Application Note

Leber’s hereditary optic neuropathy (LHON) is a classic mitochondrial disease, associated with a rapid, pain­less, acute or sub‑acute bilateral visual loss in young adults, predominantly caused by primary and secondary mutations in mitochondrial DNA (mtDNA). mtDNA is a double‑stranded 16,569‑nucleotide pair, circular molecule, consisting of one D‑Loop region and 37 genes. There is a variance in the reported incidence of known LHON causative mtDNA mutations between populations of different ethnic backgrounds.

Researchers at the Zhejiang University School of Medicine set out to investigate the spectrum and incidence of mtDNA mutations associated with LHON in a Chinese Han population in an effort to advance the current understanding of the clinical phenotype of LHON, and provide useful information for early diagnosis.

To further understand the spectrum of mutations associated with LHON in this ethnicity, the researchers focused on 46 LHON‑associated mutations distributed among 13 mitochondrial genes and performed VariantPro targeted sequencing in 275 cases of LHON as well as in 281 Chinese control subjects.

The VariantPro Mitochondrial Panel is a multi-gene panel designed for 100% amplicon coverage (16,569 bp) of the mitochondrion genome. It offers cost-effective, minimal hands-on, ultra-high resolution sequence analysis for accurate detection of rare variants in mtDNA. The VariantPro panel along with high‑throughput sequencing makes it possible to screen multi‑genes or multi‑single nucleotide polymorphisms (SNPs) for many subjects in a single sequencing run. The incidence of mutation revealed by the VariantPro panel regarding primary and secondary mutations may be indicative of disease when subjects are lacking other clinical symptoms.

Total no. of reads118,156,518
Reads mapped to the amplicons(forward primer)110,440,487
Reads mapped to the amplicons(reverse primer)108,812,285
Reads mapped to the amplicon targets105,107,193
Reads mapped to each amplicon (average)3,892,859
Reads mapped to each sample per amplicon (mean)7,001.5
Reads enrichment to the targets, (average)89%
Uniformity of coverage, (20% mean)98%
Total no. of variants among 46 point mutations363


Summary of sequencing data in the panel for 556 samples


Mean coverage of 27 amplicons. Upper: Average read coverages (log 10) for each amplicon (556 samples). Lower: Read coverages (log10) for each amplicon from one sample.

Twenty‑three mutations in the panels were absent in the patients and control cohort in the current study. Those mutations, reported as rare causative mutations in a European LHON population, were undetected in the present study. However, 6 causative mutations, were observed in 194 LHON cases. Additionally, the spectrum of secondary mutations associated with LHON was also dependent on their ethnic background, and were distinct between the Chinese and Caucasian cohorts. This panel screening demonstrated that some secondary mutations had higher frequencies in the patient cohort, as these mutations were assigned to Asian mtDNA lineage. It was confirmed that the spectrum of mutations varied between ethnic backgrounds and indicated that the selected SNPs of the panel would be optimized for a Han population in the future.

In this study, the high mapping rate and uniformity of coverage confirmed the VariantPro system works very well and sequencing results generated from VariantPro prepared libraries are reliable. The sequencing results indicate that the spectrum and incidence of mtDNA mutation‑associated LHON cases in the Han population are different to those in a Caucasian population. This finding was comparable with previous studies.

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