15812) which have accumulated sequentially in the patient harboring the 5244 mutation.The np 5244mutation could, therefore, be a recent additional mtDNA mutation which pushed this patientover the disease expression threshold, therefore servingas an example of an exacerbating genetic factor. It is not yet clear if this mutation alone in a homoplasmic state would be sufficient to cause blindness. Further determinants of

LHON could include environmental insults or mutations in nuclear genes such as X chromosome alleles. T h e detection of LHON mutations at low frequencies in the “unaffected” general population is a new observation. However, this is not necessarily at variance with studies on the 11778 mutation since significantly fewer unaffected controls have been assayed for the 11778 mutation than were used in this study. It is possible that many deleterious mtDNA mutations are maintained at low frequencies in the general population. Mitochondrial DNA mutations associated with LHON have now been foundin respiratory complexes I (ND1, ND2, ND4 and ND5) and I11 (WALLACE et al. 1988; HOWELLet al. 199 1; JOHNS and BERMAN 1991). T h e differentmitochondrial enzyme complexes implicated and thepossibility ofX chromosome involvement (VILKKI et al. 1991) notonly underscores the complex, multifactorial nature of this disease, but also provides information about the pathophysiological mechanism of the disease. A molecular defect in a single respiratory complex cannot account for all occurrences of LHON.Rather,the manifestation of clinical symptoms must relate to a common function of complex I and 111, the process of generating mitochondrial ATP. Itis therefore plausible that a number of different mtDNA mutations, either alone (if severe enough) o r acting synergistically with other mtDNA or nuclear gene mutations,result in LHON if cellular energy production falls below a maintenance threshold level. The authors would like to thank T. SCHURR and J. SHOFFNER for helpful discussionsand GREGORY KOSMORSKYfor LHON patient referrals. We would like to thank the Clinical Research Center of the Emory University School of Medicine, supported by NIH M o l RR-00039, for their assistance in the establishment of lymphoblast cell lines of individuals PS, P1, P2, P3 and CC. The continual assistance of J. HODCEin cell line maintenance, expansion, and

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storage is also gratefully acknowledged. The work was conducted under the auspices of theEmory Center for Neuromuscular Disease and was supported by National Institutes of Health Postdoctoral Fellowship Grant NS09042 (M.D.B.) and by National Institutes of Health Grant NS21468 (D.C.W.).

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