Background

The diagnostics of mitochondrial disorders (MD) remains a challenge due to clinical heterogeneity [1] and the constantly expanding amount of gene candidates [2] as well as new phenotypes of these conditions [3]. There are eight published studies evaluating diagnostic utility of next generation sequencing (NGS) in mitochondrial patient cohorts, selected either based on particular biochemical signatures of disease [4–8] or centre/cohort-based studies [9–11]. However, of these only four used whole exome sequencing (WES) [7–10].

A particular challenge is the diagnosis of MD in neonates below 3 months of age as these patients may account for up to 30 % of all MD cases [12, 13]. However, so far, this group has not been specifically focused on in terms of diagnostic effectiveness of WES. The prevailing majority (96.5 %) of cases with a molecular diagnosis of MD established at our national reference centre until 2013 included children older than 3 months, indicating considerable under-diagnosis rates in the youngest infants in the Polish population. We have achieved some improvement in neonatal MD detection by performing targeted DNA sequencing (frequently post mortem) in cases of neonates with lactic aciduria (LA-uria) found in selective GC–MS screening, including over 90 % of SCO2 [14] and DGUOK [15] deficiencies, and ~ 50 % of SURF1 deficiency [16].

The purpose of our study was to evaluate WES as a tool for diagnosis of MD depending on the disease probability assessed according to mitochondrial disease criteria (MDC) [17]. We considered both patients with full-range mitochondrial diagnostics (Leigh syndrome features in MRI and/or muscle biopsy evaluation) and those in whom only fragmentary clinical data e.g. abnormal result of GC–MS screening indicating the presence LA-uria and/or 3-methylglutaconic aciduria (3-MGA-uria) were available.

Methods

Patients

WES was performed in patients with probable or possible MD, in whom a molecular defect had not been identified within the analysed period. In the retrospective subgroup (88/113 patients) the lag time was 2–25 years (mean 7.5 +/5.9 years). Since 2013 WES has been considered in consecutive patients (25/113). To undergo WES, a patient had to fulfil at least one of the following criteria: 1/neonatal onset; 2/basal ganglia involvement (Leigh syndrome—LS, nonspecific basal ganglia involvement); 3/increased 3-MGA in urine (patients recruited from a group of >250 cases of 3-MGA aciduria identified by national selective GC–MS screening for metabolic disorders since 2000), and 4/genetic counselling demands. Access to biological material and informed consent of parents were sine qua non conditions for participation in the study. Details of criteria for patient selection and their clinical characteristics are shown in Table 1 and Additional file 1: Table S1.

Table 1 Characteristics of 113 MD suspected patients; inclusion criteria
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The study included cases with a high probability of MD and those in whom MD was considered possible. The level of probability was assessed according to the MDC score proposed by the Nijmegen mitochondrial team as follows: 2–4 points: MD possible; 5–8 points: MD probable [17]. The MDC scoring for this study did not include the results of muscle biopsy (panels A+B, without C). The mean MD score in the study group was 4.1 ± 1.5 (range 2–8). Muscle biopsy with subsequent OXPHOS evaluation was performed in 67 cases, and autopsy in 15 cases. The family history was positive in 26 cases and three couples were consanguineous.

In the retrospective group, DNA was isolated from fibroblast cultures or frozen tissue samples obtained by muscle/liver biopsy or by autopsy. Whenever possible, skeletal muscle was preferred. In the remaining cases, DNA was isolated from blood. Throughout the paper the genes were classified as MD-related if they had a connection with mitochondrial disorders documented in the literature [9] or non MD-related when this was not the case.

Parents of the patients gave informed consent for the WES analysis. The study protocol was in agreement with the Helsinki Convention and the study was approved by the Ethics Committee of The Children’s Memorial Health Institute.

Whole-exome sequencing

WES was performed using TruSeqExome Enrichment Kits according to the manufacturer’s instructions (Illumina). The samples were run on 1/4 of a lane on HiSeq 1500 using 2 × 100 bp paired-end reads. Bioinformatics analysis was performed as previously described [18]. Briefly, after initial processing with CASAVA, the sequencing reads were aligned to the hg19 reference genome with the Burrows-Wheeler Alignment Tool and further processed by Genome Analysis Toolkit [19]. Base quality score recalibration, indel realignment, duplicate removal, and SNP/INDEL calling were done as described [20]. The detected variants were annotated using Annovar and converted to MS Access format for final manual analyses. Alignments were viewed with Integrative Genomics Viewer [21, 22]. The complete results of WES, including VCF and/or FASTQ files, are available on demand to qualified researchers. All samples were sequenced so that min. 80 % of target was covered 20× or more.

