Am. J. Hum. Genet.

53:916-920, 1993

Pitfalls in the Molecular Genetic Diagnosis of Leber Hereditary Optic Neuropathy (LHON)
Donald R. Johns* and Michael J. Neufeldt
Department of Neurology, Beth Israel Hospital, Boston; and tDepartment of Neurology, Johns Hopkins University School of Medicine, Baltimore

Summary Pathogenetic mutations in mtDNA are found in the majority of patients with Leber hereditary optic neuropathy (LHON), and molecular genetic techniques to detect them are important for the diagnosis. A false-positive molecular genetic error has adverse consequences for the diagnosis of this maternally inherited disease. We found a number of mtDNA polymorphisms that occur adjacent to known LHON-associated mutations and that confound their molecular genetic detection. These transition mutations occur at mtDNA nt 11779 (SfaNI site loss, 11778 mutation), nt 3459 (BsaHI site loss, 3460 mutation), nt 15258 (AccI site loss, 15257 mutation), nt 14485 (mismatch primer Sau3AI site loss, 14484 mutation), and nt 13707 (BstNI site loss, 13708 mutation). Molecular genetic detection of the most common pathogenetic mtDNA mutations in LHON, using a single restriction enzyme, may be confounded by adjacent polymorphisms that occur with a false-positive rate of 2%-7%.

Introduction Leber hereditary optic neuropathy (LHON) is a maternally inherited form of painless, acute visual loss that occurs predominantly in young men (Nikoskelainen et al. 1987). A number of mtDNA mutations have been associated with LHON (Wallace et al. 1988; Howell et al. 1991a, 1991b; Huoponen et al. 1991; Johns and Berman 1991; Johns and Neufeld 1991; Brown et al. 1992a, 1992b; Johns et al. 1992; Mackey and Howell 1992). The 4160 mutation has been found in a single family with optic neuropathy and a variety of central nervous system abnormalities (Howell et al. 1991b). The purported LHON-associated mtDNA mutation at nt 7444 (Brown et al. 1992c) was not detected in any of our LHON probands or the Australian families reported by Mackey and Howell (1992). Molecular genetic testing is an important component of the diagnosis of LHON and allows the identification of atypical cases that could not be reliably diagnosed on the basis of purely clinical criteria. These molecular geReceived January 27, 1993; final revision received June 10, 1993. Address for correspondence and reprints: Donald R. Johns, M.D., Department of Neurology, Beth Israel Hospital, 330 Brookline Avenue, Libbey 226, Boston, MA 02215.
t 1993 by The American Society of Human Genetics. All rights reserved.

netic tests, like any other diagnostic methods, are subject to errors of sensitivity and specificity. False-positive errors with regard to a primary pathogenetic mutation have particularly adverse consequences, because they may contribute to an improper genetic diagnosis for the proband and all of his maternal relatives. We report our extensive results with molecular genetic testing of LHON, and we document mtDNA polymorphisms that create false-positive results for many of the tests currently performed.

Material and Methods

0002-9297/93/5304-0016$02.00

Molecular genetic analysis for the mtDNA mutations associated with LHON was performed on blood samples of 350 patients with acute visual loss. DNA was extracted by standard proteinase K/SDS methods, and PCR amplification was performed as referenced below for each mutation. The eight mtDNA mutations were screened for by the loss of a restriction site (fig. 1) and then were verified by means of a second molecular genetic method. All nucleotide positions are numbered according to the canonical Cambridge human mtDNA sequence (Anderson et al. 1981). The 11778 mutation (subunit 4 of NADH dehydrogenase [ND-4] gene) was screened for by the loss of an SfaNI restriction site and was verified by the creation

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Molecular Genetic Pitfalls in LHON

A. 1 1778 Mutation:
WT

917
G. 3394 Mutation:

11778 Mutation
S H I AGT CAC ATC Maeill (+) SfaNI (-)

11779 Mutati on S R I AGT CGT ATC MaeIII (-) SfaNI (-)

WT

3394 Mutation
6 H GGC CAT LI Haelll (+)

S R I AGT CGC ATC Maelll (-)

SfaNI (+)

G Y GGC TAT

Haelll (-)

H. 4160 Mutation:

B. 3460 Mutation:
WT

WT

4160

3460 Mutation D T GAC ACC BsaHI (-)

3459
Mutation

Mutation
D Q L M GAC CA CTC ATA
L
J

D A GAC GCC BsaHI (M)

C. 15257 Mutation:

D A GAT GCC BsaHI H-)

P M D Q GAC CA CCC ATA

Alul (-)

Alul (+)

