We read with interest the article by Öncül et al. about 16 pediatric patients with suspected mitochondrial disorder (MID) undergoing next generation sequencing for mtDNA variants [1]. Thirteen patients underwent mtDNA sequencing, 2 patients whole exome sequencing (WES), and 1 patient whole genome sequencing (WGS) [1]. Seven variants were found in protein-coding genes, 4 in tRNA genes, and 1 in an rRNA gene [1]. Only two variants were classified as pathogenic, three as likely pathogenic, and 7 seven as variants of unknown significance(VUS) [1]. The study is appealing but raises concerns that require discussion. 

A limitation of the study is that the pathogenicity of new mtDNA variants was assessed by in silico bioinformatic methods using the Protter and Phyre2 programs but not by functional tests. Pathogenicity of mtDNA tRNA variants is commonly assessed by application of the Yarham score relying on the number of independent reports, presence of heteroplasmy, segregation of the disease with the variant, biochemical investigations, segregation of the variant with biochemical investigations in single fiber studies, pathogenicity on trans-mitochondrial cybrid studies, normality on trans-mitochondrial cybrid studies, and the evolutionary conservation of the variant [2]. 

A further limitation is that only eight mothers were tested for mtDNA variants and that heteroplasmy rates were provided for none of these mothers tested for their offspring’s tDNA variant [1]. As heteroplasmy rates in inherited MIDs may increase in subsequent generations, it is crucial to know them for both mothers and their offspring. 

Patients 4 and 5 were negative for mtDNA variants despite suspicion of a MID. However, in the method section it is mentioned that the included patients did not have any other genetic disorders. How were other genetic disorders excluded in the two patients negative for mtDNA variants? We should know if these two patients carried mutations in nuclearly encoded genes involved in mtDNA maintenance or other mitochondrial functions.

Missing is the information how many of the 16 patients had consanguineous parents. This information is crucial as MIDs occur more frequently in offspring of consanguineous parents [3] and as consanguinity is more frequent in Turkey as compared to some other countries with a frequency of 24% [4].

We do not agree with the conclusion that patients with Leigh syndrome should be first investigated by mtDNA sequencing [1]. Since Leigh syndrome is predominantly caused by mutations in genes located on the nuclear DNA [5]. WES should be preferred. Only in patients with a maternal trait of inheritance or patients with non-informative WES, maternally inherited Leigh syndrome (MILS) should be excluded or confirmed by mtDNA sequencing.

Four patients had a visual disorder [1]. We should be told which type of visual problem was found by which means. How to explain that patients 1 and 16 had cataract surgery but now visual disorder? This discrepancy should be solved.

The term “low congenital myopathy” in table 1 is unclear [1]. We should know if the patient with suspected MID was lastly diagnosed with congenital myopathy. 

Overall, the interesting study has several limitations which challenge the results and their interpretation. Addressing these limitations may upvalue the conclusions.

Declarations

• The authors declare no conflicts of interest

• No funding was received

• Author contribution: JF: design, literature search, discussion, first draft, critical comments, FS: literature search, discussion, critical comments, final approval

• Informed consent: not applicable

• The study was approved by the institutional review board