The presence of the variants identified by WES was confirmed by Sanger sequencing.

Results

Among 67 probands, we found 99 variants in 49 different genes with a Known disease link (Table 2). They were variants in mtDNA (6 patients) and nuclear DNA (nDNA): X-linked (9 patients), autosomal dominant (5 patients), and autosomal recessive (47 patients), including 11 homozygotes. In 50.5 % (50/99) the detected variants were novel (Table 3). Sixty-six of the variants found in the study group occurred in MD-related genes, whereas 31 were found in non MD-related loci. In addition, deleterious variants in a gene not previously linked to disease in humans were identified in one proband (Table 2).

Table 2 Molecular variants identified in 67 individuals of the study group
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Table 3 Novel molecular variants identified in the study; pathogenicity status
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Mutations in MD-related genes were found in 47 probands. Identified pathogenic variants in 31 different genes included 27 located in nDNA and 4 in mtDNA (Table 2). Eleven genes were found defective more than once (PDHA1-4x, ACAD9, CLPB, and FBXL4-3x, COX10, EARS2, MTND1, MTND5, PC, RRM2B, SLC19A3-2x). The majority of these genes were not previously screened for in our mitochondrial diagnostic centre, with the exceptions of TAZ, PDHA1 [23], SCO2, and the genes encoding MTND and MTATP subunits. Below we present the results that were analysed according to selected phenotypic features (neonatal onset, basal ganglia involvement, 3-MGA) and MD likelihood.

Subgroup of neonates

WES yielded conclusive results in 63.9 % (30/47) of neonates studied (Fig. 1a). We found mutations in 23 different genes, including 16 MD-related (ACAD9, AIFM1, CLPB, FBXL4, NDUFS6, NDUFS7, PARS2, PC, PDHA1 [23], RRM2B, SERAC1, SLC19A3, SLC25A12, TAZ, TMEM126B, VARS2) and 7 non MD-related (CDKL5, CPS1, HSD17B4, MECP2, PGAP2, PRF1, SBDS). The majority of the neonates with positive WES results came from the first pregnancy of healthy unrelated parents. Twenty-nine neonates died before establishing a diagnosis; half in the early neonatal period. In 28 cases the mitochondrial testing was completed, including MR imaging and spectroscopy, muscle biopsy and fibroblast culture collection. In the remaining cases, mitochondrial diagnostics were absent or limited only to selective GC–MS screening showing increased excretion of lactate, Krebs cycle metabolites, 3-MGA and/or ketone bodies.

Fig. 1
figure 1

The percentage of detected MD-related genes, non MD-related genes and non-conclusive WES results in (a) neonates (n = 47), b patients with 3-MGA-uria (n = 16) and c patients with basal ganglia involvement (n = 28)

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Subgroup with 3-methylglutaonic aciduria

Positive WES results were obtained in seven of 16 patients with persisting 3-MGA (Fig. 1b). In two subjects [P28 and P37] we found mutations in TAZ and SERAC1 genes known to cause mitochondrial diseases with 3-MGA as a discriminative feature [24]. Ex post it was apparent that earlier some important clinical features, including hearing impairment in the patient with SERAC1 mutations and increased excretion of 3-MGA in the terminal stage in the boy with the TAZ mutation, had been overlooked.

In three unrelated 3-MGA neonates included in this study, we identified mutations in the CLPB gene, whose link to human disease was subsequently established [25]. Two of them [P5 and P27] have already been reported in the first disease description [25].

Additionally, in two 3-MGA patients [P13, P40] we found molecular variants in the CPS1, a non MD-related gene linked to urea cycle disorder. In remaining patients in whom the reason for inclusion in the study group was a single GC–MS assessment (ACAD9 and MYBPC3 patients [P15, P8]), increased excretion of 3-MGA has been apparently transient or it was within normal limits after quantitative verification (Additional file 1: Table S1). Since traces of 3-MGA excretion were also found in a number of healthy siblings and parents of the patients the transient or mild increase in patients was most likely without a causal relationship.