(F) = mismatched nucleotide in amplification primer

WT
V D S GTA GAC AGT AccI (+)
D. 14484 MutationWT
V M M AAC AT CAT

15257 Mutati on
V N S GTA AAC AGT

15258 Mutation
V G S GTA GGC AGT Accl H-)

Accl (-)

14484
Mutation
V V M AAC AC CAT

14485
Mutation M M V AAC AT TAT

Schematic representation of the nucleotide seFigure I quences surrounding eight mtDNA mutations associated with LHON, five confounding mtDNA mutations, the corresponding wild-type ("WT") sequences, and the amino acids encoded by them. The restriction enzymes used to detect each mutation are given. A plus sign (+) denotes that a restriction site is present, and a minus sign (-) denotes that a restriction site is absent. The mutant nucleotide is underlined. Amino acid abbreviations are as follows: S = serine; R = arginine; I = isoleucine; H = histidine; D = aspartic acid; A = alanine; T = threonine; V = valine; N = asparagine; G = glycine; M = methionine; L = leucine; Y = tyrosine; Q = glutamine; and P = proline.

Sau3AI (+)
(
=

Sau3AI (-)

Sau3AI (-)

mismatched nucleotide in amplification primer

E. 13708 Mutation:
WT

13708 Mutation R L T A CGC CTG ACA GCC

13707 Mutation R L A A CGC CTA GCA GCC

R L A A CGC CTG GCA GCC

BstNI (+)
Fnu4HI (+) F. 15812 Mutation: WT
V L GTA CTA Rsal (+)

BstNI (-)
Fnu4HI (-)

BstNI (-)
Fnu4HI (+)

15812
Mutation
M

L
(-)

ATA CTA

Rsal

of an MaeIII site (fig. 1A) Uohns 1990; Stone et al. 1990). The 3460 mutation (subunit 1 of NADH dehydrogenase [ND-1] gene) was screened for by the loss of a BsaHI restriction site (fig. 1B) and was verified by direct sequencing (Johns 1992). The 15257 mutation (apocytochrome b gene) was screened for by the loss of an AccI restriction site (fig. 1C) and was verified by direct sequencing (Johns and Neufeld 1991). The 14484 mutation (subunit 6 of NADH dehydrogenase gene) was screened for by a mismatch primer method that eliminated a Sau3AI restriction site in the presence of the 14484 mutation (fig. 1D) and was verified by direct sequencing (Johns et al. 1992, 1993a). The 13708 mutation (subunit 5 of NADH dehydrogenase gene) was screened for by the loss of a BstNI restriction site and was verified by the simultaneous loss of an overlapping Fnu4HI restriction site (fig. 1E) (Johns and Berman 1991). The 15812 mutation (apocytochrome b gene) was detected by the loss of an RsaI restriction site (fig. 1F) (Johns and Neufeld 1991). The 3394 mutation

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Johns and Neufeld

Discussion

(ND-1 gene) was detected by the creation of an HaeIII site (fig. 1G) (Johns et al. 1992). The 4160 mutation (ND-1 gene) was screened for by a mismatch primer method that eliminates an AluI restriction site in the presence of the 4160 mutation (fig. 1H).
Results

The 11778 mutation was detected in 87 independent LHON families by concordant loss of the SfaNI site and gain of an MaeIII site. Two other families lost the SfaNI site without gain of an MaeIII site, and direct sequence analysis revealed that both had a silent C-to-T transition at mtDNA nt 11779. One of these two families was heteroplasmic for the 11779 mutation. Another family lost an MaeIII site elsewhere in the ND-4 gene at nt 11654 because of an A-to-G transition that changed poorly conserved threonine-298 to alanine. The 3460 mutation was found in 14 independent LHON families. An additional family lost the BsaHI site but was found to harbor a silent C-to-T transition at nt 3459. The 15257 mutation was found in 16 independent LHON families. An additional proband lost the AccI restriction site but was found to have an A-to-G transition at nt 15258 which changed aspartate-171 to glycine. The 14484 mutation was detected in 19 independent LHON probands by the loss of an Sau3AI restriction site and was verified by direct sequence analysis. One sample that screened positive was found to harbor a silent C-to-T transition at nt 14485 that also eliminated the Sau3AI site with the mismatch primer. The 13708 mutation was found in 49 independent LHON families (16 had the 11778 mutation, 4 had the 3440 mutation, 12 had the 14484 mutation, and 15 had the 15257 mutation) by simultaneous loss of BstNI and Fnu4HI restriction sites. One other patient lost the BstNI site but preserved the Fnu4HI site. Direct sequence analysis revealed that he harbored a silent G-toA transition at nt 13707. Another proband had a silent A-to-G transition at nt 13824 that created a BstNI restriction site. The 3394 mutation was detected in 13 independent LHON families, and one additional patient had an HaeIII site loss nearby in the ND-1 gene because of a silent C-to-T transition at nt 3429. The 15812 mutation was detected in eight independent LHON probands, all of whom harbored the 15257 primary mutation, and no false-positive RsaI site losses were detected. The 4160 mutation was not found in any of the 350 patients.

