[Leber's hereditary optic neuropathy]. / Lebersche Optikusneuropathie.

Leber's hereditary optic neuropathy (LHON) is a rare disease primarily affecting the retinal ganglion cells. In most cases patients with LHON develop permanent visual loss with a large central scotoma in the visual field of both eyes. The optic disc becomes partially or completely pale. At the onset of the disease many patients are considered to suffer from an optic neuritis and are treated under the diagnostic and therapeutic regimen of optic neuritis. LHON is mostly only considered when high dose cortisone therapy fails to be effective or the second eye is affected. Thereafter, molecular genetic analysis will prove LHON in these cases. Detailed anamnesis including pedigree analysis in combination with observance of the peripapillary microangiopathic alterations at the fundus will help to speed up the diagnosis of LHON, but even after exact clinical and molecular genetic diagnosis of LHON some aspects of the disease still remain a mystery today.

Genomic rearrangements in OPA1 are frequent in patients with autosomal dominant optic atrophy.

INTRODUCTION: Autosomal dominant optic atrophy (ADOA) is considered as the most common form of hereditary optic neuropathy. Although genetic linkage studies point to the OPA1 locus on chromosome 3q28-q29 as by far the most common gene locus, previous screening studies-based on sequencing of the coding exons-detected OPA1 mutations in only 32-70% of ADOA patients. We therefore hypothesised that larger deletions or duplications that remained undetected in previous screening approaches may substantially contribute to the prevalence of OPA1 mutations in ADOA. METHODS: 42 independent ADOA patients were analysed for the presence of genomic rearrangements in OPA1 by means of multiplex ligation probe amplification (MLPA). Deletions or duplications were confirmed either by long distance polymerase chain reaction (PCR) and breakpoint sequencing or loss of heterozygosity analyses with flanking microsatellite markers. Patients underwent ophthalmological examination including visual acuity, colour vision testings, perimetry and funduscopy. RESULTS: We identified genomic rearrangements in 8 of 42 patients, including single exon deletions of exon 9 and exon 24, respectively, a deletion of exons 1-5, two different deletions of the complete OPA1 gene as well as a duplication of the exons 7-9, with the latter being present in three unrelated families. Patients' phenotypes were highly variable, similar to patients with point mutation in OPA1. DISCUSSION: Our findings show that gross genomic aberrations at the OPA1 gene locus are frequent in ADOA and substantially contribute to the spectrum and prevalence of OPA1 mutations in ADOA patients. They further strengthen the hypothesis that haploinsufficiency is a major pathomechanism in OPA1 associated ADOA.

[How to distinguish between autosomal dominant optic atrophy and Leber's hereditary optic neuropathy]. / Autosomal-dominant erbliche Optikusatrophie vs. Lebersche Optikusneuropathie. Wie kann man sie unterscheiden?

BACKGROUND: Patients with long-lasting bilateral optic atrophy showed typical clinical features of autosomal dominant optic atrophy (ADOA). Molecular genetic analysis identified them as atypical cases of Leber's hereditary optic neuropathy (LHON). METHOD: Three patients with bilateral optic atrophy and central scotomas of their visual fields were clinically diagnosed with ADOA. Samples of lymphocytic genomic DNA were amplified with polymerase chain reaction, and analysis of the coding exons including the flanking intron/UTR sequences of the OPA-1 gene was performed. However, no ADOA-associated mutations were found. We therefore analysed the total lymphocyte mitochondrial DNA for all common LHON mutations in these patients. RESULTS: Three patients from three unrelated pedigrees (two men, one woman) who were clinically diagnosed as suffering from ADOA did not harbor any typical mutation of the OPA-1 gene. However, analysis of their mitochondrial DNA showed that they harbored the 3460, 11778, and 14484 LHON mutations. The patients were identified as atypical cases of LHON. The pedigrees of the patients fulfilled the criteria for both dominant and mitochondrial-maternal transmission in all cases. The clinical picture of LHON differed remarkably from the classic course of LHON. CONCLUSIONS: To identify atypical LHON patients with bilateral optic atrophy and central scotomas in the visual field and to distinguish them from ADOA patients, careful molecular genetic analysis is necessary. In these rare cases, only double examinations of both the genomic and the mitochondrial DNA will allow these patients to be adequately advised.