Basal ganglia involvement (Leigh syndrome, Leigh-like, others)

In 15 of 28 patients from this group (Fig. 1c), molecular variants in LS-associated genes, including genes responsible for deficiency of complex I (MTND1, MTND3, MTND5, NDUFV1), complex IV (COX10), complex V (MTATP6), combined OXPHOS defect (EARS2, PARS2, RARS2, RRM2B, SERAC1, SLC19A3), and pyruvate dehydrogenase complex deficiency (DLD, PDHA1) [23] were identified. In the remaining 13 patients with LS or other basal ganglia involvement WES did not reveal variants in MD-related genes as listed by Neveling [9].

In three patients with basal ganglia involvement one MD-related candidate (NDUFB8) and two known non MD-related genes (ADAR, CDKL5) were identified.

Defects in non MD-related genes

In 19 patients who were included in the study because of a possible (low probability) mitochondrial disease, mutations in various non MD-related genes (ADAR, CACNA1A, CDKL5, CLN3, CPS1, DMD, DYSF, GBE1, GFAP, HSD17B4, MECP2, MYBPC3, PEX5, PGAP2, PIGN, PRF1, SBDS, SCN2A) were identified (Table 2; Additional file 1: Table S1).

New MD-related disorders

While our project was ongoing new candidate genes found by us including PARS2 [26] and CLPB have been described by other research teams [25]. The causal role of another two of our candidates has been recognized even more recently. The NAXE gene (APOA1BP according to old nomenclature), a susceptibility locus for migraine [27], in which likely pathogenic variants were found by us in two brothers with a fatal encephalitis-like disorder [P12], has been described in April 2016 as the cause of lethal infantile leukoencephalopathy in a large consanguineous family [28]. A homozygous variant in the TMEM126B gene encoding a subunit required for mitochondrial complex I assembly [29, 30], found by us in a complex I deficient girl with extra-neurological presentation [P59], has been discovered and verified functionally as a cause of the disease in a subset of other patients (ESHG 2016, Alston et al.).

The interesting remaining candidate for a novel disease gene identified in our study is NDUFB8. Compound heterozygosity for two variants in NDUFB8 was found in a boy with a typical course of LS and complex I deficiency in muscle homogenate [P26] (Additional file 1: Table S1). NDUFB8 [31] encodes a known subunit of complex I, but, to the extent of our knowledge, its association with complex I deficiency and LS in humans has not been published so far.

Mitochondrial disease criteria score

In the studied cohort there were 40 patients with high probability of MD, i.e., with an MDC score above 4 (5–8, criteria A+B, without C). Positive WES results were obtained in 36 of them (90 %). In this group, pathogenic variants were found mainly in MD-related genes (CPS1 being the exception). WES failed in four patients [P49, P62, P77, P105] with an MDC score above 4. Some of them were found to carry a deleterious variant in one of the known MD-related genes only on one allele. The definite diagnosis still remains open in these cases. Bioinformatics tools for identification of structural variants using NGS have not been applied to our data so it is possible that in some cases the disease may be caused by large deletions/duplications. The complete lists of variants detected in the subjects without fully conclusive results and/or the respective FASTQ files are available on demand to qualified researchers.

Intermediate probability of MD (MDC = 4) was associated with the occurrence of variants in both MD-related and non MD-related genes, in ten (10/31) and six (6/31) patients, respectively. MD-related genes were represented in this subset twice by ACAD9 [P15, P23] and PDHA1 [P56, P68], and in single cases by CLPB [P67], FBLX4 [P55], POLG [P113], RARS2 [P41], SLC19A3 [P109], and VARS2 [P97].

In the subgroup with low probability of MD, i.e., a MDC score of 2–3 points, positive WES results were obtained in 15 of 42 cases (36 %). Three MD-related genes (7 %) including: OPA1 [P33], TAZ [P28] and NAXE [P12] were found. Non MD-related genes were identified in 12 of 42 cases (29 %).

The percentage of positive results rose gradually as the likelihood of MD increased, as shown by the MDC score (Fig. 2). In the subset of high probability of MD (MDC above 4), the detection percentage reached 90 %. There was a broad range of MD-related genes (Table 2). Only one non MD-related gene (CPS1) was found in a neonate with a MDC score of 5.

