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  • Open Access

New perspective in diagnostics of mitochondrial disorders: two years’ experience with whole-exome sequencing at a national paediatric centre

  • 1, 2Email author,
  • 1,
  • 1,
  • 1,
  • 2,
  • 3,
  • 4,
  • 5,
  • 1,
  • 6,
  • 7,
  • 6,
  • 7,
  • 3,
  • 1 and
  • 6Email author
Contributed equally
Journal of Translational Medicine201614:174

https://doi.org/10.1186/s12967-016-0930-9

  • Received: 16 March 2016
  • Accepted: 31 May 2016
  • Published:

Abstract

Background

Whole-exome sequencing (WES) has led to an exponential increase in identification of causative variants in mitochondrial disorders (MD).

Methods

We performed WES in 113 MD suspected patients from Polish paediatric reference centre, in whom routine testing failed to identify a molecular defect. WES was performed using TruSeqExome enrichment, followed by variant prioritization, validation by Sanger sequencing, and segregation with the disease phenotype in the family.

Results

Likely causative mutations were identified in 67 (59.3 %) patients; these included variants in mtDNA (6 patients) and nDNA: X-linked (9 patients), autosomal dominant (5 patients), and autosomal recessive (47 patients, 11 homozygotes). Novel variants accounted for 50.5 % (50/99) of all detected changes. In 47 patients, changes in 31 MD-related genes (ACAD9, ADCK3, AIFM1, CLPB, COX10, DLD, EARS2, FBXL4, MTATP6, MTFMT, MTND1, MTND3, MTND5, NAXE, NDUFS6, NDUFS7, NDUFV1, OPA1, PARS2, PC, PDHA1, POLG, RARS2, RRM2B, SCO2, SERAC1, SLC19A3, SLC25A12, TAZ, TMEM126B, VARS2) were identified. The ACAD9, CLPB, FBXL4, PDHA1 genes recurred more than twice suggesting higher general/ethnic prevalence. In 19 cases, variants in 18 non-MD related genes (ADAR, CACNA1A, CDKL5, CLN3, CPS1, DMD, DYSF, GBE1, GFAP, HSD17B4, MECP2, MYBPC3, PEX5, PGAP2, PIGN, PRF1, SBDS, SCN2A) were found. The percentage of positive WES results rose gradually with increasing probability of MD according to the Mitochondrial Disease Criteria (MDC) scale (from 36 to 90 % for low and high probability, respectively). The percentage of detected MD-related genes compared with non MD-related genes also grew with the increasing MD likelihood (from 20 to 97 %). Molecular diagnosis was established in 30/47 (63.8 %) neonates and in 17/28 (60.7 %) patients with basal ganglia involvement. Mutations in CLPB, SERAC1, TAZ genes were identified in neonates with 3-methylglutaconic aciduria (3-MGA) as a discriminative feature. New MD-related candidate gene (NDUFB8) is under verification.

Conclusions

We suggest WES rather than targeted NGS as the method of choice in diagnostics of MD in children, including neonates with 3-MGA aciduria, who died without determination of disease cause and with limited availability of laboratory data. There is a strong correlation between the degree of MD diagnosis by WES and MD likelihood expressed by the MDC scale.

Keywords

  • Whole-exome sequencing
  • Mitochondrial disorders
  • Mitochondrial disease criteria scale
  • Neonates
  • Basal ganglia involvement
  • Leigh syndrome
  • 3-methylglutaconic aciduria
  • Novel mutation
  • Candidate gene

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 [48] or centre/cohort-based studies [911]. However, of these only four used whole exome sequencing (WES) [710].