OPA1 mutations in patients with autosomal dominant optic atrophy and evidence for semi-dominant inheritance.

We and others have shown recently that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein cause autosomal dominant optic atrophy (ADOA) linked to chromosome 3q28-q29. Here we report screening of the OPA1 gene in a sample of 78 independent ADOA families. OPA1 mutations were identified in 25 patients (detection rate 32.1%) including 16 novel mutations. We successfully amplified OPA1 cDNA prepared from leukocyte RNA of three patients, and found the amount of transcripts harboring the Arg366Stop mutation was significantly reduced compared with transcripts derived from the normal chromosome. Analysis of the distribution of OPA1 mutations in ADOA revealed that most missense mutations cluster within the putative GTPase domain, and that there is a preponderance of mutations, which result in premature translation termination. These observations support the notion that haploinsufficiency may represent a major pathomechanism for ADOA. In addition, we identified an ADOA patient who is a compound heterozygote for two OPA1 missense mutations. The fact that this patient is by far more severely affected than her simple heterozygotic parents and siblings implies that at least these OPA1 alleles behave semi-dominantly rather than purely dominantly. Clinical examination revealed considerable variability in disease expression among patients carrying OPA1 mutations and no strict correlation with either the position or the type of mutation.

Segregation patterns and heteroplasmy prevalence in Leber's hereditary optic neuropathy.

PURPOSE: To investigate the segregation pattern of the mitochondrial DNA mutation at nucleotide position 3460 responsible for Leber's hereditary optic neuropathy (LHON) and to determine the prevalence of heteroplasmy for the three primary LHON mutations at positions 11778, 3460, and 14484. METHODS: Segregation analysis was performed in a cross-sectional study by determining the level of heteroplasmy in blood leukocytes of 23 LHON patients and unaffected carriers from four unrelated families. One family comprising two affected and three unaffected carriers was followed over 5.5 years for a longitudinal segregation analysis of heteroplasmy. The percentage of mutant mtDNA was determined using a novel procedure of fluorescence-based primer extension and restriction fragment length polymorphism analysis. The prevalence of heteroplasmy was assessed by determining the number of genealogically unrelated LHON pedigrees with heteroplasmic maternal family members from the LHON patient records of the Department of Ophthalmology, University of Tübingen, Germany. RESULTS: The authors observed a marked variability in the degree of heteroplasmy levels within each pedigree and a tendency toward a higher mutant allele frequency in offspring generations. Disease expression was correlated with higher levels of mutant mtDNA molecules. Longitudinal analysis revealed no statistically significant decrease in the heteroplasmy level in the family studied but a reduction of 11% and 12% in one affected and one unaffected individual, respectively. In 167 genealogically unrelated LHON families the prevalence of heteroplasmy was 5.6%, 40%, and 36.4% for the 11778, 3460, and 14484 LHON mutations, respectively. CONCLUSIONS: Cross-sectional studies of heteroplasmy for the 3460 LHON mutation suggest that the genotype shifts toward a higher mutational load in offspring generations. Long-term decrease in the blood mutant load in single cases indicates negative selection of the mutant allele in the hematopoietic cell system. The prevalence of heteroplasmy varies significantly between the different primary LHON mutations, suggesting genotypical differences in disease expression.

OPA1, encoding a dynamin-related GTPase, is mutated in autosomal dominant optic atrophy linked to chromosome 3q28.

