Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening.

YMGME-05689; No. of pages: 11; 4C: Molecular Genetics and Metabolism xxx (2014) xxx–xxx

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Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme

Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening Tobias B. Haack a,b, Matteo Gorza a, Katharina Danhauser a,b, Johannes A. Mayr c, Birgit Haberberger b, Thomas Wieland a, Laura Kremer a, Valentina Strecker d, Elisabeth Graf a, Yasin Memari e, Uwe Ahting b, Robert Kopajtich a, Saskia B. Wortmann f, Richard J. Rodenburg f, Urania Kotzaeridou g, Georg F. Hoffmann g, Wolfgang Sperl c, Ilka Wittig d, Ekkehard Wilichowski h, Gudrun Schottmann i, Markus Schuelke i, Barbara Plecko j, Ulrich Stephani k, Tim M. Strom a,b, Thomas Meitinger a,b, Holger Prokisch a,b, Peter Freisinger l,⁎ a

Institute of Human Genetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany Institute of Human Genetics, Technische Universität München, 81675 Munich, Germany Department of Pediatrics, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria d Functional Proteomics, SFB 815 core unit, Faculty of Medicine, Goethe-University, 60590 Frankfurt am Main, Germany e Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom f Nijmegen Center for Mitochondrial Disorders, Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen 6500 HB, The Netherlands g Department of General Pediatrics, Division of Inherited Metabolic Diseases, University Children's Hospital, 69120 Heidelberg, Germany h Department of Pediatrics and Pediatric Neurology, Universitätsmedizin Göttingen, 37075 Göttingen, Germany i Department of Neuropediatrics and NeuroCure Clinical Research Center, Charité Universitätsmedizin Berlin, 13125 Berlin, Germany j Department of Neurology, Kinderspital Zürich, Zürich, Switzerland k Department of Neuropediatrics, University Hospital, 24105 Kiel, Germany l Department of Pediatrics, Inherited Metabolic Disease Centre, Klinikum Reutlingen, 72764 Reutlingen, Germany b c

a r t i c l e

i n f o

Article history: Received 18 December 2013 Accepted 18 December 2013 Available online xxxx Keywords: MTFMT Mitochondrial translation OXPHOS deficiency Exome sequencing Leigh syndrome

a b s t r a c t Defects of mitochondrial oxidative phosphorylation (OXPHOS) are associated with a wide range of clinical phenotypes and time courses. Combined OXPHOS deficiencies are mainly caused by mutations of nuclear genes that are involved in mitochondrial protein translation. Due to their genetic heterogeneity it is almost impossible to diagnose OXPHOS patients on clinical grounds alone. Hence next generation sequencing (NGS) provides a distinct advantage over candidate gene sequencing to discover the underlying genetic defect in a timely manner. One recent example is the identification of mutations in MTFMT that impair mitochondrial protein translation through decreased formylation of Met-tRNAMet. Here we report the results of a combined exome sequencing and candidate gene screening study. We identified nine additional MTFMT patients from eight families who were affected with Leigh encephalopathy or white matter disease, microcephaly, mental retardation, ataxia, and muscular hypotonia. In four patients, the causal mutations were identified by exome sequencing followed by stringent bioinformatic filtering. In one index case, exome sequencing identified a single heterozygous mutation leading to Sanger sequencing which identified a second mutation in the non-covered first exon. High-resolution melting curve-based MTFMT screening in 350 OXPHPOS patients identified pathogenic mutations in another three index cases. Mutations in one of them were not covered by previous exome sequencing. All novel mutations predict a loss-of-function or result in a severe decrease in MTFMT protein in patients' fibroblasts accompanied by reduced steady-state levels of complex I and IV subunits. Being present in 11 out of 13 index cases the c.626C N T mutation is one of the most frequent disease alleles underlying OXPHOS disorders. We provide detailed clinical descriptions on eleven MTFMT patients and review five previously reported cases. © 2013 Elsevier Inc. All rights reserved.

1. Introduction ⁎ Corresponding author at: Department of Pediatrics Klinikum Reutlingen Steinenbergstrasse 31 72764 Reutlingen, Germany. Fax: + 49 7121 200 4481. E-mail address: [email protected] (P. Freisinger).

The human OXPHOS system is composed of about 90 structural proteins. Protein subunits encoded by the nuclear DNA are imported into the mitochondrion after translation in the cytoplasm. The 13 proteins

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Please cite this article as: T.B. Haack, et al., Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening, Mol. Genet. Metab. (2014), http://dx.doi.org/10.1016/j.ymgme.2013.12.010

2

Patient ID

Sex AO

Age

Variations Nucleotide

OXPHOS activities in muscle Amino acid

RCC

% of lower control range

MRI

Clinical features

Slight developmental delay in childhood, episode of fatigue and disturbed vision at age 17 y, ventricular septum defect, lactic acidosis in CSF, deceased at age 17 y after cardiosurgery. Developmental delay in childhood, short stature and growth hormone deficiency, at age 15 y acquired strabism of the right eye, unstable gait and muscular hypotonia. Increased serum and CSF lactate levels. No COX-negative fibers in histochemistry. Clinically stable condition at age 22 y. Microcephaly, “metabolic stroke”, convulsions, episode with severe lactic acidosis; deceased at age 14 m. Histochemistry mild lipid accumulation in type I fibers. Microcephaly, severe muscular hypotonia and developmental delay; deceased at age 19 m due to respiratory failure during pneumonia. No COX-negative firbes in histochemistry.

#33009a

M

17 y

17 yb

c.146_153del p.Arg49Leufs*58 c.626C N T p.Arg181Serfs*5

I IV

65% Normal

T2-hyperintensities in midbrain and crus cerebri

#33467a

M

15 y

22 y

c.146_153del p.Arg49Leufs*58 c.626C N T p.Arg181Serfs*5

I IV

76%, 50%b 100%, 81%b

T2-hyperintensity in midbrain and patchy signal changes in white matter

#49728

F

1m

#52181

F

2m

14 mb c.626C N T c.878G N A 19 mb c.219_222del c.626C N T

p.Arg181Serfs*5 p.Ser293Asn p.Glu74Lysfs*3 p.Arg181Serfs*5

I IV I IV

21% 49% 21% Normal

#54502

M

3m

6y

c.626C N T c.994C N T

p.Arg181fs*5 p.Arg332*

I IV

#56713

M

8m

6y

#56902

M

20 m 5 y

c.73C N T c.626C N T c.452C N T c.994C N T

p.Gln25* p.Arg181Serfs*5 p.Pro151Leu p.Arg332*

I IV I IV

65%, 21%c 45%, Normalc 64% 100% 89% 80%

Symmetric T2-hyperintensities in putamen, globus pallidus, and brainstem Symmetric T2-hyperintensities of the nucleus caudatus, globus pallidus and mesencephalon; extensive damage of the white matter with cystic lesions T2-hyperintensities in subcortical frontal and parietal white matter, splenium callosum

#73922

M

Birth

15 n

#8432723

M

Birth

12 y

#44409 (Haack et al)

M

3y

24 y

#61606 (Haack et al) P1 (Tucker et al)

F

16 m 6.5 y

F

9y

21 y

P1 cousin

M

9y

P2 (Tucker et al)

F

P1 (Neeve et al.) P2 (sister P1) (Neeve et al.)

c.452C c.626C c.626C c.766C c.626C c.994C

N N N N N N

T T T T T T

p.Pro151Leu p.Arg181Serfs*5 p.Arg181Serfs*5 p.Gln256* p.Arg181Serfs*5 p.Arg332*

c.626C c.626C c.382C c.626C

N N N N

T T T T

p.Arg181Serfs*5 p.Arg181Serfs*5 p.Arg128* p.Arg181Serfs*5

18 y

c.382C N T c.626C N T

p.Arg128* p.Arg181Serfs*5

5y

5 yb

c.374C N T c.626C N T

p.Ser125Leu p.Arg181Serfs*5

F

3y

16 y

c.452C N T c.994C N T

p.Pro151Leu p.Arg332*

F

5y

6y

c.425C N T c.994C N T

p.Pro151Leu p.Arg332*

Developmental delay, muscular hypotonia, intermittent strabism. Lactic acidosis. No COX-negative fibers in histochemistry.