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

ID patient

Sex

Date of birth (year)

Neonatal onset

3-MGA in urine

Basal ganglia involvement

Death

MDC score

Muscle biopsy

Period from onset to WES (year)

1

F

2009

+

  

+

5

 

5

2

F

2013

+

  

+

4

Autopsy

0

3

M

2012

+

   

5

+

2

4

F

2007

    

4

+

0

5

F

2013

+

+

 

+

5

 

0

6

F

2011

    

4

 

0

7

M

2006

  

+

 

5

+

7

8

M

2008

 

+

  

2

 

0

9

M

2011

   

+

6

+

2

10

M

2004

  

+

 

5

+

7

11

M

2005

    

2

 

2

12

M

2005

   

+

3

Autopsy

7

13

M

2014

+

+

 

+

4

Autopsy

0

14

F

2006

   

+

3

+ Autopsy

7

15

F

2008

+

+

  

4

+

5

16

M

2012

  

+

 

3

 

0

17

F

1992

  

+

 

3

+

21

18

F

2003

  

+

 

3

+

7

19

M

2009

    

5

+

3

20

M

2009

    

4

 

2

21

F

2006

   

+

6

+

8

22

M

2010

 

+

+

 

8

+

2

23

M

2011

+

  

+

4

+

3

24

F

2008

+

   

6

+

4

25

M

2010

+

  

+

7

+

3

26

M

2011

+

 

+

 

8

+

2

27

M

2008

+

+

 

+

5

 

6

28

M

2004

+

+

 

+

3

+

11

29

F

2007

+

   

5

+

7

30

F

2002

+

  

+

2

+

13

31

F

2005

  

+

 

6

+

9

32

M

2002

  

+

+

5

Autopsy

3

33

F

2006

    

3

 

2

34

M

2006

  

+

 

6

+

4

35

M

2012

   

+

6

+

2

36

M

2006

  

+

+

5

+

6

37

M

2003

+

+

+

 

7

+

12

38

M

1985

    

3

+

12

39

M

1996

    

3

+

11

40

M

2010

+

+

 

+

5

Autopsy

4

41

F

2011

  

+

 

4

+

3

42

F

2013

    

2

 

0

43

M

1967

   

+

2

 

10

44

F

1956

    

4

 

3

45

F

1995

    

2

+

11

46

M

2009

    

3

+

4

47

M

2013

    

2

 

0

48

F

2007

    

2

 

4

49

M

2012

+

  

+

6

Autopsy

2

50

M

2009

+

  

+

2

Autopsy

5

51

M

2003

+

 

+

+

5

+

12

52

F

2011

+

   

5

 

3

53

M

2007

    

6

+

7

54

M

1990

   

+

6

+

25

55

F

1981

    

4

+

21

56

F

2012

    

4

 

0

57

M

2010

  

+

 

6

+

0

58

M

2012

  

+

 

6

+

0

59

F

2010

+

   

6

+

4

60

M

2003

+

 

+

+

6

+

10

61

M

1989

   

+

8

+

23

62

M

1997

+

  

+

6

+

18

63

F

1989

    

4

+

16

64

F

2012

  

+

+

6

+

2

65

M

1991

+

 

+

 

4

+

23

66

F

2012

+

   

5

+

2

67

F

2014

+

+

  

4

 

0

68

M

2012

  

+

 

4

+

0

69

M

2013

    

3

 

0

70

F

2004

   

+

5

+

11

71

M

2001

+

  

+

5

+

14

72

M

2011

+

  

+

4

Autopsy

3

73

F

2002

+

   

3

+

11

74

F

1989

    

4

+

12

75

M

2008

+

  

+

5

+

6

76

F

2003

  

+

 

4

+

6

77

F

2011

+

 

+

+

6

+

3

78

M

1994

 

+

  

3

 

17

79

M

2004

    

3

+

6

80

F

2012

 

+

 

+

2

 

0

81

F

1990

+

  

+

4

+ Autopsy

21

82

F

2000

  

+

 

3

 

2

83

F

2003

+

  

+

4

+

12

84

M

2010

+

   

3

+

4

85

F

2013

  

+

+

3

 

0

86

M

2008

   

+

2

 

5

87

M

2010

    

3

 

0

88

M

1997

    

2

 

0

89

F

2004

+

 

+

+

4

+

11

90

M

2002

   

+

4

+

13

91

M

2009

   

+

6

+

5

92

M

1995

 

+

  

2

 

5

93

M

2011

+

  

+

3

Autopsy

3

94

F

2010

+

+

  

4

 

3

95

F

2011

+

   

4

+

3

96

M

2011

   