Autosomal dominant optic atrophy (ADOA) is the most prevalent hereditary optic neuropathy resulting in progressive loss of visual acuity, centrocoecal scotoma and bilateral temporal atrophy of the optic nerve with an onset within the first two decades of life. The predominant locus for this disorder (OPA1; MIM 165500) has been mapped to a 1.4-cM interval on chromosome 3q28-q29 flanked by markers D3S3669 and D3S3562 (ref. 3). We established a PAC contig covering the entire OPA1 candidate region of approximately 1 Mb and a sequence skimming approach allowed us to identify a gene encoding a polypeptide of 960 amino acids with homology to dynamin-related GTPases. The gene comprises 28 coding exons and spans more than 40 kb of genomic sequence. Upon sequence analysis, we identified mutations in seven independent families with ADOA. The mutations include missense and nonsense alterations, deletions and insertions, which all segregate with the disease in these families. Because most mutations probably represent null alleles, dominant inheritance of the disease may result from haploinsufficiency of OPA1. OPA1 is widely expressed and is most abundant in the retina. The presence of consensus signal peptide sequences suggests that the product of the gene OPA1 is targeted to mitochondria and may exert its function in mitochondrial biogenesis and stabilization of mitochondrial membrane integrity.

[Leber optic neuropathy with clinical improvement]. / Lebersche Optikusneuropathie mit klinischer Besserung.

BACKGROUND: Spontaneous recovery in Leber's hereditary optic neuropathy is rare. Does the clinical course of Leber's hereditary optic neuropathy (LHON) differ between patients with and without spontaneous recovery? MATERIALS AND METHODS: We compared the clinical and molecular genetic characteristics of 12 visually symptomatic patients having the classical clinical course of LHON who recovered spontaneously with those of 60 who did not. RESULTS: Classical fundus findings and typical visual field defects were comparable in the two groups; vision improved within 18 months in all cases. The worst visual acuity during the acute stage of LHON was 0.03 in the recovery group. Patients with the 3460 and especially the 14484 mutation had a better chance of recovery. No patient with the 11778 mutation who recovered had secondary mutations. Among patients who recovered women were underrepresented and heteroplasmy was more common. Some families showed a raised rate of clinically affected members with recovery. CONCLUSIONS: The clinical picture of LHON remains the same regardless of whether the patient recovers spontaneously. A higher rate of spontaneous recovery characterizes some families. Spontaneous recovery is rare in women. Heteroplasmy is frequent in patients with recovery. Our results show a better clinical course of LHON in patients with the 11778 mutation without secondary mutations. Prognosis is better if the peripapillary microangiopathy is seen for a relatively long period, and there is only partial optic atrophy.

Leber's hereditary optic neuropathy: clinical and molecular genetic findings in a patient with a new mutation in the ND6 gene.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited ocular disease associated with mutations in the mitochondrial DNA (mtDNA). We describe the clinical and molecular genetic findings in a LHON patient and his family with a new mtDNA mutation at np14568 in the ND6 gene. METHODS: Ophthalmological examination was performed in one affected male and two maternal relatives. Direct sequence analysis of the complete mtDNA protein coding region was initiated in the affected patient. Four unaffected maternal relatives also underwent molecular genetic evaluation. RESULTS: Clinical examination of the affected male showed typical features of LHON. In his unaffected mother slight peripapillary microangiopathy was found. Molecular analysis did not show any of the common LHON mutations. A nucleotide exchange was detected at position 14568 replacing a glycine by serine in the ND6 gene. This mutation was the only new mutation found within the entire protein and tRNA coding region of the patient's mitochondrial genome. This novel mutation was also present in four non-affected maternal family members, but absent in 60 other LHON lineages and 175 unrelated controls. CONCLUSION: The new mutation at nucleotide position 14568 lies in the close vicinity of other LHON-related mutations (np14459, np14484, np14498, np14596) within the evolutionarily most conserved region of the ND6 gene. Since no other mutation was detected throughout the mtDNA coding region and the new alteration was excluded in controls, our clinical and molecular genetic findings suggest that the novel point mutation at np14568 is responsible for LHON in this family.

Pupillary light reflexes in patients with Leber's hereditary optic neuropathy.

