New perspective in diagnostics of mitochondrial disorders: two years’ experience with whole-exome sequencing at a national paediatric centre
Our results confirm that the implementation of WES led to a significant breakthrough in the diagnostics of MD in children [32]. This is expressed by both the increased number of identified genes and faster establishment of final diagnosis. The total number of genes with likely causative defects found in the present work was 47, a very satisfactory diagnostic yield when compared with 8 genes identified by us by single-gene Sanger sequencing before the introduction of WES (203 such diagnoses per ~1200 patients studied in the period from 1996 to 2013).
In our study we observed a pronounced upward trend in the detection of the molecular background of mitochondrial diseases that was associated with increased MD probability (Fig. 2). According to the MDC scale that we used, a final genetic diagnosis was achieved in over 90 % of patients with the highest MDC scores (5–8 points). In all such cases (with one exception for a neonate with CPS1 mutation), variants were found exclusively in MD-related genes. The diagnostic yield was the lowest (36 %) in the patients with low MD suspicion (MDC score 2–3), and most of the variants in this group were present in non MD-related genes.
A similar correlation between detection rate and the level of MD probability was described recently in a similar patient group studied by WES at the Nijmegen Mitochondrial Centre [10]. However, our results differed from that study in terms of the scope of detected defects. In our cohort, mutations in MTO1, TK2, C12orf65, COA6, TUFM, GFM1 were absent and the defects in nuclear encoded complex I subunits are different. This may be a result of random patient selection, but we should also take into account ethnic differences among European populations, e.g., the Slavonic vs. north-western European populations.
In addition, we identified six rare mtDNA pathogenic variants, not included in the common mutations screening i.e. m.9185TC in MTATP6 [33–35] and in mitochondrial DNA genes encoding complex I subunits, MTND1 [36–38], MTND3 and MTND5 [39–42].
One-third (15/47) of the identified gene defects were discovered during last 10 years and relatively poorly characterized in terms of phenotype. These included PGAP2 [43, 44], ACAD9 [45, 46], EARS2 [47], SERAC1 [48], SLC19A3 [49, 50], MTFMT [51], SLC25A12 [52] as well as VARS2 [53], AIFM1 [54], RARS2 [55], RRM2B [56], PIGN [44, 57], ADCK3 [58, 59] which were described in just individual cases. Notably, most of these genes are generally absent from commercial NGS panels available at present.
It is worth emphasizing that in some cases WES allowed for a diagnosis in statu nascendi, that is, at the time of the first publication of the new gene. This concerned, for example, mutations in CLPB [25, 60], PARS2 [26], FBXL4 [61, 62] and recently added TMEM126B (data published on ESHG 2016 by Alston et al.), and NAXE [28] In one of the patients with the MD phenotype we identified potentially pathogenic variants in candidate NDUFB8 which role in human pathology is under verification [Piekutowska-Abramczuk et al. submitted to SSIEM 2016].
According to published literature, every third paediatric MD case (approximately 30 % of all MD diagnoses in this age group) manifests clinically shortly after birth [12, 13]. The fatal outcome in such cases precludes transport to a reference centre and proper mitochondrial diagnostics. We have previously shown significantly reduced (up to ten times, about 3 % of all diagnoses) recognition of MD in this age group in Poland [16]. Therefore, neonates with suspected MD intentionally constituted a significant proportion of patients (47/113) undergoing WES in the present study.
Surprisingly, in the neonatal subgroup WES proved to be particularly useful, allowing identification of pathogenic variants in 24 various genes in 63.8 % of patients, including those without muscle biopsy or even autopsy. Our results extend the list recommended by Honzik [13] for neonatal MD diagnostics by at least 15 genes (MD-related: RRM2B, CLPB, ACAD9, FBXL4, PC, AIFM1, SLC25A12, MTND5, NDUFS6 and non MD-related: CPS1, PGAP2 and more).
In the LS subgroup WES expanded the set of patients from our centre diagnosed with complex I deficiency by three known genes: NDUFS6 [63, 64], NDUFV1 [65, 66], NDUFS7 [67], a new candidate NDUFB8 [68] and five MTNDs mentioned above. Despite this, complex I deficiency continues to be underrepresented in our cohort in relation to complex IV deficiency because of the high carriage rate of SURF1 mutations in Poland [69]. In a number of cases with basal ganglia brain changes, WES failed to show mutations in known LS-associated genes. This was especially the case in patients without lactic acidaemia and MDC scores below 5 (MD possible but not likely). We speculate that other, still unknown, genes or non-genetic factors might influence the occurrence of LS-brain changes.
Taken together, our results indicate that WES rather than targeted NGS should be the method of choice for MD testing, at least until all MD-associated genes are identified. Furthermore, the rationale for choosing WES in MD-suspected neonates is the non-specificity of symptoms and overlapping results of biochemical tests with non-mitochondrial errors of metabolism.
In 50.5 % the molecular variants were novel (Table 3). However, a number of recurrent rare pathogenic variants found in some recently discovered MD genes (p.Arg22* in FBLX4, p.Arg518Cys in ACAD9, p.Arg417* in CLPB and c.1822_1828+10delinsACCAACAGG in SERAC1) may extend the ethnic specificity of MD in the Polish population reported earlier by us for variants p.Glu140Lys in SCO2 [14] and c.845_846delCT in SURF1 genes [69]. Confirmation of these findings could facilitate in-house diagnostics in selected suspected cases.