+

2

 

2

97

M

2005

+

  

ND

4

+

10

98

F

2012

+

+

 

+

2

Autopsy

2

99

F

1974

    

2

 

0

100

M

2009

   

+

3

+

5

101

M

2012

  

+

 

4

 

0

102

F

2006

  

+

 

3

 

0

103

F

2008

+

+

 

+

3

Autopsy

4

104

F

1988

  

+

 

4

+

18

105

F

2014

+

  

+

5

 

0

106

M

2011

+

   

3

+

2

107

M

2006

    

4

+

8

108

M

2012

+

   

3

+

2

109

M

1997

+

  

+

4

Autopsy

18

110

M

2010

    

2

+

4

111

F

2014

+

  

+

4

 

0

112

F

2010

+

  

+

3

Autopsy

4

113

M

2013

   

+

4

+

0

F female, M male

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

Gene

Chromosome:RefSeq

Variant 1

Variant 2

Zygosity status

Mode

ID patient

Type

Status

Origin

Type

Status

Origin

Mitochondrial disease gene

 ACAD9

chr3:NM_014049.4

c.514G>A/p.Gly172Arg

Novel

mat

c.803C>T/p.Ser268Phe

Novel

pat

comphtz

AR

15

 ACAD9

chr3:NM_014049.4

c.1552C>T/p.Arg518Cys

Known

mat

c.1553G>A/p.Arg518His

Known

pat

comphtz

AR

23

 ACAD9

chr3:NM_014049.4

c.728C>G/p.Thr243Arg

Novel

ND

c.1552C>T/p.Arg518Cys

Known

mat

comphtz

AR

53

 ADCK3

chr1:NM_020247.4

c.827A>G/p.Lys276Arg

Novel

mat

c.1702delG/p.Gly568Argfs

Novel

pat

comp htz

AR

61

 AIFM1

chrX:NM_004208.3

c.1474T>C/p.Tyr492His

Novel

mat

 

hemi

XLR

25

 CLPB

chr11:NM_030813.4

c.2045T>A/p.Ile682Asn

Known

pat

c.1937_1938insG/p.645Gly_646Cysfs

Known

mat

comphtz

AR

5

CLPB

chr11:NM_030813.4

c.1249C>T/p.Arg417a

Known

pat

c.748C>T/p.Arg250a

Known

mat

comphtz

AR

27

 CLPB

chr11:NM_030813.4

c.1249C>T/p.Arg417a

Known

pat

c.1222A>G/p.Arg408Gly

Known

mat

comphtz

AR

67

 COX10

chr17:NM_001303.3

c.1030A>G/p.Met344Val

Novel

pat

c.1270dupC/p.Leu424Profs

Novel

mat

comphtz

AR

9

 COX10

chr17:NM_001303

c.674C>T/p.Pro225Leu

Known

mat

c.674C>T/p.Pro225Leu

Known

pat

hom

AR

36

 DLD

chr7:NM_000108.4

c.1123G>A/p.Glu375Lys

Known

mat

c.1123G>A/p.Glu375Lys

Known 

pat

hom

AR

31

 EARS2

chr16:NM_001083614.1

c.164G>A/p.Arg55His

Known

mat

c.325G>C/p.Gly109Arg

Novel

pat

comphtz

AR

7

 EARS2

chr16:NM_001083614.1

c.164G>A/p.Arg55His

Known

pat

c.1256C>T/p.Pro419Leu

Novel

mat

comphtz

AR

70

 FBXL4

chr6:NM_012160.4

c.858+1G>T/p.?

Novel

pat

c.585+5G>C/p.?

Novel

mat

comphtz

AR

3

 FBXL4

chr6:NM_012160.4

c.1303C>T/p.Arg435a

Known

ND

c.64C>T/p.Arg22a

Novel

mat

comphtz

AR

52

 FBXL4

chr6:NM_012160.4

c.64C>T/p.Arg22a

Novel

mat

c.64C>T/p.Arg22a

Novel

pat

hom

AR

55

 MTATP6

chrM:NC_012920.1

m.9185T>C/p.Leo220Pro

Known

mat

 

hompl

M

32

 MTFMT

chr15:NM_139242.3

c.994C>T/p.Arg332a

Known

ND

c.626C>T/p.Ser209Leu

Known

ND

comphtz

AR

91

 MTND1

chrM:NC_012920.1

m.3902_3908invACCTTGC/p.?

