Suppl. Data; Cámara et al; 1

SUPPLEMENTAL DATA

METHODS

Patients

Five patients suffering from mitochondrial myopathy associated with multiple

mtDNA deletions were included in the study. Mutations in TK2 (RefSeq

NP_004605.4) were identified for all 5 patients. Patient 1 (P1) was compound

heterozygote for the mutations p.K202del and p.T108M; patient 2, 3 and 4 (P2,

P3 and P4) were homozygous for the p.K202del mutation; patient 5 (P5) and

patient 6 (P6) were homozygous for the p.T108M mutation. Patient 7 (P7) was

compound heterozygote for the mutations p.R192K and p.T108M. Besides the

adult patients, we also studied skin fibroblasts from 3 healthy controls (C) and

four infantile patients suffering from severe mtDNA depletion: patient 8 (P8,

described in (1)) was compound heterozygote for the mutations p.K202del and

p.S51Ifs*99; patient 9 (P9) was compound heterozygote for the mutations

p.R130W and p.Y208C; patient 10 (P10) was compound heterozygote for the

mutations p.R192K and p.H121N; and patient 11 (P11, described in (2)) was

compound heterozygote for the mutations p.K202del and p.R183G. All

mutations but p.Y208C in P9 had been previously described (3, 4). This novel

mutation affects a highly conserved residue and scores as probably damaging

on functionality prediction softwares (PolyPhen-2; score:1). Pathogenicity of the

p.Y208C mutation was finally proved after observing a severe reduction of TK2

activity (16% residual activity) in fibroblasts derived from P9. All subjects gave

informed consent in accordance with our Institutional Review Boards and the

Declaration of Helsinki.

Suppl. Data; Cámara et al; 2

Biochemical and Histological analysis and Mitochondrial DNA

investigations

Biochemical, histological and molecular investigations were performed on

skeletal muscle biopsies. Measurement of respiratory chain complexes activity,

biochemical determinations and histology analysis were performed as

previously described (5, 6). mtDNA deletions were investigated by long-range

PCR and/or Southern blot and mtDNA copy number was assessed by

quantitative PCR as previously described (7, 8).

Genetic analysis

For patients 1, 2, 4 and 5, the coding region of 13 genes that had been related

with mtDNA maintenance (DGUOK, MFN2, MPV17, OPA1, POLG, POLG2,

RRM2B, SLC25A4, SUCLA2, SUCLG1, TK2, PEO1 and TYMP) was PCR

enriched by an IonAmpliSeqTM Custom panel (Life Technologies). “In silico”

panel coverage (exons and exon-intron boundaries) was 97,39%.

The pool of PCR products was followed by DNA libraries preparation using the

Ion AmpliSeq Library kit 2.0 and massive parallel sequencing by Ion PGM

instrument (Ion Torrent, Life technologies). Sequence alignment, variant calling

and annotation were performed by Ion Torrent Suite Software™ and Annovar

software (9). Whole-exome sequences from patient 3 were captured by

SureSelect XT HumanAllExon 50Mb (Agilent) and later sequenced with Illumina

HiSeq2000 (Illumina). Variants have been selected by the following criteria: i)

1000 genomes MAF lower than 0.0050, ii) deleterious effects (nonsynonymous

SNV, frameshift mutation, stoploss SNV, etc) and iii) presence in “Mitocarta”

gene list (10).

Suppl. Data; Cámara et al; 3

TK2 activity determination

Primary cultured fibroblasts were obtained from skin biopsies of patients 3 and

4. Cells were seeded in 3 plates of 100 mm diameter in DMEM with 4.5 g/L

glucose, supplemented with 2 mM L-glutamine, 100 U/mL penicillin and

streptomycin, and 10% dialyzed FBS (Invitrogen) in a humidified incubator at

37ºC and 5% CO2. After tight confluence was reached, FBS was reduced to

0.1% to induce quiescence. Twelve days later cells were washed with ice-cold

PBS and collected using a cell scraper. Cells were pelleted and stored at -80ºC

until further use. We measured TK2 activity following a published method (11).

250 µl of extraction buffer (0.5% Triton X, 2 mM EDTA, 1 mM DTT, 10 mM Tris

HCl pH 7.5 supplemented with protease inhibitors Complete Mini, Roche) were

added to the cell pellets, plus a further addition of NaCl up to 0.2 M. After vortex

homogenization, extracts were centrifuged at 19,000 g and 4ºC for 20 min.

Protein concentration in the supernatants was determined by a BCA Protein

Assay (Bio-Rad). Seven µg of extracted protein in a final volume of 10 µl were

mixed with 10 µl of 2x dilution buffer (1 mg/ml BSA, 10 mM Tris HCl pH 7.5, 2

mM DTT, 5 mM ATP) and 20 µl of 2x reaction mix (100 mM Tris HCl pH 7.5, 10

mM ATP, 10 mM MgCl2, 1 mg/ml BSA, 10 mM NaF, 4 mM DTT, 1 mM 5-

Bromouracil (TP inhibitor) and 5 µM of 1,000 cpm/pmol substrate, [5′-3H]5-(2-

bromovinyl)-2′-deoxyuridine (Moravek biochemicals). After 60 min at 37ºC, 30 µl

of reaction was spotted on Whatman DE81 filters and air dried. Subsequently,

filters were washed three times for 5 min in 5 mM ammonium formate and

placed in vials to elute with 2 ml of 0.1 M HCl, 0.2 M NaCl for 30 min at room

temperature. Radioactivity in the eluate was counted after adding 7 ml of BCS

Biodegradable Counting Scintillant Liquid (Amersham Biosciences) in a LS

Suppl. Data; Cámara et al; 4

6000 scintillation counter (Beckman). Enzyme activity was calculated as pmol

product/min/mg protein.

