Introduction and purpose

Mitochondrial diseases (MD), caused by mutations in nuclear or mitochondrial genomes (mtDNA), are the most common group of inherited metabolic diseases and affect approximately 1/4300 people (1). Features such as bigenomic control of mitochondrial functions, heteroplasmy and maternal inheritance, and the dynamic organelle structure involving more than a thousand proteins encoded by nuclear DNA (nDNA) make the molecular diagnosis of mitochondrial diseases difficult. Developments in next-generation sequencing (NGS) techniques and the acceleration of their use for diagnostic purposes in genetic laboratories, MH introduced the "genetics first" approach, which is a noninvasive method in diagnosis (2). Whole exome sequencing (WES) has significantly improved diagnostic yield, enabling the diagnosis of monogenic diseases that mimic mitochondria. This study primarily aimed to evaluate the diagnostic effectiveness of NGS in cases with clinical suspicion of mitochondrial disease.

Materials and Methods

The study included 24 different families, who were followed up with a preliminary diagnosis of mitochondrial disease based on clinical, radiological and laboratory findings by Istanbul Faculty of Medicine, Department of Child Health and Diseases, Department of Nutrition and Metabolism, in 2018-2019, and were referred to the Department of Medical Genetics for molecular genetic research. A total of 30 cases were included. Family trees were drawn by questioning at least three generations of the cases, which were evaluated holistically with detailed history, physical examination, radiological and laboratory findings. Single, double or triple whole exome sequencing and mtDNA sequence analysis were performed depending on the consanguinity between the parents and the presence of an affected or healthy sibling. Other laboratory and imaging data were obtained from patient follow-up files. Study, I.U. It was approved by the Istanbul Faculty of Medicine Clinical Research Ethics Committee with date and number 08.06.2018/872 and supported by Istanbul University Scientific Research Projects Unit (Project no: TDK-2018-32544). Wet laboratory studies of WES and whole mtDNA sequencing with the new generation sequencing method were performed on the Thermo Scientific™ Ion Gene S5 System platform in the Medical Genetics AD laboratory of Istanbul University, Istanbul Faculty of Medicine. For mtDNA long PCR, 4 pairs of primers were designed to fragmentally amplify mtDNA consisting of 16,569 base pairs. After sequencing, data were analyzed by automatically transferring to Ion Reporter™ software via Torrent Suite™ software using the IonReporterUploader plug-in.

Results

According to the results of molecular analysis, three of them were mtDNA (MT-ATP6 and MT-TL1) and nine were nuclear (TK2, POLG, EARS2, AARS2, MICOS13, NDUFAF6, FBXL4, ECHS1, OXCT1) and eight of them were novel, detected in mitochondrial disease-related genes. 14 pathogenic variants were reported. Segregation studies confirmed the gene-disease relationship in accordance with the inheritance model. In one case, the c.260C>T (p.Thr87Ile) change detected in the COX10 gene and reported as VUS in the literature could not explain the etiology because it was also detected as homozygous in the healthy sibling. 50% of the selected cohort resulted in mitochondrial disease (15/30, 50%) and 20% (6/30) resulted in Limb Girdle Muscular Dystrophy with known mutations in CAPN3, DYSF, TCAP genes. The molecular basis of 30% (9/30) of the cases could not be elucidated. With the bigenomic next-generation sequencing approach strategy (WES and mtDNA) applied to this cohort, which included 30 affected cases from 24 unrelated families, a diagnostic success rate of 67% (16/24) on an index basis and 70% (21/30) on a patient basis was achieved (Figure one).

Discussion and Conclusion

The increasing use of new generation sequencing techniques in routine laboratories has increased the diagnostic success of mitochondrial diseases, and the non-invasive, bigenomic sequencing approach has led to a change in the algorithm as the step before biopsy in the diagnosis of mitochondrial diseases. Biochemical and pathological examinations requiring muscle biopsy are now performed in unresolved cases or when the diagnosis is not clear after NGS studies (4).

In this last decade, where molecular knowledge about mitochondria is increasing, the concept of "Mitochondrial Disease" is being reconsidered and a new classification system based on genetic origin rather than biochemical deficiency is proposed (5). The increasing use of NGS in the diagnosis of patients with clinical findings suggestive of mitochondrial dysfunction is also clarifying the molecular etiology of other single gene diseases, especially those with neuromuscular findings.

In diagnostic studies conducted by various groups in recent years, the success rate of NGS varies between 45% and 77%. Similar to our results, some studies appear to provide molecular diagnosis for non-mitochondrial diseases due to clinical overlap. In these studies, the diagnostic yield and the detection rate of causative pathogenic variants in non-mitochondrial disease-related genes are respectively; 68% (80/117) and 15% (18/117) (3), 45.70% (53/116) and 9.48% (11/116) (4), 57% (16/28) and 42% (12) /28) (6), 59.3% (67/113) and 17% (19/113) (7), 77.9% (46/59) and 28.8% (17/59) (8) It was reported as.