Known

de novo

 

hetpl

M

22

 MTND1

chrM:NC_012920.1

m.3688G>A/p.Ala128Thr

Known

ND

 

hompl

M

64

 MTND3

chrM:NC_012920.1

m.10254G>A/p.Asp66Asn

Known

de novo

 

hetpl

M

57

 MTND5

chrM:NC_012920.1

m.12706T>C/p.Phe124Leu

Known

de novo

 

hetpl

M

34

 MTND5

chrM:NC_012920.1

m.13513G>A/p.Asp393Asn

Known

de novo

 

hetpl

M

35

 NAXE

chr1:NM_144772.2

c.653A>T/p.Asp218Val

Known

mat

c.743_744delC/p.247Ala_248Thrfs

Known

pat 

comphtz

AR

12

 NDUFS6

chr5:NM_004553.4

c.313_315delAAAG/p.104Lys_106Thrfs

Novel

pat

c.334_359del26ins13/p.Glu112 fs

Novel

mat

comphtz

AR

1

 NDUFS7

chr19:NM_024407.4

c.376C>T/p.Leu126Phe

Novel

ND

c.504G>C/p.Arg168Ser

Novel

ND

het

AR

75

 NDUFV1

chr11:NM_007103.3

c.733G>A/p.Val245Met

Novel

pat

c.383G>T/p.Arg128Leu

Novel

mat

comphtz

AR

10

 OPA1

chr3:NM_015560.2

c.1146A>G/p.Ile382Met

Known

mat

 

htz

AD

33

 PARS2

chr1:NM_152268.3

c.1091C>G/p.Pro364Arg

Novel

mat

c.239T>C/p.Ile80Thr

Novel

pat

comphtz

AR

60

 PC

chr11:NM_000920.3

c.808C>T/p.Arg270Trp

Known

pat

c.2381_2383delTGG/p.Val794del

Novel

mat

comphtz

AR

29

 PC

chr11:NM_000920.3

c.1487G>A/p.Arg496Gln

Novel

ND

c.584C>T/p.Ala195Val

Novel

ND

comphtz

AR

71

 PDHA1

chr X:NM_000284.3

c.262C>T/p.Arg88Cys

Known

mat

 

hemi

XLD

19

 PDHA1

chrX:NM_000284.3

c.856_859dupACTT/p. Arg288Leufs

Novel

de novo

 

htz

XLD

56

 PDHA1

chrX:NM_000284.3

c.933_935del/p.Arg311del l

Known

de novo

 

htz

XLD

66

 PDHA1

chrX:NM_000284.3

c.291G>A/p.?

Novel

de novo

 