Symmetric T2-signal intensive lesions of the putamen and globus pallidus. Pinealis cyst Periventricular T2-signal intense lesions, confluent in the centrum semiovale, lesion in the splenium callosum

Microcephaly, truncal hypotonia, ataxia. At 4 y cognitive and speech development retarded by about 1.5 y.. Loss of gait during infection, severe muscular hypotonia. Microcephaly, growth retardation. Serum and CSF lactic acidosis. Non-compaction cardiomyopathy. Merosin-deficient but no SDH or COX-negative fibers in histochemistry. I 7% Mild cortical atrophy and myelination delay at age Muscular hypotonia, lactic acidosis. Mild hypertrophic cardiomyopathy. Body height, IV 92% 7m length and head circumference 1st-10th percentile. I 43% Periductal T2-signal intense gray matter lesions and Severe developmental delay, abnormal breathing pattern; short stature and IV normal white matter involvement at the centrum semiovale microcephaly; lactic acidosis; relative COX-deficiency in muscle histochemistry. T2-signal intensive lesions of caudate nucleus, 38% External ophthalmoplegia, partial optic atrophy, mental retardation, tetra-spasticity I Normal IV putamen, medulla oblongata and periventricular white (lower N upper limbs), neurosensoral bladder dysfunction, stable condition. 75% PDHc matter I 12% T2-signal intensive bilateral lesions of the putamen and Microcephaly, ataxia, muscular hypotonia. Retardation of gross a fine motor IV Normal in the fronto-parietal white matter development and social skills. T2-hyperintensities in the Nn lentiformis, caudatus, and Acquired strabism (internuclear ophthalmoplegia), pale optic disks, developmental/ Decreased I Low normal ruber, midbrain tectum, corpus callosum, and mental retardation, elevated lactic acid and pyruvate levels, Wolff–Parkinson–White (III) Decreased syndrome. IV subcortical white matter I Decreased “Signal abnormality” affecting periaqueductal gray Developmental delay, optic atrophy, mild bilateral pyramidal tract signs, impaired IV Decreased matter of midbrain and upper pons visual acuity, mildly dysmorphic facies, elevated CSF lactic acid levels, Wolff– Parkinson–White syndrome, episode of acute deterioration with weakness and ataxia, good recovery. Hyperintensities in putamen, globus pallidus and Cushing disease due to pituitary adenoma, elevated lactic acid levels in CSF, seizures Decreased I brainstem following anesthesia, neurologic deterioration. Decreased III Decreased IV T2-hyperintensities in nucleus caudatus, putamen. 59% I Developmental delay, muscular hypotonia, atxic gait, dysarthria. Subsacrolemmal 78% IV Abnormalities in dorsal periventricular white matter. accumulation of mitochondria, no COX-negative or ragged red fibers. IV (Fib) Normal N.d. Normal at 4 y of age Developmental delay, muscular hypotonia, tremor.

M, male; F, female; m, months; N.d., not determined; y, years; RCC, mitochondrial respiratory chain complex; PDHc, pyruvate dehydrogenase complex; AO, age at onset; CSF, cerebrospinal fluid; MRI, magnetic resonance imaging. Reduced activity values of respiratory chain complexes (RCC) I, II, III, and IV are given in relation to citrate synthase (CS) activity in mUnit/mUnit CS in percent of the lower control range. a These individuals are siblings. b Patient deceased.

T.B. Haack et al. / Molecular Genetics and Metabolism xxx (2014) xxx–xxx


Table 1 Genetic and phenotypic findings in MTMFT-mutant individuals.


#33467

#44409

#52181

#54502

A

C

E

G

B

D

F

H

#56713

#56902

3

#61606

#8432723

I

K

M

O

J

L

N

P

Fig. 1. Brain MRI studies in MTMFT-mutant individuals #33467 (A, B; T2-weighted axial scans at age 16 years), #44409 (C, D; T2-weighted axial scans at age 24 years), #52181 (E, F; T2weighted axial and T2 flair-weighted coronal scans at age 14 months), #54502 (G, H; T2 flair-weighted axial scans at age 2 years), #56713 (I, J; T2-weighted axial and coronal scans at age 4 years), #56902 (K, L; T2-weighted axial scans at age 20 months), #61606 (M, N; T2-weighted axial and coronal scans at age 4 years), and #8432723 (O, P, T2-weighted axial scans at age 5 years). There is a marked difference in the size and localization of the lesions affecting basal ganglia and the white matter.

that are encoded by the mitochondrial DNA (mtDNA) are translated within the organelle. Besides the 13 structural protein subunits of OXPHOS complexes I, III, IV, and V the mtDNA also codes for the RNA components of the mitochondrial translation machinery comprising 22 mitochondrial transfer RNAs (mt-tRNAs) and 2 ribosomal RNAs (mt-rRNAs). In addition, mitochondrial translation requires a presently unknown number of additional nuclear-encoded factors. Dysfunction of mitochondrial protein synthesis typically results in combined deficiencies of OXPHOS complexes although isolated defects, mainly of complex I, might occur as well [1]. The responsible genetic defects are located in genes that function at different levels of mitochondrial translation. These include mt-tRNAs and mt-rRNAs [2], nuclear genes encoding mitochondrial ribosomal proteins (MRPL3 [3], MRPL44 [4], MRPS16 [5], MRPS22 [6]), tRNA modifying proteins (MTO1 [7], PUS1 [8], TRMU [9]), a growing list of aminoacyl-tRNA synthetases (AARS2 [10], DARS2 [11], EARS2 [12], FARS2 [13], HARS2 [14], MARS2 [15], RARS2 [16], SARS2 [17], YARS2 [18]), translation elongation

factors (GFM1 [19], TUFM [20], TSFM [21]), a gene involved in mitochondrial RNA processing (ELAC2 [22]), and a gene of unknown function (C12orf65 [23]). Due to the large number of genes involved NGS-based genetic analysis provides substantial advantages over sequential testing of candidate genes, which leaves many patients without a molecular diagnosis in routine clinical diagnostics. The unbiased analysis of either a large set of a priori candidates, all coding exons, or the whole genome is a powerful tool to identify new disease genes and mutations. However, previous experience suggests that even such approaches fail to unravel the underlying genetic defect in about half of the cases. Among many other reasons, one possible explanation is the failure to capture the genetic region of interest. Therefore, in some cases, hypothesis-driven analysis of poorly covered regions using alternative methods such as Sanger sequencing is warranted to complement NGS studies. Impaired mitochondrial translation can be caused by mutations in MTFMT, which encodes a mitochondrial methionyl-tRNA formyl-


4

T.B. Haack et al. / Molecular Genetics and Metabolism xxx (2014) xxx–xxx 38 Mb 50 Mb V1 50 Mb V4 50 Mb V5

Coverage x-fold

300

200

100

20 0 Exon NM_139242.3 c.73C>T p.Gln25*

1

2

3

c.382C>T p.Arg128*

c.219_222del c.374C>T c.146_153del p.Arg49Leufs*58 p.Glu74Lysfs*3 p.Ser125Leu

4

5

c.626C>T p.Arg181Serfs*5 (p.Ser209Leu) c.452C>T p.Pro151Leu

6

7

c.766C>T p.Gln256*

c.878G>A p.Ser293Asp Homo sapiens (NP_640335.2) Pan troglodytes (XP_510478.2) Canis lupus familiarisf (XP_853) Mus musculusf (NP_081410.2) Gallus gallus (XP_413901.3)

... ... ... ... ...