BACKGROUND: According to a recent pupillographic study, patients with Leber's hereditary optic neuropathy (LHON) show the same pupillary behaviour as normals. Because this raises many questions concerning the real nature of LHON and challenges our concept of the afferent pupillary system, we tried to verify the results of this study. METHODS: Pupillary function was assessed in 34 normal subjects and 40 patients with LHON. Pupillary light reflexes were recorded by means of the Compact Integrated Pupillograph (CIP, AMTech). Under mesopic conditions 200-ms stimuli were presented at two different stimulus intensities. Latency, constriction amplitude and baseline diameter were defined automatically. Pupil light reflexes were compared between LHON patients and normals and between the better and the worse eye in 20 LHON patients with different visual acuities. RESULTS: For both stimuli there were significant differences in latency between LHON patients and controls. The latency of the pupil light reflex proved to be about 20 ms longer for LHON patients, and the amplitude was significantly smaller for the bright stimulus. Within LHON patients, the eyes with the worse visual acuity had a significantly smaller constriction amplitude than the eyes with the better visual acuity. CONCLUSION: The results of our study confirm that LHON really is an optic nerve disease and that the pupillary light reflexes are not normal.

Mapping and genomic characterization of the gene encoding diacylglycerol kinase gamma (DAGK3): assessment of its role in dominant optic atrophy (OPA1).

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27-28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1.

[Leber optic neuropathy in women and children]. / Lebersche Optikusneuropathie (LHON) bei Frauen und Kindern.

BACKGROUND: Leber's hereditary optic neuropathy is associated with point mutations in the mitochondrial DNA (mtDNA) that appear to be pathogenetic for this disease. These mutations affect nucleotide positions 3460, 11778 and 14484. Does the clinical course of LHON differ between men, women and children? MATERIALS AND METHODS: We reviewed the clinical and molecular genetic characteristics of 15 visually symptomatic patients with the clinical diagnosis of LHON (11 women and 4 male children) and compared them with 66 men with LHON. RESULTS: LHON was confirmed clinically and with molecular genetic methods in all cases. Men, women and children showed no differences: Classic fundus findings and typical visual field defects were equally found in both sexes. However, age at the beginning of the disease, severity of LHON and rate of spontaneous recovery differed between groups. Women were older (19-55 years, average 31.3 years) than men (15-53 years, average 24.3 years) at the beginning of the disease. Women suffered more severely from LHON. Spontaneous recovery of vision in women was extremely rare. Many more women had a LHON-affected mother than men. All the affected children (9-14, average 11.7 years at the beginning of the disease) did not have a good visual outcome. CONCLUSIONS: There are some differences in the course of LHON between men and women, concerning age, severity of LHON and rate of spontaneous recovery. Children may also have an unfavorable prognosis.

Ocular symptoms in association with antiphospholipid antibodies.

OBJECTIVE: To describe the clinical and serologic findings of 50 antiphospholipid antibody (APA)-positive patients within a retrospective study. METHODS: Measurement of visual acuity, slit-lamp biomicroscopy, tonometry, fundus examination and perimetry. Laboratory tests were performed for detection of APA against thromboplastin and cardiolipin. Antinuclear antibodies (ANA), antibodies to dsDNA, antithyroidal and antiparietal antibodies were also tested. RESULTS: A combination of both transient and permanent visual disturbances was noticed in more than half of the patients. Transient visual disturbances included transient blurred vision, partial defects of the visual fields and amaurosis fugax. The most frequent permanent abnormalities were optic atrophy in 20 patients, due to AION in 9 cases, and disturbances of the choroidal circulation in 17 patients. Fourty-six patients had positive levels of thromboplastin APA; cardiolipin APA were found to be increased in 36 patients. CONCLUSIONS: We did not find a clear correlation between APA activity or the immunoglobulin classes in the individual and the severity of the ocular disease. The benefit from a therapy with the antiplatelet agent acetylsalicylic acid was evident in a reduction of the patients' transient visual disturbances and, in most cases, no further progression of permanent visual field defects was observed.

[Diagnostic error in Leber's optic neuropathy. Value of clinical and molecular genetic studies]. / Fehldiagnose Lebersche Optikusneuropathie (LHON). Stellenwert klinischer und molekulargenetischer Untersuchungen.