hemi, mosaic

XLD

68

 POLG

chr15:NM_001126131.1

c.2639C>A/p.Ala880Asp

Novel

pat

c.2243G>C/p.Trp748Ser

Known

mat

comphtz

AR

113

 RARS2

chr6:NM_020320.3

c.1026G>A/p.Met342Ile

Novel

mat

c.622C>T/p.Gln208a

Novel

pat

comphtz

AR

41

 RRM2B

chr8:NM_015713.4

c.414_415delCA/p.Tyr138a

Novel

mat

c.414_415delCA/p.Tyr138a

 Novel

ND

hom

AR

21

 RRM2B

chr8:NM_015713.4

c.686G>T/p.Gly229Val

Known

mat

c.686G>T/p.Gly229Val

Known

pat

hom

AR

51

 SCO2

chr22:NM_005138.2

c.418G>A/p.Glu140Lys

Known

ND

c.418G>A/p.Glu140Lys

Known

ND

hom

AR

54

 SERAC1

chr6:NM_032861.3

c.1822_1828+10delinsACCAACAGG

Known

ND

c.1822_1828+10delinsACCAACAGG

Known

ND

hom

AR

37

 SLC19A3

chr2:NM_025243.3

c.68G>T/p.Gly23Val

Known

Pending

c.68G>T/p.Gly23Val

Known 

Pending

hom

AR

58

 SLC19A3

chr2:NM_025243.3

c.74dupT/p.Ser26Leufs

Known

ND

c.74dupT/p.Ser26Leufs

Known 

ND

hom

AR

109

 SLC25A12

chr2:NM_003705.4

c.1335C>A/p.Asn445Lys

Novel

mat

c.1335C>A/p.Asn445Lys

Novel

pat

hom

AR

24

 TAZ

chrX:NM_000116.3

c.684_685insC/p.227Phe_228Profs

Novel

ND

 

hemi

XLR

28

 TMEM126B a

chr11:NM_018480.4

c.635G>T/p.Gly212Val

Known

mat

c.635G>T/p.Gly212Val

Known 

pat

hom

AR

59

 VARS2

chr6:NM_001167734.1.5

c.1100C>T/p.Thr367Ile

Known

Pending

c.1490G>A/p.Arg497His

Novel

Pending

comphtz

AR

97

Non mitochondrial disease gene

 ADAR

chr1:NM_001111.4

c.3202+1G>A/p.?

Novel

ND

c.577C>G/p.Pro193Ala

Known

ND

comphtz

AR

18

 CACNA1A

chr19:NM_001127221.1

c.1997C>T/p.Thr666Met

Known

mat

 

htz

AD

39

 CDKL5

chrX:NM_003159.2

c.1942C>T/p.Gln648a

Novel

mat

 

hemi

XLD

65

 CLN3

chr16:NM_001042432.1

c.954_962+18del27/p.Leu313_Trp321del

Known

pat

c.461-280_677+382del966

Known

Pending 

comphtz

AR

88

 CPS1

chr2:NM_001875.4

c.1837-8A>G/p.?

Known

mat

c.3691G>C/p.Ala1231Pro

Novel

Paternal

comphtz

AR

13

 CPS1

chr2:NM_001875.4

c.1289C>G/p.Ser430a

Novel

mat

c.3971_3972delT/p.1323Ile_1324Leufs

Novel

pat 

comphtz

AR

40

 DMD

chr X:NM_004006

c.31+1G>A/p.?

Novel

mat

 

hemi

XLR

38

 DYSF

chr2:NM_003494.3

c.1180+5G>A/p.?

Known

ND

c.6124C>T/p.Arg2042Cys

Known

ND

comphtz

AR

45

 GBE1

chr3:NM_000158.3

c.1621A>T/p.Asn541Tyr

Novel

mat

c.263G>A/p.Cys88Tyr

Novel

pat

comphtz

AR

14

 GFAP

chr17:NM_002055.4

c.1100G>C/p.Arg367Thr

Novel

de novo

 

htz

AD

42

 HSD17B4

chr5:NM_000414.3

c.46G>A/p.Gly16Ser

Known

ND

c.367C>T/p.His123Tyr

Novel

ND

comphtz

AR

30

 MECP2

chrX:NM_004992.3

c.89delA/p.Lys30Argfs

Novel

de novo

 

hemi

XLD

106

 MYBPC3

chr11:NM_000256.3

c.1351+1G>A/p.?

Known

pat

 

htz

AD

8

 PEX5

chr12:NM_001131025.1

c.1669C>T/p.Arg557Trp

Known

mat

c.1799C>T/p.Ser600Leu

Novel

pat

comphtz

AR

20

 PGAP2

chr11:NM_001256240.1

c.2T>G/p.Met1?

Known

mat

c.221G>A/p.Arg74His

Known

pat

comphtz

AR

73

 PIGN

chr18:NM_176787.4

c.932T>G/p.Leu311Trp

Known

mat

c.790G>A/p.Gly264Arg

Known

pat

comphtz

AR

6

 PRF1

chr10:NM_001083116.1

c.808_812delGGCAG/p.Gly270 fs

Novel

mat

c.658G>A/p.Gly220Ser

Known

pat

comphtz

AR

2

 SBDS

chr7:NM_016038.2

c.258+2T>C/p.?