Molecular genetic techniques can now detect pathogenetic mtDNA mutations in the majority of patients with LHON, and these techniques are an important component of the diagnosis of LHON. Our data deal exclusively with false-positive diagnostic errors in the molecular genetic techniques themselves, but there are much broader considerations in terms of the role of mtDNA mutations in the causation of LHON. The pathogenetic role of the LHON-associated mtDNA mutations appears to be variable. A primary mutation should occur exclusively in multiple LHON families, while a secondary mutation may be found in both normal families and at a statistically significant increased frequency in LHON families (Johns et al. 1992). Secondary mutations are usually found in conjunction with primary mutations or other secondary mutations. Thus the 11778 (Wallace et al. 1988), 3460 (Howell et al. 1991a; Huoponen et al. 1991), and 14484 (Johns et al. 1992; Mackey and Howell 1992) mutations appear to be primary mutations, and the 3394 (Brown et al. 1992a; Johns et al. 1992), 13708 (Johns and Berman 1991; Johns and Neufeld 1991; Brown et al. 1992a, 1992b; Johns et al. 1992), and 15812 (Johns and Neufeld 1991; Brown et al. 1992b) mutations appear to be secondary mutations. In a recent comprehensive review of the molecular genetics of LHON, Newman (1993) concludes that the 11778, 3460, 14484, and 15257 mutations are primary mutations. The 15257 mutation is strongly associated with LHON, but not to the absolute degree that the three primary mutations are associated with LHON. The 15257 mutation has been found in 20 independent LHON families, but it is only 99.7% specific for LHON (Brown et al. 1992b; Johns et al. 1993b; Newman 1993). The extraordinary power of molecular genetic testing is tempered by the adverse consequences of a falsepositive result for a primary mutation. Molecular genetic data must be properly integrated into the entire clinical context of the patient's visual loss in order to arrive at a diagnosis of LHON. This limitation applies most stringently to the secondary mtDNA mutations, and a patient should not be given the diagnosis of LHON solely on the basis of their presence. mtDNA is highly polymorphic, and thus the high incidence of false-positive restriction digests noted is expected. The 11778 mutation, which is the primary pathogenetic mutation in the majority of LHON patients, is usually detected by the loss of an SfaNI restriction site.

Molecular Genetic Pitfalls in LHON

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Since a mutation in any one of the five nucleotides that constitute this site will eliminate it, a simultaneous gain in an MaeIII site was proposed as a means to avoid a false-positive result (Johns 1990; Stone et al. 1990). The two families reported here with the 11779 mutation establish the need for this dual restriction-digestion method and prove its efficacy. By properly detecting the confounding silent polymorphism, misdiagnosis of the probands as having LHON was avoided, and thus the consequences of a false-positive diagnosis for their entire maternal lineages were obviated. The ratio of false-positive:true-positive results is 2:87 (2.3%). The 3460 mutation, like the 11778 primary mutation, has been found only in LHON probands or their maternal relatives. The silent polymorphism at nt 3459 also eliminates the BsaHI site and could have caused a false-positive diagnosis. The ratio of false-positive: true-positive results is 1:14 (6.7%). Molecular genetic detection of the 15257 mutation solely on the basis of the loss of an AccI restriction site has a ratio of false-positive:true-positive results of 1:16 (5.9%). The 14484 mutation can be screened for by a mismatch primer technique that has a ratio of false-positive: truepositive results of 1:19 (5%). The 13708 mutation, which is second only to the 11778 primary mutation in frequency in the LHON population (Johns et al. 1992), is reliably detected by the simultaneous losses of BstNI and Fnu4HI restriction sites that overlap only at nt 13708 (Johns and Berman 1991). The false-positive rate for the BstNI digestion alone is 1:50 (2%). The presence of restriction site polymorphisms near the region of interest could potentially complicate the molecular genetic analysis of the 11778 mutation (MaeIII site loss), the 13708 mutation (BstNI site gain), and the 3394 mutation (HaeIII site

techniques that create novel restriction enzyme recognition sites. Mismatch primer methods should be designed such that the mutation of interest is detected by the loss of a restriction site that is present with the wild-type sequence. Any patients who screen positive are then investigated with direct sequence analysis of this mtDNA region to verify the mutation.

