naica® Droplet Chip Digital PCR Detects Mitochondrial Heteroplasmy after Nuclear Transfer, Obtaining Results in 2.5 Hours

Mitochondrial diseases are a type of genetic disorder caused by genetic mutations in the mitochondrial genome (mtDNA), which are transmitted only through female lineages. Usually, mutations in over 60% of mitochondrial DNA in cells can lead to diseases, and the more mitochondrial DNA mutations a person has, the more severe their disease becomes.

Currently, nuclear transfer (NT), also known as mitochondrial donation, has received increasing attention as a strategy to prevent mitochondrial diseases from being transmitted from diseased mothers to their offspring. However, due to the presence of a small amount of cytoplasmic derived mtDNA in nuclear transplantation, which mediates heterogeneity and amplification of mtDNA in the recipient, accurate quantification of mtDNA mutation load is required. The limitations of current NGS sequencing methods are high cost, long time consumption, complex data processing, and low signal-to-noise ratio.

Leber hereditary optic neuropathy (LHON) is the most common maternal inherited mitochondrial disease. A team of experts from the Department of Biology at Ghent University in Belgium used the digital PCR (dPCR) platform to detect the m.11778 G>A mutation site related to LHON, and compared it with the NGS method to explore the applicability of digital PCR in mtDNA heterogeneity quantification. The research results were published in the renowned journal Clinical Chemistry.

To evaluate the applicability of dPCR in heterogeneity assessment, three types of samples were set up: (i) 13 samples from patients with high mutation burden; (ii) Three homogeneous wild-type samples donated by healthy volunteers; (iii) Samples treated with NT carry low mutation load due to mtDNA residue, totaling 6 samples.

Testing method:

By processing, the sample mutation load range is set at 50% to 0.01% for analysis and validation.

Experimental conclusion:

The mutation rates observed in dPCR and NGS results showed good consistency.

Compared to NGS, dPCR has lower background noise. Using Naica® droplet chip digital PCR system showed no positive signal for mutant alleles in non patient samples, as expected.

Compared with the dPCR results, the secondary allele frequencies of almost all heterogeneous samples were overestimated in the NGS results. It is preliminarily speculated that incorrect sequences may have been introduced in the NGS experimental process, and the secondary allele frequencies were altered through PCR amplification.

Compared to NGS, digital PCR has lower cost, simpler operation, more intuitive results, and is more suitable for low-frequency mutation detection.

The digital PCR method is suitable for quantitative detection of mitochondrial heterogeneity after nuclear transplantation.