Known

pat

c.184A>T/p.Lys62a

Novel

mat

comphtz

AR

95

 SCN2A

chr2:NM_021007.2

c.2948T>G/p.Leu983Trp

Novel

de novo

 

htz

AD

47

New candidate gene for mitochondrial disease

 NDUFB8

chr10:NM_005004.3

c.432C>G/p.Cys144Trp

Novel

mat

c.227C>A/p.Pro76Gln

Novel

pat

comphtz

AR

26

mat maternal, pat paternal, ND not determined (DNA not available), hom homozygote, htz heterozygote, comp htz compound heterozygote, hemi hemizygote, hompl homoplasmic, hetpl heteroplasmic, AR autosomal recessive inheritance, AD autosomal dominant inheritance, XLR X-linked recessive inheritance, XLD X-linked dominant inheritance, M mitochondrial inheritance

aData published on ESHG 2016 by Alston et al.

Table 3

Novel molecular variants identified in the study; pathogenicity status

Gene

Variant

MAF

Pathogenicity statusa

Genotype–Phenotype correlationb

Parental results status

Family history

ID patient

1000 G

POL 400

ACAD9

c.514G>A/p.Gly172Arg

0

0

Pathogenic

Moderate

in-trans

Negative

15

ACAD9

c.803C>T/p.Ser268Phe

0

0

Pathogenic

Moderate

in-trans

Negative

15

ACAD9

c.728C>G/p.Thr243Arg

0

0

Pathogenic

Low

in-trans

Negative

53

ADAR

c.3202+1G>A/p.?

0

0.0014

Pathogenic

Moderate

ND

Affected brother

18

ADCK3

c.827A>G/p.Lys276Arg

0

0

Pathogenic

High

in-trans

Negative

61

ADCK3

c.1702delG/p.Gly568Argfs

0

0

Pathogenic

High

in-trans

Negative

61

AIFM1

c.1474T>C/p.Tyr492His

0

0

Pathogenic

Moderate

X-linked

Negative

25

CDKL5

c.1942C>T/p.Gln648a

0

0

Pathogenic

Moderate

X-linked

Negative

65

COX10

c.1030A>G/p.Met344Val

0

0.0007

Pathogenic

Moderate

in-trans

Negative

9

COX10

c.1270dupC/p.Leu424Profs

0

0

Pathogenic

Moderate

in-trans

Negative

9

CPS1

c.3691G>C/p.Ala1231Pro

0

0.0014

Pathogenic

Low

In-trans

Affected sister

13

CPS1

c.1289C>G/p.Ser430a

0

0.0014

Pathogenic

Moderate

in-trans

Affected brother

40

CPS1

c.3971_3972delT/p.1323Ile_1324Leufs

0

0.0014

Pathogenic

Moderate

in-trans

Affected brother

40

DMD

c.31+1G>A/p.?

0

0

Pathogenic

Low

X-linked

Affected many males

38

EARS2

c.325G>C/p.Gly109Arg

0

0.0014

Likely pathogenic

High

in-trans

Negative

7

EARS2

c.1256C>T/p.Pro419Leu

0

0

Likely pathogenic

Moderate

in-trans

Negative

70

FBXL4

c.858+1G>T/p.?

0

0

Pathogenic

High

in-trans

Miscarriage

3

FBXL4

c.585+5G>C/p.?