Acknowledgments
The authors wish to thank the numerous ophthalmologists and neurologists who submitted blood samples from their patients with visual loss. We would also like to thank David Mackey, M.D., Wilmer Ophthalmological Institute, for sharing the mismatch primer sequences, and we thank Paul Rothberg, Ph.D., University of Kansas School of Medicine, for providing DNA from the patient with the 11654 mutation. This work was supported by National Institutes of Health grant NS 01359. D.R.J. is the recipient of a Clinical Investigator Developmental Award from the National Institute of Neurological Disorders and Stroke and of a Basil O'Connor Starter Award 5-FY92-1033 from the March of Dimes Birth Defects Foundation.

References
Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457-465 Brown MD, Voljavec AS, Lott MT, McDonald I, Wallace DC (1992a) Leber's hereditary optic neuropathy: a model for mitochondrial neurodegenerative diseases. FASEB J 6:
2791-2799 Brown MD, Voljavec AS, Lott MT, Torroni A, Yang C-C, Wallace DC (1992b) Mitochondrial DNA complex I and III mutations associated with Leber's hereditary optic neuropathy. Genetics 130:163-173 Brown MD, Yang C-C, Trounce I, Torroni A, Lott MT, Wallace DC (1992c) A mitochondrial DNA variant, identified in Leber hereditary optic neuropathy patients, which extends the amino acid sequence of cytochrome c oxidase subunit I. Am J Hum Genet 51:378-385 Howell N, Bindoff L, McCullough DA, Kubacka I, Poulton J, Mackey D, Taylor K, et al (1991a) Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees. Am J Hum Genet 49:939-950 Howell N. Kubacka I, Xu M, McCullough DA (1991b) Leber hereditary optic neuropathy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation. Am J Hum Genet 48:935-942 Huoponen K, Vilkki J, Aula P, Nikoskelainen EK, Savontaus M-L (1991) A new mtDNA mutation associated with

loss). The false-positive molecular genetic results cited above are due to transition mutations immediately adjacent to, or near, the nucleotide position of interest. Any molecular genetic techniques used to detect LHON-associated mutations must rely on unambiguous restriction enzyme digestions (e.g., combined SfaNI site loss and MaeIII site gain to detect the 11778 primary mutation or simultaneous BstNI and Fnu4HI site losses to detect the 13708 secondary mutation) or on direct sequence analysis of the nucleotide of interest after a restriction site loss is detected by screening (e.g., BsaHI site loss to detect 3460 primary mutation or AccI site loss to detect 15257 mutation). Pathogenetic mutations that do not alter a native restriction site are readily detected by mismatch primer

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Leber hereditary optic neuroretinopathy. Am J Hum Genet 48:1147-1153 Johns DR (1990) Improved molecular genetic diagnosis of Leber's hereditary optic neuropathy. N Engl J Med 323: 1488-1489 (1992) Mitochondrial ND-1 mutation in Leber hereditary optic neuropathy. Am J Hum Genet 50:872-874 Johns DR, Berman J (1991) Alternative, simultaneous complex I mitochondrial DNA mutations in Leber's hereditary optic neuropathy. Biochem Biophys Res Commun 174: 1324-1330 Johns DR, Heher KL, Miller NR, Smith KH (1993a) Leber's hereditary optic neuropathy: clinical manifestations of the 14484 mutation. Arch Ophthalmol 111:495-498 Johns DR, Neufeld MJ (1991) Cytochrome b mutations in Leber hereditary optic neuropathy. Biochem Biophys Res Commun 181:1358-1364 Johns DR, Neufeld MJ, Park RD (1992) An ND-6 mitochondrial DNA mutation associated with Leber hereditary optic neuropathy. Biochem Biophys Res Commun 187:15511557

Johns and Neufeld

Johns DR, Smith KH, Savino PJ, Miller NR (1993b) Leber's hereditary optic neuropathy: clinical manifestations of the 15257 mutation. Ophthalmology 100:981-986 Mackey D, Howell N (1992) A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual genetic etiology. Am J Hum Genet 51:12181228 Newman NJ (1993) Leber's hereditary optic neuropathy: new genetic considerations. Arch Neurol 50:540-548 Nikoskelainen E, Savontaus M-L, Wanne OP, Katila MJ, Nummelin KU (1987) Leber's hereditary optic neuroretinopathy, a maternally inherited disease: a genealogic study in four pedigrees. Arch Ophthalmol 105:665-671 Stone EM, Coppinger JM, Kardon RH, Donelson J (1990) Mae III positively detects the mitochondrial mutation associated with type I Leber's hereditary optic neuropathy. Arch Ophthalmol 108:1417-1420 Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AMS, Elsas LJ II, et al (1988) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242:1427-1430