K K K K K

L L L L L

LDLV LDLV LDLV LDLV LD FV

8

EV N S SV - L A D P K EV N S SV - L A D P K EV N S S I - L T D P K EV N N S I - L A D P K E V D S I L G S A DQ V

9 c.994C>T p.Arg332* L ... L ... L ... L ... L ...

Fig. 2. Gene structure of MTFMT and position of the identified mutations. Bold face script indicates newly identified pathogenic mutations. The coverage of MTFMT in patient #52181 is visualized with the Integrated Genomics Viewer demonstrating the failure of enrichment and/or sequencing of exon 1. Colored lines indicate the average coverage per exon calculated from 10 samples processed with different versions of the enrichment kit. Error bars indicated ±one standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

transferase. These have been identified by targeted analysis of the “MitoExome” (1034 nuclear-encoded mitochondrial-associated genes and the mtDNA [24]) as well as by whole exome sequencing [1]. In contrast to bacteria, metazoan mitochondria contain only a single tRNAMet that functions in both — translation initiation and elongation. The functional determination of tRNAMet strongly depends on an additional modification after aminoacetylation: MTFMT uses a portion of MettRNAMet to produce formylated Met-tRNAMet (fMet-tRNAMet). fMettRNAMet has increased affinity toward the mitochondrial initiation factor (IF2mt), which is then delivered to the P site of the ribosome for translation initiation. In contrast, translation elongation utilizes MettRNAMet, which is recruited to the ribosomal A site by the mitochondrial elongation factor (EFTUmt). Decreased levels of fMet-tRNAMet due to mutant MTFMT have been shown to impair mitochondrial translation resulting in reduced activities of multiple OXPHOS complexes in two families [24]. Another three index cases have been reported thereafter [1,25] for a total of five index patients. We now report on the identification of MTFMT mutations in nine additional cases from eight families together with detailed biochemical and clinical findings.

mild developmental delay at 6.5 years he was able to attend normal school until the age of 16 years. At 17 years he presented with a four day episode of fatigue, disturbed vision, and uncoordinated eye movements. At that time his body length was on the 5th and weight on the 70th percentile. The examination of his inner organs was normal apart from a 3/6 systolic heart murmur. Echocardiography revealed a 3rd degree aortic valve regurgitation and a ventricular septum defect (VSD). Neurologic examination revealed impaired fine motor skills, unstable gait, an upbeat nystagmus, and limited ocular motility. Cranial MRI (cMRI) showed symmetrical T2-weighted hyperintensities in the midbrain and crus cerebri. Auditory evoked potentials showed reduced amplitudes of the wave V on both sides. Lactate levels in cerebrospinal fluid (CSF) were increased to 4.0 mmol/l (N b 2.0 mmol/), while serum levels were normal. In frozen muscle tissue the activity of respiratory chain (RC) complex I was reduced to 0.11 mU/mU citrate synthase (CS) (N 0.17-0.56). Activities of other RC-complexes were normal. Progressing cardiac insufficiency required surgical closure of the VSD. He died on the fifth postoperative day from a massive intracranial hemorrhage.

2. Patients, materials and methods

2.1.2. Patient #33467, c.[146_153del];[c.626C N T], p.[Arg49Leufs*58]; [Arg181Serfs*5] The younger brother of patient #33009, had normal growth parameters at birth. His development was delayed and he went to a specialized school. Starting from the age of 6 years he was treated with growth hormone due to short stature and growth hormone deficiency. At the age of 15 years he developed a squint of the right eye. Neurologic investigation revealed unstable gait and reduced muscle tone. CSF and serum lactate were increased (both values 3.8 mmol/l), as was CSF alanine (49 μmol/l; N 29–39). cMRI revealed symmetrically increased T2-weighted signal intensities in the midbrain and several patchy signal changes in the white matter (Figs. 1A and B). Biochemical investigation of a muscle biopsy specimen showed an isolated RC-defect of complex I with a residual activity of 0.13 mU/mU CS (N 0.17-0.56). Histological and electron microscopic investigation did not reveal any pathologic changes, only a faint subsarcolemmal accumulation of mitochondria. The patient was treated with coenzyme Q10 (7 mg/kg BW/d), riboflavin (10 mg/kg BW/d), and a fat enriched diet (70% of caloric intake). At his present age of 22 years, his clinical condition is stable and he works in a sheltered workshop.

2.1. Clinical data We applied whole exome sequencing and candidate gene screening to identify MTFMT mutations in eight index cases with encephalopathy, lactic acidosis and OXPHOS-defects. Two additional patients (#44409 and #61606) carrying mutations in MTFMT have been reported previously [1]. Informed written consent was obtained from all participants or their guardians. The Ethics Committee of the Technische Universität München approved the study [2341/09]. Clinical and biochemical findings of MTFMT patients are summarized in Table 1 and additional detailed biochemical information is provided in Supplementary Table 1. Abnormal MRI findings are shown in Fig. 1. 2.1.1. Patient #33009, c.[146_153del];[c.626C N T], p.[Arg49Leufs*58]; [Arg181Serfs*5] The boy was born at term as the first child of healthy unrelated Caucasian parents. He was small for gestational age (birth weight 2250 g) and his psychomotor development was delayed with first independent steps at 18 months of age and first words at 24 months of age. Despite a