0

0

Pathogenic

High

in-trans

Miscarriage

3

FBXL4

c.64C>T/p.Arg22a

0

0

Pathogenic

Moderate

in-trans

Empty ovum

52

FBXL4

c.64C>T/p.Arg22a

0

0

Pathogenic

Moderate

in-trans

Negative

55

GBE1

c.1621A>T/p.Asn541Tyr

0

0

Pathogenic

Moderate

in-trans

Negative

14

GBE1

c.263G>A/p.Cys88Tyr

0

0

Possibly pathogenic

Moderate

in-trans

Negative

14

GFAP

c.1100G>C/p.Arg367Thr

0

0

Pathogenic

Moderate

de novo

Negative

42

HSD17B4

c.367C>T/p.His123Tyr

0

0.0014

Pathogenic

Moderate

ND

Affected brother

30

MECP2

c.89delA/p.Lys30Argfs

0

0.0

Pathogenic

High

de novo

Negative

106

NDUFB8

c.432C>G/p.Cys144Trp

0

0.0014

Possibly pathogenic

Moderate

in-trans

Negative

26

NDUFB8

c.227C>A/p.Pro76Gln

0

0

Pathogenic

Moderate

in-trans

Negative

26

NDUFS6

c.313_315delAAAG/p.104Lys_106Thrfs

0

0

Pathogenic

Moderate

in-trans

Affected brother

1

NDUFS6

c.334_359del26ins13/p.Glu112 fs

0

0

Pathogenic

Moderate

in-trans

Affected brother

1

NDUFS7

c.376C>T/p.Leu126Phe

0

0

Pathogenic

Moderate

ND

Similar symptoms in brother

75

NDUFS7

c.504G>C/p.Arg168Ser

0

0

Likely Pathogenic

Moderate

ND

Similar symptoms in brother

75

NDUFV1

c.733G>A/p.Val245Met

0.0005

0

Pathogenic

High

in-trans

Negative

10

NDUFV1

c.383G>T/p.Arg128Leu

0

0

Pathogenic

High

in-trans

Negative

10

PARS2

c.1091C>G/p.Pro364Arg

0.0014

0.003

Pathogenic

Moderate

in trans

Affected sibs

60

PARS2

c.239T>C/p.Ile80Thr

0

0

Pathogenic

Moderate

in trans

Affected sibs

60

PC

c.2381_2383delTGG/p.Val794del

0

0

uncertain Pathogenic

High

in-trans

Affected brother

29

PC

c.1487G>A/p.Arg496Gln

0

0

Pathogenic

High

ND

Negative

71

PC

c.584C>T/p.Ala195Val

0

0

Pathogenic

High

ND

Negative

71

PDHA1

c.856_859dupACTT/p. Arg288Leufs

0

0

Pathogenic

High

de novo

Negative

56

PDHA1

c.291G>A/p.?

0

0.0000

Uncertain pathogenic

Moderate

de novo

Negative

68

PEX5

c.1799C>T/p.Ser600Leu

0

0

Pathogenic

Low

in-trans

Negative

20

POLG

c.2639C>A/p.Ala880Asp

0

0

Pathogenic

Moderate

in-trans

Negative

113

PRF1

c.808_812delGGCAG/p.Gly270 fs

0

0.0000

Pathogenic

Low

in trans

Negative

2

RARS2

c.1026G>A/p.Met342Ile

0

0

Likely pathogenic

Moderate

in-trans

Affected brother

41

RARS2

c.622C>T/p.Gln208a

0

0.0014

Pathogenic

Moderate

in-trans

Affected brother

41

RRM2B

c.414_415delCA/p.Tyr138a

0

0.0014

Pathogenic

High

ND

Negative

21

SBDS

c.184A>T/p.Lys62a

0

0.002

Pathogenic

Low

in-trans

PI neural tube defect

95

SCN2A

c.2948T>G/p.Leu983Trp

0

0.0013

Pathogenic

High

de novo

Negative

47

SLC25A12

c.1335C>A/p.Asn445Lys

0

0

Pathogenic

Moderate

in-trans

Negative

24

TAZ

c.684_685insC/p.227Phe_228Profs

0

0.0012

Pathogenic

Low

ND

Negative

28

VARS2

c.1490G>A/p.Arg497His

0

0

Pathogenic

Low

ND

Similar disease in sibs

97

ND not determined due to lack of clinical data or DNA not available

aPathogenicity status evaluated according to in silico prediction algorithms (CADD, MetaSVM, Polyphen2 HDIV, Polyphen HVAR, mutation assessor, LRT, MetaLR, SIFT, mutationtaster) and classified as: pathogenic—nonsense, frameshift, splicesite and missense variants with pathogenic status at least in 7 of used algorithms; likely pathogenic - missense variants with pathogenic status in 4–6 of used algorithms; possibly pathogenic—missense variants with pathogenic status <4 of used algorithms

bGenotype-Phenotypecorrelationassessed by two independent specialists in clinical genetics and metabolic medicine