BACKGROUND: Leber's hereditary optic neuropathy is associated with point mutations in the mitochondrial DNA (mtDNA) that appear to be pathogenic for this disease. These mutations affect nucleotide positions 3460, 11,778 and 14,484. MATERIALS AND METHODS: We reviewed the clinical and molecular genetic characteristics of 29 visually symptomatic patients with the clinical diagnosis of LHON. RESULTS: Nine patients really suffered from LHON, but in 20 patients other ocular diseases could be proven. Degeneration of the retina and choroid was most common (seven patients), followed by vascular optic disease (six patients). Three patients suffered from tobacco-alcohol amblyopia, two from optic neuritis and two from autosomal dominant optic neuropathy. CONCLUSIONS: The clinical diagnosis of LHON is strengthened by a proven maternal inheritance and clinical signs such as a severe decrease in visual acuity, central or centrocecal scotomas in the perimetry and pseudoedema of the optic disc, followed by optic atrophy. Pathognomonic clinical signs of LHON are twisted vessels and ectatic capillaries in the fundus of these patients and their relatives of the maternal line, i.e., peripapillary microangiopathy. A careful analysis of the patients' pedigrees, anamnesis and the functional and morphological results of the clinical examinations helps to avoid misdiagnosis of the disease. However, the expensive and time-consuming molecular genetic analysis is always necessary to confirm or exclude the diagnosis of LHON.

Mutation analysis of the ND6 gene in patients with Lebers hereditary optic neuropathy.

DNA sequence analysis of the gene encoding subunit 6 of the NADH-ubiquinone-oxidoreductase complex (ND6) in human mitochondria was performed in 25 independent patients who suffer from Lebers hereditary optic neuropathy (LHON). In 10 cases the well-known LHON mutation at nucleotide position (np) 14484 was detected. Furthermore, silent substitutions at np14167 and np14527 and missense mutations at np14498, np14564, np14568, and np14582 were found in individual patients. The np14498 and np14568 mutations were found in patients who present a typical clinical picture and course of LHON but lack any of the canonical mtDNA mutations. The np14568 mutation, which replaces a moderately conserved glycine by a serine residue, was observed in a single male patient and subsequently excluded in 175 independent controls. The mutation at np14498, which replaces an evolutionarily highly conserved tyrosine with a cysteine, was found in a multigeneration family with four affected members, the eldest carrying a heteroplasmic mixture of mutated and wildtype mtDNA molecules. None of 170 analyzed control subjects carried this mutation. These findings provide evidence that several allelic ND6 gene mutations may be involved in Lebers hereditary optic neuropathy.

Leber's hereditary optic neuropathy: clinical and molecular genetic results obtained in a family with a new point mutation at nucleotide position 14498 in the ND 6 gene.

Mitochondrial DNA mutations at nucleotide position (np) 3460 in the ND 1 gene, np 11778 in the ND 4 gene, and np 14484 in the ND 6 gene are commonly considered to be associated with the clinical features of Leber's hereditary optic neuropathy (LHON) and account for the majority of LHON cases. Recently, a further mutation in the mtDNA at np 14459 was detected. Herein we report the clinical and the most relevant molecular genetic findings obtained in a LHON family with a new mitochondrial DNA mutations at np 14498 in the ND 6 gene. Clinical and historical data were collected over four generations on three affected and five yet unaffected relatives of the maternal line in this family. All three patients and four of their relatives underwent molecular genetic examination. Two patients and five relatives were also studied clinically. All patients exhibited typical clinical features of LHON. In all yet unaffected relatives, various degrees of peripapillary microangiopathy were found. Molecular analysis did not reveal any of the common LHON mutations. Sequence analysis of the mtDNA of one patient was performed and showed a thymine-to-cytosine exchange at np 14498 in the ND 6 gene, leading to the replacement of an evolutionary highly conserved tyrosine by a cysteine residue. The mutation was not found among 70 other LHON lineages and 180 controls. The new mutation at np 14498 lies in the vicinity of the LHON-related mutations at np 14484 and of the recently described mutation at np 14459, in a region constituting the most evolutionarily conserved part of this polypeptide. That the new mutation at np 14498 is found within this highly conserved region and was not present in any controls implies that this mutation is responsible for LHON in this family.

[Correlation between clinical and molecular genetic findings in Leber's optic atrophy]. / Korrelation klinischer und molekulargenetischer Befunde bei Leberscher Optikusneuropathie (LHON).