2.1.3. Patient #49728, c.[626C N T];[878G N A], p.[Arg181Serfs*5]; [Ser293Asn] The girl is the third child of healthy, unrelated parents. Two brothers (8 and 11 years old) are healthy. At the age of 2 months lack of fixation and microcephaly (1 cm b 3rd percentile) was noted. Ophthalmologic examination was normal. At the age of 4 months she had a sudden loss of muscle tone while drinking and had to be intubated and mechanically ventilated due to respiratory insufficiency. On admission she had metabolic acidosis with highly elevated lactate levels (14 mmol/l, N b 2.1 mmol/l) and transaminases (ALAT 275 U/l, ASAT 189 U/l). After recovery transaminases normalized, but lactate levels remained elevated (4 mmol/l). T2-weighted cMRI revealed diffuse signal hyperintensities in the basal ganglia and the brainstem. Echocardiography showed mild thickening of the ventricular septum. CSF lactate and serum amino acids levels were normal, but urinary organic acid analysis revealed highly elevated lactate and pyruvate. Biochemical assessment of a muscle biopsy specimen revealed a combined deficiency of RCcomplexes I (0.03 mU/mU CS, N 0.14-0.35) and IV (0.4 mU/mU CS, N 0.82-2.04). Concordant with this biochemical signature, quantification of fluorescent-labeled mitochondrial complexes in patient's fibroblasts using 2D BN/SDS-PAGE showed a clear decrease in complex Icontaining supercomplexes as well as in complex IV (Fig. 2). However, histochemical investigation of skeletal muscle detected only a mild lipid accumulation in type I muscle fibers but no COX-negative fibers. The girl developed generalized tonic–clonic seizures resistant to antiepileptic drugs and died during a febrile illness. 2.1.4. Patient #52181, c.[219_222del];[626C N T], p.[Glu74Lysfs*3]; [Arg181Serfs*5] The girl was born to unrelated healthy Caucasian parents at 41 weeks of gestation. Mild developmental delay was noted at the age of 2 months. Marked muscular hypotonia and feeding difficulties were present at 6 months. From there on, development stopped and she regressed. At 1 year, she was unable to sit and control her head. She was microcephalic with a head circumference 1 cm below the 3rd percentile. Plantar reflexes were upward on both sides. cMRI showed extensive white matter lesions with cysts. Diffusion-weighted images revealed cytotoxic edema in nucleus caudatus, pallidum, and mesencephalon (Figs. 1E and F). MR-spectroscopy discovered a significant lactate-peak in pallidum and white matter. Lactate levels were elevated in serum (5.4 mmol/l) and CSF (4.9 mmol/l), as were alanine levels. Urinary lactate and pyruvate were increased. Complex I activity in muscle was reduced to 0.03 mU/mU CS (N 0.14-0.35). Citrate synthase activity was elevated to 365 mU/mg protein (N 150–338). Other enzyme activities were within normal range. Histochemistry showed a mildly inhomogeneous distribution of mitochondria but no evidence for a diffuse COX-deficiency or COX-negative/SDH-positive fibers. She was set on a ketogenic diet (3:1) and supplemented with riboflavin (10 mg/kg BW/d), and coenzyme Q10 (10 mg/kg BW/d). The patient died at 19 months of age from pneumonia with respiratory insufficiency. 2.1.5. Patient #54502, c.[626C N T];[994C N T], p.[Arg181Serfs*5]; [Arg332*] The boy was born at 35 weeks of gestation as the first child of healthy unrelated parents. His birth weight was 2550 g, length 47 cm, and head circumference 32 cm (all at the 25th percentile). At 3 months a global retardation and muscular hypotonia were noticed. His developmental progress continued to be delayed with unsupported sitting at 14 months, standing at 18 months, and first steps at 22 months. He started to speak at 2 years and spoke 10 words at 3 years. When last seen at the age of 6 years he showed muscular hypotonia with normal reflexes. He had a mild intermittent strabismus. His gait was clumsy and slightly broad based, but he was able to walk about 1.5 km. His speech was slurry but he spoke in short sentences and understood well. His fine motor skills were retarded by 3 years.

5

Echocardiography at 3 years revealed a mild hypertrophy of both ventricles with normal function and no progression over the last three years. cMRI at 2 years showed several subcortical frontal and parietal T2-hyperintense lesions (Figs. 1G and H). Some of them showed an abnormal diffusion restriction pattern. Lactate levels were repeatedly elevated up to 6 mmol/l in serum (N b 2.2 mmol/l) and in CSF. A metabolic workup was unremarkable apart from elevated alanine concentrations in serum (650 μmol/l, N b 420 μmol/l). A muscle biopsy performed at age 31 months did not show significant histological or histochemical abnormalities. However, complex I enzyme activity was reduced in two independent analysis (0.05 mU/mU CS, N 0.140.35 mU/mU CS and 0.11 mU/mU CS, N 0.17-0.56 mU/mU CS) with an additional defect in complex IV (0.5 mU/mU CS, N 0.11-5.0 mU/mU CS) in one of them (Supplementary Table 1). At that time a treatment with coenzyme Q10 and riboflavin (each 10 mg/kg BW/d) as well as a fat rich diet (70% of total caloric supply) were started. According to the parents he improved significantly with this treatment and was more resilient. Since that time lactate levels never exceeded 3.5 mmol/l. 2.1.6. Patient #56713, c.[73C N T];[626C N T], p.[Gln25*];[Arg181Serfs*5] The boy is the only child of unrelated healthy Caucasian parents. He was born at term after a normal pregnancy. At the age of 8 months he presented with truncal hypotonia, mild spasticity of the upper limbs, and developmental delay of ≈2 months. His head circumference was on the 3rd percentile whereas body length and weight were between the 50th and 75th percentiles. At 11 months he was able to sit and at 15 months he started to walk. At 3 years, his parents observed unstable gait, ataxia and frequent falls. He had difficulties in social interaction, avoided contact with other children and was very sensitive to noise. At 4 years his cognitive and speech development was retarded by ≈ 1.5 years. cMRI revealed a pineal cyst and a bilateral diffusionweighted and T2-hyperintensity in the globus pallidus (Figs. 1I and J). CSF lactate levels were increased (4.2 mmol/l). Extensive metabolic workup (organic acids, amino acids, acylcarnitines, purine, pyrimidine, VLCFA, screening for CDG syndrome) and genetic tests (karyotyping, array-CGH, and FMR1 mutation screening) were normal as were ECG, echocardiography and several EEGs. Biochemical investigation of a muscle biopsy specimen revealed isolated RC-complex I deficiency (0.09 mU/mU CS, N 0.14-0.35). He was set on a fat rich diet (70% of kcal) and supplemented with riboflavin (10 mg/kg BW/d) and Coenzyme Q10. The treatment failed to show beneficial effects and was stopped by the parents after several months. A control cMRI at 6 years did not show any novel pathological changes. He was able to walk unaided with a broad-based gait and attended a special school. 2.1.7. Patient #56902, c.[452C N T];[994C N T], p.[Pro151Leu]; [Arg332*] The boy was born as the first child of healthy unrelated parents at 34 weeks of gestation. His birth weight was 1684 g (100 g below 10th percentile). During a bacterial infection at the age of 20 months he developed progressive gait instability and loss of independent ambulation within one week. His body weight, length, and head circumference were all below the 3rd percentile. Serum lactate and pyruvate levels were increased to 7 mmol/l (N b 2.2 mmol/l) and 239 μmol/l (N b 102 μmol/l), respectively. Increased lactate levels were also documented in the CSF (4.9 mmol/l, N b 2.2 mmol/l), as well as elevated alanine concentrations (73 μmol/l, N 12.6-45 μmol/l). cMRI revealed T2-signal intensive lesions of the periventricular white matter, centrum semiovale and spelium callosum (Figs. 1K and L). MRI spectroscopy showed increased periventricular lactate peaks. Echocardiography indicated a left ventricular non-compaction cardiomyopathy although size and function of the left ventricle were still within normal range. Biochemical investigation of a muscle biopsy showed a combined deficiency of complex I (0.075 mU/mU CS, N 0.084-0.273) and IV (0.416 mU/mU CS, N 0.045-0.187) which was subsequently also confirmed in fibroblasts (Table 1). Histochemical studies indicated a disproportion of type I b type 2 fibers and merosin-deficient but no SDH or


6


COX-negative fibers. LAMA2 and mitochondrial tRNA sequencing failed to detect potentially disease-causal mutations. His psychomotor developmental progress continued to be delayed and he displayed a marked generalized muscular hypotonia at the age of 28 months.