Fig. 2
figure 2

Efficacy of WES in 113 patients with possible or probable mitochondrial pathology depending on the level of probability expressed by MDC Nijmegen score

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The participation of detected MD-related genes as compared with non MD-related genes also grew as the likelihood of MD probability increased (from 20 to 97 %, data not shown).

WES diagnostics of current cases vs. archival DNA samples

Characteristics of the patients stratified by the waiting period between disease onset and WES qualification into archival material and current diagnostics subset is shown in Table 4. WES efficacy assessed as percentage of molecularly confirmed diagnoses was comparable being higher than 50 % in both subsets. Contribution of MD-related genes expressed by the ratio of MD-related/non MD-related genes was higher in the archival than current subset (3.4 vs. 1.0, respectively) indicating that this subset contained more patients with non-mitochondrial genetic disorders and that our current qualification for WES became less demanding.

Table 4 WES results related to the origin of the qualified material and to the specific inclusion criteria
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Muscle biopsy findings

OXPHOS assessment available for 67 muscle homogenates showed isolated complex I deficiency in 16 cases, complex IV deficiency in 6 cases and combined OXPHOS defect in 10 cases. There were unspecific changes in 22 bioptates and normal OXPHOS activity in 10. The results were not conclusive in three cases due to technical problems (too small muscle specimen, low protein concentration, low citric synthase activity).

Complex I deficiency was found in 11 patients with molecular variants in MD-related genes (ACAD9 [P15, P23, P53], NDUFV1 [P10], NDUFS7 [P75], MTND1 [P64], MTND3 [P57], EARS2 [P7], SLC19A3 [P58], TMEM126B [P59]) and in one candidate (NDUFB8 [P26]. In one patient [P95] a defect in non MD-related gene (SBDS) was found. In 4 patients WES results were not conclusive.

In the subset with complex IV deficiency molecular defects were confirmed in three patients including COX10 [P9, P36] and EARS2 [P70]) while three WES analyses were not conclusive.

Combined OXPHOS defect occurred in 8 patients with variants identified in MD-related genes (FBXL4 [P3], ADCK3 [P61], RRM2B [P21, P51], AIFM1 [P25], TAZ [P28], PC [P71], MTND5 [P34]). In two cases WES results were not conclusive.

Histological and histochemical data of the patients with positive WES showed presence of ragged red fibers in four cases (ADCK3 [P61], ACAD9 [P15, P23, P53]), “lipid storage myopathy” in four (PC [P71, P29], MTND5 [P35], PDHA1 [P66]) and SMA-like pattern in three (AIFM1 [P25], SCO2 [P54], RRM2B [P51]).

Depletion of mitochondrial DNA (<30 % of reference value) was revealed in tissues of 8 patients. Molecular defect was established by WES in four of them (COX10 [P9], FBXL4 [3], RRM2B [P21, P51]).

Verification of mitochondrial genome variants

Interestingly, in six patients with typical MD phenotype the search for pathogenic variants in MD-related nuclear genes by WES was negative yet pathogenic variants were found in mtDNA. Each mtDNA variant identified by WES, was subsequently verified by Sanger sequencing using specific primers for mitochondrial genome. All detected changes are known and have been repeatedly reported. Examination of different tissues in probands and maternally related family members showed varying levels of heteroplasmy (Fig. 3).

Fig. 3
figure 3

Family study in six probands with mtDNA known mutations

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Discussion

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.9185T>C 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.

Conclusions

  1. 1.

    In a nationwide reference centre, WES provided positive results in >90 % of children with high likelihood of MD (MDC score above 4);

  2. 2.

    WES should be recommended for diagnostics of mitochondrial pathology considering remarkable representation of non MD-related genes among causal factors in patients with lower likelihood of MD, as well as a possibility to discover new mitochondrial genes;

  3. 3.

    WES significantly improves recognition of MD in newborns, even in the case of limited availability of appropriate diagnostic procedures;

  4. 4.

    Despite being a sine qua non for certain diagnoses 3-MGA is not a universal marker of mitochondrial dysfunction;

  5. 5.

    Recurrent variants recognized in some relatively new MD genes (FBLX4, ACAD9, and CLPB) may extend the known ethnic specificity of MD in the Polish population reported earlier for SCO2 and SURF1 variants.