The mitochondrial 12S rRNA A1555G mutation is a hot spot for deafness-associated mutations in the Chinese population . This mutation has been detected in up to 60% of hearing-impaired patients with previous exposure to aminoglycosides and in 0.09–0.70% of the general population [9, 10]. The incidence of this mutation is much lower in nonsyndromic hearing loss than in aminoglycoside-induced hearing impairment. In a Chinese pediatric population, Lu et al. reported that the incidence of the A1555G mutation was 1.43% in nonsyndromic hearing loss and 10.41% in aminoglycoside-induced hearing loss . Although the contribution of the mtDNA A1555G mutation to congenital (prelingual, early childhood onset) deafness is minor, mitochondrial involvement is seen in patients with postlingual hearing impairment, a much larger population.
There is no treatment for mitochondrial hearing impairment, suggesting that testing for the mutation should be performed routinely before administering aminoglycoside antibiotics. Detection of the A1555G mutation is an important part of deafness gene screening. Sanger sequencing was long the gold standard for identifying unknown mtDNA point mutations before the advent of massively parallel sequencing analysis. However, the conventional Sanger sequencing does not have the sensitivity to detect heteroplasmic mutations below about 20%. NGS technologies have the capability of massively parallel sequencing and offer a robust platform for comprehensive analysis of mtDNA . The small size of the mitochondrial genome resulting in high coverage at each nucleotide position enable more rapid, sensitive, and accurate quantification of low-level heteroplasmy. NGS is a powerful tool for detecting low-level heteroplasmy variants in the mitochondrial genome, which has greatly improved the ability to distinguish carriers from non-carriers. The sensitive detection and accurate quantification of pathogenic heteroplasmic changes are helpful for risk prediction and genetic counseling.
In our study, targeted NGS showed that subject II:2 is a heterogeneous carrier of the mitochondrial 12S rRNA A1555G mutation at a level of 28.68% heteroplasmy. The son of II:2 (III:2) is also a heterogeneous mutation carrier at a level of 7.25% heteroplasmy. The brother (II:1) and mother (I:1) had 0.23% and 0.03% A1555G heteroplasmy. The level of mitochondrial 12S rRNA A1555G heteroplasmy tested by NGS in the 100 cases with normal hearing and 100 patients with severe sensorineural hearing loss negative for GJB2, SLC26A4, and mtDNA 12S rRNA was less than 1% and did not differ significantly (P > 0.05) between these two groups.
The presence of the mtDNA mutation in subject II:2 but not in her mother has three possible explanations: (1) other family members in the maternal lineage harbor the mutation, but at levels below the detection limit; (2) there is no biological relationship between subject II:2 and other family members tested; or (3) a de novo mutation occurred. We investigated all three possibilities.
To investigate the possibility that the mutation is present at a level below the sensitivity of the sequencing method, we sequenced the mtDNA at an average depth of 5000 × using NGS technology. The Ion Proton sequencing platform is sensitive enough to detect point mutations present with heteroplasmy levels as low as 1%. In the control groups, the level of mitochondrial 12S rRNA A1555G heteroplasmy was less than 1%, confirming the accuracy of this method. The probability of multiple false-negative results in the brother (II:1) and mother (I:1) is very low. Therefore, it is unlikely that other family members in the maternal lineage also carry the mutation at heteroplasmy levels below the detection limit.
To investigate non-kinship as the potential cause, we confirmed kinship from the mothers within the family. The paternity of the three generations (I:1, II:1, II:2, and III:2) was confirmed by genotype analysis of 15 informative STRs, yielding a probability of paternity of 0.999999, assuming a prior probability of 0.50. Having excluded kinship and sensitivity issues, we conclude that the A1555G mutation most likely appeared de novo in the family.
The A1555G mutation has been detected in patients with different haplotypes, indicating that de novo appearance of the A1555G mutation has occurred frequently in the past. MtDNA is solely inherited maternally and does not recombine, consequently, mutations accumulate in maternal lineages. According to most literature reports, mitochondria have relatively less sophisticated DNA protection and repair systems and high mutation rates . But there are very few reports about de novo mutation in mitochondria, so the frequency of de novo mutation in mitochondria is not clear. In our previous study, we found that among 193 families carrying mitochondrial A1555G mutation, only one family had de novo mutation. So the frequency of de novo mutation in mitochondrial A1555G is 0.52% based on our data. Although the mechanism behind the de novo appearance of the A1555G mutation is unknown, we speculate that the mutation likely occurred during oogenesis (during embryonic development of the mother) or during early embryonic development of subject II:2.
There has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. This study did not verify the mitochondrial genetic maternal model. In the future research, we will supplement this part of work. In Fig. 1, we show the genetic relationship between I:1/II:2 and II:2/III:2 according to the law of maternal inheritance.
The observation of a de novo A1555G mutation is relevant for both diagnostic investigations and genetic counselling. First, even when there is no (maternal) family history in patients with deafness, A1555G mutation screening should still be performed to identify the cause of the disease. Second, screening in maternally related family members is recommended to provide reliable counselling for these families, given that the A1555G mutation may have arisen de novo. A genetic diagnosis of the A1555G mutation in an isolated patient does not necessarily mean that others in the maternal lineage also harbor the mutation. Although the vast majority of A1555G mutations are inherited maternally, a thorough family investigation should always be performed. Genetic counselling for deafness caused by the A1555G mutation is complicated. In individuals treated with aminoglycosides and thus at risk of hearing loss, mtDNA analysis can help predict hearing loss and the need to take precautions before symptom onset, as well as enable more accurate genetic counseling.