2.1.8. Patient #73922, c.[452C N T];[626C N T], p.[Pro151Leu]; [Arg181Serfs*5] The boy was born at term as the first child of healthy unrelated Caucasian parents. He was small for gestational age (birth weight 2030 g). Psychomotor development progressed within the normal range. Neurological assessment revealed mild muscular hypotonia. Since birth lactate levels in serum were found to be constantly increased (lactate 4.41 mmol/l, N 0.86-2.19), as were alanine levels (alanine 701 μmol/l, N 100–439). Organic acid analysis was normal. cMRI at 7 months showed a mild cortical atrophy and myelination delay. MRI spectroscopy was normal. Echocardiography at 7 months revealed a mild hypertrophy of the right ventricle with normal function. At the present age of 15 months, his clinical condition is stable. His body weight, length, and head circumference are all between 1st and 10th percentiles. The patient is treated with coenzyme Q10 (10 mg/kg BW/d), riboflavin (10 mg/kg BW/d), and carnitine (70 mg/kg BW/d).

2.1.9. Patient #8432723, c.[626C N T];[766C N T], p.[Arg181Serfs*5]; [Gln256*] The male patient is the third child of unrelated Dutch parents, three sibs are healthy. He was born prematurely (32 weeks of gestation, birth weight 1320 g) due to maternal HELLP syndrome. The neonatal course was complicated by frequent apnea and bradycardia, however, artificial ventilation was never necessary. The parents reported excessive fatigue and exercise intolerance from the neonatal period onwards. The motor milestones were delayed (e.g. walking with two years) as was the speech development (two word sentences with five years). The patient never learned climbing the stairs or cycling, he also made no further progress on speech and intellectual development. He developed an epileptic state at age four years. Since then no more seizures have been witnessed without anti-epileptic treatment. At the same time an abnormal breathing pattern became obvious with periods of hypoalternating with hyperventilation. Chronic nocturnal hypoventilation necessitated nocturnal non-invasive ventilation from age five onwards. Serum lactate (up to 9 mmol/l, N b 2 mmol/l) and alanine (609 mmol/l, N b 450 mmol/l) were elevated, urinary organic acids normal. The cerebral MRI at age five years showed white matter alterations in both hemispheres as well a bilateral periductal gray matter alterations suggestive for Leigh syndrome (Figs. 1O and P). On repetitive MRI at age eight and nine years these alterations were not progressive. Immunochemistry of the muscle showed a relative COX-deficiency, a predominance of small type I muscle fibers with an increase of fat between fibers. The mitochondrial ATP + CrP production from pyruvate was reduced (21.4 nmolCO2/h.mUCS, N 42.1–81.2 nmolCO2/h.mUCS). The results of enzyme activity measurements in muscle and fibroblasts are given in Supplementary Table 1. The clinical course has been very stable. He is currently aged 12 years and at the developmental level of a 2.5 year old when tested. He is very social and has a friendly and open character. He still suffers from frequent upper airway infections which take him long time to overcome. He has a short stature (height − 2.5 SD, weight for height +1 SD, head circumference −1.5 SD). He has a parkinson-like phenotype with paucity of facial expressions, very slow (“freezed”) movements and a tippling and insecure, slightly broad based gait. The legs are somewhat hypertonic and show hyperreflexia. He is able to walk independently and uses a wheelchair for longer distances. Besides severe constipation no other organ involvement has been observed yet.

2.1.10. Patient #44409, c.[626C N T];[994C N T], p.[Arg181Serfs*5]; [Arg332*] The boy was born at term to healthy unrelated parents. From 3 years of life he showed psychomotor developmental delay. At 5 years he presented with acute, leftsided, central nerve VII palsy. At 12 years his motor, speech and mental capabilities (IQ 75) regressed and he developed ataxia, progressive spastic paraplegia with knee and ankle contractures. Now, at 24 years he is wheelchair-dependent with severe spastic tetraplegia and strabism with diplopia and vertical gaze palsy, neurogenic bladder dysfunction, depression, and chronic fatigue. He never had seizures. Serial cMRIs revealed progressive T2-signal intensive lesions of the, putamen, periventricular white matter, medulla oblongata and the head of the nucleus caudatus (Figs. 1C and D). Lactate levels in serum (1.9-3.3 mM) and CSF (4.6 mM) were elevated. Urine excretion of organic and amino acids was always normal. Muscle histology revealed a variation fiber size and increased number of fat droplets. COX- and SDH-staining were normal and ragged-red-fibers were absent. Biochemical investigations revealed complex I deficiency with 0.026 mU/mU CS (N 0.07-0.25) residual activity and reduced activity of PDHc (71% of norm). Treatment with riboflavin, coenzyme Q10, creatine monohydrate, vitamins C and K, and succinate did not alter his disease course. 2.1.11. Patient #61606, c.[626C N T];[626C N T], p.[Arg181Serfs*5]; [Arg181Serfs*5] The girl is the first child of healthy, unrelated parents. She was born after a normal pregnancy at 39 weeks of gestation. Psychomotor developmental progress was in the low normal range and she was able to walk independently at 16 months of age. Her gait was always stiff and unstable with frequent falls and poor endurance. She started to speak at 12 months. At 3 years her sentences consisted of 2–3 words only. At 3.5 years her weight was 13 kg (10th percentile), length 93 cm (3rd percentile) and she was microcephalic with a head circumference of 46.5 cm (b 3rd percentile). Neurologic assessment revealed persistent mouth opening, reduced muscle mass and generalized mild muscular hypotonia. Her gait was unsteady, broad-based with dystonic inward rotation of her right leg and she was unable to stand on one leg. The Achilles tendon reflex was weak and other deep tendon reflexes were absent. The Denver test at age 40 months revealed a gross motor development of 20 months, fine motor skills of 24 months, verbal skills of 36 months, and social skills of about 30 months. When last seen at the age of 6 years, she still made continuous developmental progress but had to attend a special school for mentally disabled children. cMRI at 38 months revealed bilateral lesions of the putamen and T2-signal intensive lesions in the fronto-parietal white matter (Figs. 1M and N). MR-spectroscopy at the putamen revealed a decreased NAA/creatine ratio and a lactate peak. Biochemical investigation of a muscle biopsy specimen revealed isolated complex I deficiency with residual activity of 0.03 mU/mU CS (N 0.14-0.35). Therapy with riboflavin, L-carnitine, and coenzyme Q10 over one year did not have any beneficial effects and an Atkins diet was not tolerated. 2.2. Exome sequencing and HRMA screen We used a dual approach of exome sequencing and high resolution melting curve analysis (HRMA) to analyze a cohort of patients with suspected mitochondrial disorders in order to identify pathogenic mutations in MTFMT in nine patients from eight families. Four index patients (#44409, #56713, #56902, and #61606) were investigated by exome sequencing using the SureSelect Human All Exon 50 Mb Kit (Agilent) for enrichment and a HiSeq2000 (Illumina) for sequencing. The average coverage was 152 × (#44409), 157 × (#56713), 66 × (#56902), and 121 × (#61606). More than 91% of the exome was covered at least 20× allowing for high-confidence variant calls. One individual (#52181) was investigated with the SureSelect Human All Exon 38 Mb Kit (Agilent) and sequenced to an average 68 × coverage



7

exomes and public databases. Based on the autosomal recessive pattern of inheritance and the assumption that mitochondrial respiratory chain defects are likely to be associated with mitochondrial proteins, we than applied different prioritization filters for (i) two known pathogenic variants, (ii) two novel potentially pathogenic variants in a structural gene of complex I, and (iii) presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein [26]. In individual #8432723 exome sequencing and bioinformatic analysis were performed as described before [27].