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
Fig. 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)

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
Fig. 2

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

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

Subgroups of patients

MD or non-MD genes loci of variants

Diagnostics based on archival material

Current diagnostics

Total

Disease onset (year)

1996–2012

2013–2014

1996–2014

Number of patients

88 (5.5/year)

25 (12.5/year)

113

Period from onset to WES qualification (years)

2–25 (mean 5.5 ± 5.9 )

0

0–25

MDC scale (A+B, without C)

4.2 ± 1.5 (2–8)

3.6 ± 1.2 (2–6)

4.1 ± 1.5

Ratio of MD-related/non MD related genes

3.4

1.0

2.4

Patients deceased

Total no.

41

8

44 %

MD

51.2 % (21)

2

47 % (23)

non MD

(3)

2

(5)

Patients with neonatal onset

Total no.

41

6

42 %

MD

53.7 % (22)

2

51 % (24)

non MD

(5)

2

(7)

Patients with LS or other basal ganglia involvement

Total no.

21

7

25 %

MD

61.9 % (13)

3

57 % (16)

non MD

(2)

0

(2)

3-methylglutaconic aciduria

Total no.

13

3

14 %

MD

53.8 % (7)

2

53 % (9)

non MD

0

1

(1)

Muscle biopsy

Total no.

62

5

67/113

MD

56.4 % (35)

(4)

58 % (39)

non MD

(10)

(0)

(10)

Percentage of muscle biopsy

70 %

20 %

59 %

aItalics in brackets indicates the number of patients in the given subset

LS Leigh syndrome, MD mitochondrial disorder, MD/non MD MD-related/non MD-related genes wherein variants were identified

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
Fig. 3

Family study in six probands with mtDNA known mutations

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 [3335] and in mitochondrial DNA genes encoding complex I subunits, MTND1 [3638], MTND3 and MTND5 [3942].

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.

     

Notes

Abbreviations

MD: 

mitochondrial disorders

WES: 

whole-exome sequencing

MDC: 

mitochondrial disease criteria

NGS: 

next generation sequencing

LA-uria: 

lactic aciduria

3-MGA-uria: 

3-methylglutaconic aciduria

nDNA: 

nuclear DNA

Declarations

Authors’ contributions

Conception and design: EP, RP, EC, DPA, JT, DR. Analysis and interpretation of data: EP, DR, DPA, EC, JT, AKW, MPa, EJ, JK, AP, MR, MPr. Coordination and drafting the article: DPA, EC, JT, PH, EP, AP. Bioinformatic analysis: PS, RP. Revising article critically for important intellectual content EP, MPr, MKW, RP. All authors read and approved the final manuscript.

Acknowledgements

We thank all of the physicians who referred affected children to our mitochondrial centre, especially Hanna Mierzewska, Jacek Pilch, Ewa Jamroz, Jolanta Wierzba, Maria Giżewska, and others.

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

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. Parents of the patients gave informed consent for the WES analysis.

Funding

The study was supported by CMHI projects no. S136/13, no. 126/12, no. 216/12, no. 217/12, no. S134/13, no. S211/10, and by grants from the National Science Centre, 2012/05/B/NZ2/01627, 1154/B/P01/2011/40, 2857/B/P01/2010/39, and EU Structural Funds Project POIG.02.01.00-14-059/09.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Medical Genetics, The Children’s Memorial Health Institute, 04-730 Warsaw, Poland
(2)
Department of Paediatrics, Nutrition and Metabolic Diseases,, The Children’s Memorial Health Institute, Warsaw, Poland
(3)
Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland
(4)
Department of Biochemistry and Experimental Medicine, The Children’s Memorial Health Institute, Warsaw, Poland
(5)
Department of Radiology, The Children’s Memorial Health Institute, Warsaw, Poland
(6)
Department of Medical Genetics, Warsaw Medical University, Pawińskiego str, 02-106 Warsaw, Poland
(7)
Department of Genetics, Institute of Physiology and Pathology of Hearing, Nadarzyn, Poland

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