Leber's hereditary optic neuropathy (LHON) is associated with point mutations of mitochondrial DNA (mtDNA) that appear to be pathogenetic for this disease. These mutations affect nucleotide positions 3460, 4160, 11,778, 14,484, and possibly 15,257. The pathogenetic significance of other mtDNA point mutations (secondary mutations) is less clear. We reviewed the clinical and molecular genetic characteristics of 29 visually symptomatic patients from 26 families. In addition, we studied 54 relatives of the maternal line of these patients. Sixteen of them underwent clinical and molecular genetic examination; 38 underwent only molecular genetic examination. The 29 affected individuals showed a male predominance of 93.1% (27/29) and ages of onset of visual loss ranging from 15 to 55 years. The time interval between affected eyes was never longer than 1 year. Tobacco and/or alcohol abuse was common. Peripapillary microangiopathy was found in 20.7% (6/29) of our patients. The number of patients with peripapillary microangiopathy seems to be small, but we could not examine all patients early after onset of the disease: the time of first examination is critical for the diagnosis of peripapillary microangiopathy. From the 16 relatives who underwent clinical examination, 62.5% (10/16) also had peripapillary microangiopathy. Eighteen patients were analyzed by brain computed tomography or magnetic resonance imaging. Four definitely pathological results seem remarkably high in comparison with the results of other authors. Our LHON patients and their relatives invariably had an identical pattern of point mutations, both primary as well as secondary. Of the LHON patients, 79.3% had the primary mutation at position 11,778, 20.7% at position 3460. Different numbers and combinations of secondary mutations were observed in a large portion of both groups. Three patients with the 11,778 mutation noticed remarkable visual recovery. There was no clear correlation between the type and number of point mutations in the individual and the severity of the disease.

[The antiphospholipid syndrome. The neurological complications and the therapeutic possibilities]. / Antiphospholipid-Syndrom. Neurologische Komplikationen und therapeutische Möglichkeiten.

For several months a 23-year-old woman had been suffering from increasingly frequent attacks of bilateral amaurosis fugax. They affected only individual segments of a monocular visual field and finally occurred several times daily. Physical and cardiological examinations as well as echocardiography were within normal limits. Laboratory tests revealed a slight increase in erythrocyte sedimentation rate and high titres of serum antibodies against phospholipids. Fundoscopy did not show any vascular changes. Because vasculitis--possibly as an expression of systemic lupus erythematodes--was suspected, high-dosage treatment with steroids was commenced, but failed to influence the visual disorder. While steroid dosage was gradually decreased, administration of acetylsalicylic acid (for three months 100 mg daily, then three times daily 100 mg) brought about complete disappearance of the visual signs. Their cause was probably a reversible platelet aggregation induced by antiphospholipid antibodies which, because of the physiological characteristics of the terminal retinal vascular bed brought about the isolated sign of recurrent amaurosis fugax.

Detection of the G to A mitochondrial DNA mutation at position 11778 in German families with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is characterized by acute or subacute bilateral (usually permanent) loss of central vision, caused by neuroretinal degeneration. The maternal inheritance is explained by the mitochondrial origin of the disease. Recently, a single mitochondrial DNA (mtDNA) mutation, a G to A substitution at position 11778 that converts a highly conserved arginine to histidine, has been associated with LHON. The mutation eliminates an SfaNI restriction enzyme recognition site and thus provides a method for detection of the mutation by amplification, enzyme digestion and agarose gel electropheresis of polymerase chain reaction (PCR) products. Leukocyte mtDNA from 7 German families with LHON, diagnosed by clinical criteria, was tested for the presence of the G to A mutation at bp 11778. The mtDNa mutation, detected as a loss of the SfaNI site, was seen in one family. The G to A mtDNA mutation is the only known gene alteration associated with LHON so far. It has been identified in patients of different ethnic origin and recent reports strongly support the hypothesis that it represents the most frequent cause of LHON. Identification of the mtDNA replacement mutation using PCR and restriction enzyme digestion requires only a small amount of blood and can be performed rapidly. This method is thus a useful tool in the diagnosis of LHON.