corresponding to 78% of the exome covered at least 20×. Another individual (#54502) was investigated with Agilent 38 Mb as well as the 50 Mb kit with 86% of the target being covered at least 20 × in the second experiment. We used BWA (version 0.5.8) for read alignment to the human reference assembly (hg19). Single-nucleotide variants (SNVs) and small insertions and deletions were detected with SAMtools (version 0.1.7). The detailed filtering strategy leading to the identification of the causal mutations in patients #44409 and #61606 has been reported previously [1]. In brief, we first excluded variants present with a frequency higher than 0.2% in 2000 control

MTFMT expression levels in % ofcontrol

A 100 90 80 70 60 50 40 30 20 10

0.25

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#49728

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97 #4

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28

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ne HF

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67 #5

43 kDa

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81 21

28 #4

#5

97

34 #3

ND

HF

67

ne

o

o

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MTFMT longer exposure TOM20

09 44

#4

#5

67

13

81 21

97

28 #5

#4

67 34

#3

ND

HF

ne

o

C

TOM20

NDHFneo #33467

ATP5a

#49728 #52181 #56713 COX4

#44409

SDHA

NDUFB8

0

0.5

1

1.5

x-fold protein levels relative to NDHFneo Fig. 3. Quantification of MTFMT expression by qPCR (A) and immunoblot investigation of steady state levels of MTFMT (B) and respiratory chain complexes (C). qPCR values are mean of two measurements and are given relative to the mean of three controls. Values of quantified OXPHOS subunits have been corrected for loading of mitochondrial proteins (TOM20) and represent the mean of three experiments relative to a control. Error bars indicate ±1 standard deviation.


8


HRMA was performed as described previously [28] with intronic primers covering all coding exons of MTFMT. Primer sequences used for HRMA and mutation confirmation are given in Supplementary Table 2. 2.3. Quantification of MTFMT expression by qPCR For qPCR total RNA was isolated from muscle of individuals #49728, c.[626C N T];[878G N A], #56713, c.[73C N T];[626C N T], #33467, c.[146_153del];[626C N T], #52181, c.[219_222del];[626C N T] as well as from 3 controls. Following DNase treatment (Turbo DNase, Ambion), RNA was reverse transcribed using random hexamer primers (Maxima RT, Thermo Scientific) and used for subsequent qPCR analysis. PCR reactions were set up using B-R SYBR® Green SuperMix for iQ™ (Quanta Biosciences), covered with a layer of mineral oil and performed in an iCycler iQ5 (BioRad Laboratories). The oligonucleotides for the MTFMT-transcript were MTFMT-E4-F 5′-GGCCCAATTCTCAAACAAGA-3′ and MTFMT-E8-R 5′-CCTGTCCCGTTAATTTTGGA-3′ starting from exon 4 and exon 8, respectively. Ct values were normalized to the mean of two housekeeping genes, HPRT1 (HPRT1-F 5′-TTCCTTGGTCAGGCAGTA TAATC-3′, HPRT1-R 5′-GGGCATATCCTACAACAAACTTG-3′) and RPL27 (RPL27-F 5′-GCTGGAATTGACCGCTACC-3′, RPL27-R 5′-TCTCTGAAGACA TCCTTATTGACG-3′). The normalized expression of MTFMT of the patients was related to the mean of the 3 controls (ΔΔCt) and the relative amount of MTFMT expression was calculated as 2 − −ΔΔCt. 2.4. Immunoblot analysis of MTFMT and respiratory chain complexes Analysis of mitochondrial respiratory chain complexes was performed on whole cell lysates. MTFMT quantification was done in mitochondria-enriched fractions. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), blotted onto PVDF membrane (GE-Healthcare, Buckinghamshire, UK) for subsequent incubation with primary antibodies and probing with appropriate secondary antibodies. Chemiluminescence was documented on a Fusion FX7 system (Peqlab, Erlangen, Germany) and

quantified using the BIO-1D software. The following antibodies were used for western blotting: mouse-anti-SDHA (ab-14715, 1:1000), mouse anti-NDUFB8 (ab-110242, 1:1000), mouse anti-ATP5a (ab14748, 1:1000), rabbit anti-COX IV (ab-16056, 1:1000), and mouse anti-MTFMT (ab-119022 1:100) were purchased from Abcam (Cambridge, UK). Rabbit anti-TOM20 (sc-11415, 1:1000) was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Mouse anti-beta actin (A-5441, 1:10,000) was purchased from Sigma-Aldrich (St. Louis, USA). HRP-conjugated secondary antibodies for western blot were obtained from Jackson Immunoresearch Laboratories (USA) (1:15,000). 2.5. 2D BN/SDS-PAGE Separation and quantification of fluorescent-labeled mitochondrial complexes and 3D visualization were performed as described previously [29,30]. Briefly, sedimented enriched mitochondrial membranes from homogenized fibroblasts were labeled with NHS-Fluorescein (Thermo Scientific) and solubilized with the mild detergent digitonin. Fluorescence signals from the 2D Blue native electrophoresis (BNE)/SDSPAGE separation of mitochondrial complexes [31] were quantified by DeCyder 7.0 (GE Healthcare) software. 3. Results Exome sequencing and bioinformatic filtering identified MTFMT (NM_139242.3) mutations in two patients reported previously ([1]; #44409, c.[626C N T];[994C N T], p.[Arg181Serfs*5];[Arg332*], and #61606, c.[626C N T];[626C N T]; p.[Arg181Serfs*5];[Arg181Serfs*5]) and a two patients newly reported in this study (#56902, c.[452C N T]; [994C N T], p.[Pro151Leu];[Arg332*], and #8432723, c.[626C N T]; [766C N T], p.[Arg181Serfs*5];[Gln256*]). In patient #56902, the change c.452C N T (p.Pro151Leu) affected the maternal and the c.993C N T (p.Arg332*) mutation the paternal allele. Both mutations have been reported only recently in the same combination in two sisters [25]. The pathogenicity of these mutations has been established by western blots analyses showing a severe decrease in MTFMT protein in patient's

Fig. 4. Mito-Panorama: separation and quantification of fluorescent-labeled mitochondrial complexes by 2D BN/SDS-PAGE in control and patient #49728 fibroblasts (A). Silver stained 2Dgels are shown for comparison. 3D visualization “MitoPanorama” (B) of quantified mitochondrial membrane complexes from control and patient fibroblasts (C). Assignment of complexes: S, supercomplexes composed of respiratory chain complexes I, III, and IV; VM, complex V or ATP synthase; III2, complex III or cytochrome c reductase; IV, complex IV or cytochrome c oxidase; II, complex II or succinate dehydrogenase.



