We included 30 subjects in the study cohort: 28 participants belonged to 10 families; the remaining 2 participating were unrelated (
Supplementary Fig. S1). Clinical findings (when available) including sex, molecular genetic testing results, LHON risk factors, recovery of vision and response to Idebenone therapy, are reported in
Table 1. In 12 subjects (male:female ratio, 10:2) the diagnosis of LHON was based on unilateral and severe visual decline followed, within a few weeks, by declining vision in the contralateral eye. Ophthalmologic examinations revealed the presence of a centrocecal scotoma, the presence of an edematous, hyperemic optic nerve head, tortuosity and telangiectasia of vasculature and, finally, the absence of leakage and staining using fluorescein angiography. Visual acuity ranged between light perception and 20/25 (
Table 1). Patients with these features comprised the Affected cohort; those patients lacking these features were Carriers. The 90 unrelated subjects (male:female ratio, 47:43), the Control group, exhibited no history of retinal disease, eye trauma or surgery, nor any evidence of systemic or neurologic disease. The diagnosis of LHON was confirmed by mitochondrial genetics testing: the m.11778G>A mutation was identified in 14 subjects (2 homoplasmic; 12 heteroplasmic) and the m.3460G>A in 16 subjects (1 homoplasmic and 17 heteroplasmic) (
Table 1;
Supplementary Fig. S2). None carried the m.14484T>C primary mutation. The mean frequency of the appearance of mutant alleles for both mutations of heteroplasmic subjects was 70% for the Affected (range, 15%–95%) and 53% for the Carriers (8%–95% range) (
Table 1). The penetrance value of both LHON mutations was 40% (12/30) in our LHON cohort, 71% (10/14) in male and 12% (2/16) in female. Among the 14 subjects (8 males; 6 females) who harbored the m.11778G>A mutation, six (5 male; 1 female) developed typical optic neuropathy with a total of 43% phenotype penetrance. In this group, the penetrance value of 83% in men was five times higher than that for woman. Among the 16 subjects (6 males; 10 females) who harbored m.3460G>A mutation, six (5 males; 1 female) were affected with a total of 37% phenotype penetrance. In this group, the penetrance value of 83% in men was eight times higher than that for women. One m.11778G>A patient recovered vision without Idebenone treatment (
Table 1). The determination of mtDNA copy numbers was performed independently from the type of LHON primary mutations in all the heteroplasmic subjects (5 Affected and 18 Carriers) and all compared with Controls. We could not quantify the mtDNA content for three homoplasmic Affected (II-1 FAM-A2; III-2 FAM-A4; III-1 FAM-B4;
Table 1) and for four heteroplasmic Affected (II-1 FAM-A1; II-3 FAM-A3; I-1 FAM-A5; II-1 FAM-B3;
Table 1) due to the insufficient quantity of useful genetic material. The mtDNA copy number distribution was highly variable between the Affected and Carriers. Using very strict criterion, the Carriers showed a different distribution with respect to either the Affected or the Controls who could not be distinguished (i.e., the probability of the null hypothesis < 0.001) (
Table 2). Conversely, when the distribution of the two LHON populations was compared with the heteroplasmy of mutations, no statistically significant differences were observed (not shown). Panel A of the Figure shows that all the Affected fall in the same range of mtDNA copy number as the Controls. The mean value of the mtDNA copy number showed that the peak of mtDNA content shifted progressively toward higher values from Controls (210 ± 66) to Affected (293 ± 85) to Carriers (488 ± 140) (Controls versus Carriers
P < 0.001; Affected versus Carriers
P < 0.001; Controls versus Affected did not reach statistical significance; ANOVA test) (
Fig. panel B). It is important to note that two affected subjects (II-5 FAM-A4; I-1 FAM-B7) having the lowest quantities of mtDNA had been exposed to ethanol, tobacco, and controlled substances, all of which have been shown to decrease mtDNA amounts and considered as risk factors for the development of LHON.
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