FIGURE LEGENDS

Figure e-1: Histochemical findings in muscle biopsies Histochemical

analysis of muscle biopsies evidenced the existence of COX negative that were

SDH-hyper-reactive fibers in all patients. Ragged-red fibers were also present in

most of the patients. Atrophy or necrosis affecting isolated fibers were less

frequently observed (i.e. HE stain for P1). Some scattered regenerative fibers

were also identified. HE: hematoxylin-eosin stain; COX(SDH): Cytochrome

oxidase stain alone or in combination with Succinate-dehydrogenase stain; GT:

Gomori trichrome stain. (*) helps localizing those COX-negative fibers that are

otherwise hardly noticeable.

**

*

P1 P2 P3 P4 P5 P6 P7

HE

CO

X(S

DH

)S

DH

GT

Figure e-1

Suppl. Data; Cámara et al; 5

Figure e-2: mtDNA characterization and TK2 activity determination

Multiple deletions were observed in mtDNA of all patients by long range PCR

(A) which were positive also by Southern blot analysis for all patients except P4

(B). TK2 activity in skin fibroblasts from patients and healthy controls. TK2

activity was measured in quiescent fibroblasts obtained from 2 adult (P3 and

P4) patients, 3 age-matched healthy controls and 4 paediatric patients (P8-

P11). All results correspond to the average value obtained in 2-3 independent

determinations. The error bar indicates standard deviation of the values

obtained for the three controls (C).

TK

2 a

ctivity

C

0.0

2.0

4.0

6.0

8.0

10.0

12.0

C P3 P4P8 P9 P10

P11

(pm

ols

of

pro

duct/

min

/mg)

Figure e-2

B

C C CP3CP1 P4P2 C P5 C P6 C P7

A

C C CP3CP1 P4P2 C P5 C P6 C P

7

Suppl. Data; Cámara et al; 6

SUPPLEMENTAL TABLE

Table e-1: Genetic and phenotypic description of previously reported TK2-

deficient patients with mild forms of the disease

a) Patient identification (Id) in the original publication (Pa-Pc appear as P1-P3 (12)) b)

Mutation according to RefSeq NP_004605.4 c) Residual levels of mtDNA in muscle

biopsies as quantified by real-time PCR. Results are shown as percentage of control

samples. Histochemical findings (d) and Respiratory chain complexes activity (e) on

muscle biopsies are indicated when available. f) Onset of the clinical manifestations is

indicated in years (y) or months (m) when available. g) TK2 activity expressed as

percentage of control samples CS: Citrate Synthase. NA: Information not available.

Suppl. Data; Cámara et al; 7

REF

Patient

Ida

Mutationb

mtDNA

Levelsc

Main Clinical Features Histochemical Findingsd

Respiratory Chain

Activitiese

Onsetf;

Progression

TK2

Activityg

6 -- p.T108M 45% Muscle weakness; Respiratory insuficiency; Dysarthria;

Dysphagia 50% COX-

CI deficiency;

Increased CS

Infancy (24m);

Slow NA

12

Pa p.T108M 30% Muscle weakness; Restrictive respiratory failure 7% COX- and RRF; Type 1 are predominant;

Nuclear internalization

Combined CI, CIII and

CIV deficiency Infancy; Slow 40%

Pb

p.R90C

p.W4VfsX40

30% Ptosis; Dysarthria; muscle weakness; Mild restrictive

respiratory failure

4% COX- and RRF; Type 1 are predominant;

Nuclear internalization

Combined CI, CIII and

CIV deficiency

Adolescence;

Slow 4%

Pc p.T108M 30% Progressive muscle weakness; Moderate restrictive

respiratory failure

20% COX- and RRF; Type 1 are predominant;

Nuclear internalization

Combined CI, CIII and

CIV deficiency Infancy; Slow NA

8

II4

p.R183W

p.T188A

60% Ptosis; PEO; Proximal muscle weakness and atrophy;

mild dysphagia 10% COX- and RRF; Fibrosis in late stage NA Adulthood (40y) 25%

II5

p.R183W

p.T188A

NA Ptosis; PEO; Proximal muscle weakness and atrophy;

Mild dysphagia; Mild dysarthria 10% COX-; RRF; Fibrosis in late stage NA Adulthood (40y) 48%

Suppl. Data; Cámara et al; 8

4 --

p.Q35*

p.K194N

185% PEO; Limb-gridle dystrophy-like; Progressive muscle

weakness; Rapidly progressive respiratory failure 20% COX- ; RRF NA

Late adulthood

(74y) NA

Suppl. Data; Cámara et al; 9

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