tissues. In patient #8432723, the previously reported c.626C N T (p.Arg181Serfs*5) mutation was inherited from the father and the novel predicted truncating c.766C N T (p.Gln256*) mutation (MutationTaster: disease causing, score 1) from the mother. However, exome sequencing and stringent variant filtering failed in individuals #52181 and #56713 as well as the first analysis of patient #54502. In individual #56713, we identified rare compound heterozygous or homozygous variants in 9 candidate genes (HAGHL, ZNF407, WDR18, ZNF497, TMEM129, FGF2, NIPAL2, CLCN5, and KDM5C). None of these genes had been linked to mitochondrial function. Assuming that we might have detected one mutation but missed the second, we next searched for heterozygous variants in genes predicted to code for mitochondrial proteins. This analysis identified 17 heterozygous rare variants: 2 nonsense variants, 1 splice variant, and 14 missense variants including a mutation in MTFMT, c.626C N T (p.Ser209Leu). Tucker et al. had demonstrated that this presumed missense mutation, in fact causes a loss of function via impaired splicing which led to skipping of exon 4 and a premature truncation of the protein, p.Arg181Serfs*5 [24]. Given the strong evidence for a detrimental functional impact of the mutation and its causative role in respiratory chain dysfunction together with the fact that mutant MTFMT could explain the phenotype of individual #56713, we manually inspected the coverage of MTFMT in our exome sequencing analysis. We realized that exon 1 of MTFMT was poorly covered (Fig. 2) and accordingly repeated DNA sequence analysis by Sanger sequencing. We discovered a previously unreported heterozygous nonsense mutation, c.73C N T (p.Gln25*), predicting a premature truncation of more than 90% of the protein. Sequence analysis of the parents confirmed the compound heterozygous state of the mutations with the c.73C N T mutation being located on the paternal allele and the c.626C N T mutation on the maternal allele. In patient #54502, an initial exome sequencing experiment using the Agilent 38 Mb kit failed to reveal any rare mutations likely to cause the disease phenotype. However, a second exome analysis based on the Agilent 50 Mb V5 kit detected compound heterozygous MTFMT mutations, c.[626C N T];[994C N T], p.[Arg181Serfs*5]; [Arg332*], with the mother being a heterozygous carrier of the c.626C N T (p.Arg181Serfs*5) mutation. The pathogenic character of both mutations has been established previously [24,25]. In patient #52181, searching for genes carrying predicted homozygous or compound heterozygous mutations failed to reveal obvious candidate genes. A search for heterozygous mutations identified missense mutations in three genes previously associated with respiratory chain defects, NUBPL, POLG, and AIFM1. Sanger sequencing did not unravel any additional rare variant. The heterozygous mutation c.952G N A (p.Ala318Thr) in X-chromosomal AIFM1 (NM_004208.3) was found in the hemizygous state in a healthy relative, thereby excluding its pathogenicity in our patient. We next screened a cohort of 350 patients with a suspected mitochondrial disorder and with isolated or combined OXPHOS defects for genetic variation in MTFMT. This analysis identified MTFMT mutations in four additional patients, including two brothers (#33009 and #33467) and patient #52181, who was previously investigated by exome sequencing. In the latter, both mutations in MTFMT (c.[219_222del];[626C N T], p.[Glu74Lysfs*3];[Arg181Serfs*5]) were missed by exome sequencing due to poor coverage of the entire gene. The mother was a heterozygous carrier of the c.626C N T mutation and the father of the predicted truncating c.219_222del mutation (MutationTaster: disease causing, score 1). In patient #49728 we detected the known c.626C N T mutation, (p.Arg181Serfs*5) and a heterozygous c.878G N A missense mutation (p.Ser293Asn). The c.878G N A variant was not observed in 2500 control exomes analyzed in Munich and the Exome Variant Server. However, in silico prediction suggested that the amino acid change affecting a moderately conserved residue (Fig. 2) might be benign (MutationTaster: polymorphism, score 0.99; SIFT: tolerated, score 0.7; PolyPhen-2: benign, score 0.001). We therefore performed experiments to provide additional

9

evidence that defective MTFMT is indeed causal for the disease phenotype in patient #49728. We quantified the amount of MTFMTexpression in muscle which was reduced to b1% of control levels in this patient (Fig. 3). Sequencing of the primary transcript and the mRNA showed only wild type sequences at c.878 indicating that the expected mRNA is not expressed (Supplementary Fig. 1). In the two brothers (#33009 and #33467) we identified the c.626C N T mutation (p.Arg181Serfs*5) together with a heterozygous 8 bp deletion (c.146_153del, p.Arg49Lysfs*58) predicting a frameshift and premature truncation of more than 70% of the protein. As in patient #49728, no material of the parents was available for carrier testing. We next investigated the impact of novel MTFMT mutations on steady state MTFMT protein levels. Testing of available mutant fibroblast cell lines showed a severe decrease in MTFMT in all including patient #49728 carrying the c.878G N A allele (Fig. 3). These results provide strong evidence for the pathogenic character of identified mutations and argue for a de facto loss-of-function mechanism causing the MTFMT-associated phenotype. Therefore, MTFMT protein analysis in patient-derived tissues and cell lines is a helpful assay to test the pathogenicity of newly identified MTFMT variants. Analysis of steady state levels of different subunits of RC-complexes in fibroblasts suggested a severe decrease in the amounts of complexes I and IV (Fig. 3). Quantification of fluorescent-labeled mitochondrial complexes in patient's fibroblasts (#49728) using 2D BN/SDS-PAGE showed a clear decrease in complex I-containing supercomplexes as well as in complex IV (Fig. 4). Biochemical investigations of skeletal muscle specimen showed a complex I defect in all cases. In at least half of them an additional decrease in complex IV activity was observed (Table 1 and Supplementary Table 1). Investigations in fibroblasts from five patients indicated the same pattern with complex I being more severely affected than complex IV. 4. Discussion We report the identification of five novel mutations in MTFMT in compound heterozygosity with a frequent mutation (c.626C N T) present in eleven out of thirteen index cases. The pathogenic character of newly identified mutations is supported by a severe decrease in MTFMT levels in mutant fibroblasts indicating that the mutations affect the stability of the protein. All eleven patients presented with an encephalomyopathic phenotype characterized by ataxia, muscular hypotonia and psychomotor retardation. The severity of the disease is variable ranging from early death with 16 months to predominantly motor handicap and spasticity at the age of 24 years. One patient with a severe phenotype had a metabolic stroke with peracute deterioration and respiratory failure. At least seven out of eleven patients had a microcephaly with a head circumference below or on the 3rd percentile. Many of the mitochondrial encephalomyopathies with onset in childhood and infancy show involvement of one or more visceral organs. Though none of our patients presented significant hepatic, renal, cardiac intestinal or hematologic involvement, we cannot exclude that the patients with a more severe phenotype who had died early, might have developed such symptoms. Five patients from three families reported by Tucker et al. and Neeve et al. also show an encephalomyopathic phenotype [24]. Two of them (cousins) had Wolff–Parkinson–White syndrome (WPWS), a cardiac re-entry arrhythmia, which can be due to a mitochondrial disorder but can also be coincidental as WPWS is not rare in the normal population. cMRI of seven out of eleven patients showed symmetrical lesions in the basal ganglia predominantly of putamen and globus pallidus. Two of them also had lesions in the medulla oblongata. This pattern corresponds to classical Leigh syndrome and has also been documented in the three index cases reported by other groups [24,25]. Four patients (#33467, #44409, #61606 and #52181) presented with additional lesions of the


10


cerebral white matter, which were particularly extensive in patient #52181 with multiple cystic lesions at an early age (10 months). In the other three patients the white matter lesions appeared after the abnormalities in the basal ganglia and later in the disease course. However, individuals #54502, #56902, and #8432723 showed lesions in the periventricular white matter and splenium callosum (and #8432723 in the brain stem) but no alterations of the basal ganglia. Patient #73922 showed only a mild cortical atrophy and myelination delay by the age of 15 months. Together, our data suggests that, although MTFMT mutations mainly manifest with Leigh syndrome, the localization and size of the lesions is variable. Exome sequencing is an efficient tool for the molecular diagnostic workup of mitochondrial disorders characterized by vast clinical and genetic heterogeneity. However, in a substantial fraction of cases this method fails to identify the causal mutations. In this study we provide two examples. In the first instance, we used the first available exome enrichment kit from Agilent covering 38 Mb. With this kit MTFMT was not covered at all probably due to an unbalanced enrichment and only 80% of the target being covered more than 20×. In the second case the coverage of more than 92% of the target by at least 20 × fulfilled our internal standards and the enrichment of MTFMT was substantially improved in the Agilent 50 Mb V1 kit (Fig. 2). However, only one mutation was detected. While missed by exome sequencing due to systematically insufficient capture of the first exon of MTFMT, reinvestigation of the heterozygous variants and subsequent Sanger sequencing identified the second heterozygous mutation in exon one. This study highlights the fact that a candidate gene approach using traditional methods still remains a valuable tool to complement NGS-based studies in case the gene of interest is not fully covered and to find the genetic diagnosis in additional patients. Furthermore, our study suggests to eventually re-investigate unsolved cases from earlier experiments with an updated version of the enrichment kit which not only allows for the identification of previously uncovered variants but also facilitates the discovery of large deletions and insertions due to a more balanced and reproducible distribution of the sequence reads. In addition, also with regards to the need of reporting negative results in a diagnostic context, systematic ascertainment of poorly covered regions in known disease genes (n N 220 for OXPHOS disorders) should be included in the bioinformatic readout. These lists provide the option for subsequent complementation of exome investigations by more focused single gene studies. Our study provides an example for both, power and limitation of enrichment-based sequencing in terms of unbiased and cost-efficient detection of disease-causing mutations on one side and incomplete sensitivity for the whole exome on the other. With 90% coverage the sensitivity to detect compound heterozygous mutations is about 80% for exomic variants in annotated genes captured by the enrichment kit. One option to address this issue is to replace targeted re-sequencing approaches by whole genome sequencing. While data interpretation is challenging, falling sequencing costs and faster protocols make whole genome sequencing attractive [32]. Of note, nine out of ten index cases presented in this study and also the two index patients reported by Tucker et al. carry the c.626C N T mutation. This mutation was present in the heterozygous state in 4 out of 2000 (MAF 0.1%) mainly German controls analyzed by us and in the Exome variant server in 9 out of 4104 European American exomes (MAF 0.11%) while being absent from 1867 African American exomes. With a MAF of about 0.1% in the European population this mutation is likely to be the most frequent loss-of-function mutation in an OXPHOS-associated gene. MTFMT mutations might be an underdiagnosed cause of OXPHOS defects and are among the most frequent causes of impaired mitochondrial translation. Biochemically, at least five out of twelve MTFMT index cases reported previously and in this study, have an isolated complex I deficiency and seven had a combined complex I and IV defect in skeletal muscle. Steady state levels of subunits of RC-complexes I and IV were significantly decreased in patient-derived fibroblasts, with complex I

being more severely affected than complex IV. The notion that complex I seems to be a sensitive marker for OXPHOS dysfunction, especially when arising from perturbed mitochondrial protein synthesis, has been discussed previously [1]. Potential explanations include the fact that seven out of 13 mtDNA-encoded RC subunits have been assigned to complex I, the largest and most intricate complex of the OXPHOS machinery. In summary, we reported detailed clinical, biochemical, and genetic data on nine new patients with MTFMT mutations, interestingly, apart from one exception, all in compound heterozygosity with a known disease mutation. While our study strengthens the application of exome sequencing as an early diagnostic tool in mitochondrial disease it also points out the need to investigate unsolved cases by alternative strategies and methods. Additional factors contributing to the success are up-to-date mutational databases and joint efforts in biobanking of rare phenotypes.

Web resources The URLs for data presented herein are as follows: MitoP2, http://www.mitop.de MITOPRED, http://bioapps.rit.albany.edu/MITOPRED/ TargetP, http://www.cbs.dtu.dk/services/TargetP/ Online Mendelian Inheritance in Man (OMIM), http://www.ncbi. nlm.nih.gov/Omim/ Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA URL: http://evs.gs.washington.edu/EVS/ Conflict of interest statement Nothing to report.

Acknowledgments We are grateful to the patients and their family for their participation in this study and especially to E. Botz, C. Fischer, and R. Hellinger and for their technical support. S.B.W. and R.J.R. would like to thank the technicians of the muscle, DNA, and tissue culture labs of the NCMD/LGEM for excellent technical assistance. T.M. and H.P. were supported by the Impulse and Networking Fund of the Helmholtz Association in the framework of the Helmholtz Alliance for Mental Health in an Ageing Society (HA-215) and the German Federal Ministry of Education and Research (BMBF) funded German Center for Diabetes Research (DZD e.V.) and Systems Biology of Metabotypes grant (SysMBo #0315494A). H.P. was supported by the grant RF-INN-2007-634163 of the Italian Ministry of Health. T.M., P.F., M.S. and H.P. were supported by the BMBF funded German Network for Mitochondrial Disorders (mitoNET #01GM1113C/D). H.P., T.M., J.A.M. and W.S. were supported by the E-Rare project GENOMIT (01GM1207 and FWF I 920-B13). J.A.M. and W.S. were supported by the Vereinigung zur Förderung Pädiatrischer Forschung und Fortbildung Salzburg. I.W. was supported by the BMBF funded German Network for Mitochondrial Disorders (mitoNET #01GM1113B) and by the Cluster of Excellence “Macromolecular Complexes” at the Goethe University Frankfurt (EXC 115). The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ymgme.2013.12.010.



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Novel PSEN1 mutations (H214N and R220P) associated with familial Alzheimer's disease identified by targeted exome sequencing.

Spectrum of gene mutations detected by next generation exome sequencing in brain metastases of lung adenocarcinoma.

Exome sequencing extends the phenotypic spectrum for ABHD12 mutations: from syndromic to nonsyndromic retinal degeneration.

Mutational spectrum and clinical features of patients with ACTG1 mutations identified by massively parallel DNA sequencing.

Exome sequencing identified new mutations in a Marfan syndrome family.

Next-generation Sequencing Extends the Phenotypic Spectrum for LCA5 Mutations: Novel LCA5 Mutations in Cone Dystrophy.

Targeted Exome Sequencing of Deafness Genes After Failure of Auditory Phenotype-Driven Candidate Gene Screening.

PNKP Mutations Identified by Whole-Exome Sequencing in a Norwegian Patient with Sporadic Ataxia and Edema.

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis.

Identification of a novel DMD duplication identified by a combination of MLPA and targeted exome sequencing.

Identification of novel mutations by exome sequencing in African American colorectal cancer patients.

Novel somatic mutations identified by whole-exome sequencing in muscle-invasive transitional cell carcinoma of the bladder.

Novel pathogenic variants and genes for myopathies identified by whole exome sequencing.

Candidate gene discovery in autoimmunity by using extreme phenotypes, next generation sequencing and whole exome capture.

Exome sequencing covers >98% of mutations identified on targeted next generation sequencing panels.

SIPA1L3 identified by linkage analysis and whole-exome sequencing as a novel gene for autosomal recessive congenital cataract.

Late-onset CORD in a patient with RDH12 mutations identified by whole exome sequencing.

Phenotypic Variability and Newly Identified Mutations of the IVD Gene in Japanese Patients with Isovaleric Acidemia.

Whole exome sequencing identified novel CRB1 mutations in Chinese and Indian populations with autosomal recessive retinitis pigmentosa.

Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing.

Whole-exome sequencing revealed two novel mutations in Usher syndrome.

Novel TBL1XR1, EPHA7 and SLFN12 mutations in a Sezary syndrome patient discovered by whole exome sequencing.

Candidate gene analysis and exome sequencing confirm LBX1 as a susceptibility gene for idiopathic scoliosis.

Identification of Candidate Gene Variants in Korean MODY Families by Whole-Exome Sequencing.