Mutations in the gene encoding PDGF-B cause brain ... · Mutations in the gene encoding PDGF-B...

Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice

Annika Keller, Ana Westenberger, Maria J. Sobrido, Maria García-Murias, Aloysius Domingo, Renee L.

Sears, Roberta R. Lemos, Andres Ordoñez-Ugalde, Gael Nicolas, José E. Gomes da Cunha, Elisabeth J.

Rushing, Michael Hugelshofer, Moritz C. Wurnig, Andres Kaech, Regina Reimann, Katja Lohmann,

Valerija Dobričić, Angel Carracedo, Igor Petrović, Janis M. Miyasaki, Irina Abakumova, Maarja Andaloussi

Mäe, Elisabeth Raschperger, Mayana Zatz, Katja Zschiedrich, Jörg Klepper, Elizabeth Spiteri, Jose M.

Prieto, Inmaculada Navas, Michael Preuss, Carmen Dering, Milena Janković, Martin Paucar, Per

Svenningsson, Kioomars Saliminejad, Hamid R.K. Khorshid, Ivana Novaković, Adriano Aguzzi, Andreas

Boss, Isabelle Le Ber, Gilles Defer, Dider Hannequin, Vladimir S. Kostić, Dominique Campion, Daniel

Geschwind, Giovanni Coppola, Christer Betsholtz, Christine Klein, Joao R.M. Oliveira

Supplementary Information

Nature Genetics: doi:10.1038/ng.2723

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Supplementary Figure 1. Computed tomography (CT) scans of IBGC patients. Examples show patients

displaying mild (A), moderate (B), and severe (C) brain calcifications. (A) Patient 7, Family B. (B) Patient

4, Family S. (C) Patient 5, Family S.


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Supplementary Figure 2. Stereomicroscope images brain calcifications in PDGF-B deficient mice. One

mm thick coronal brain sections of a Pdgfbret/ret mouse shows bilateral nodules in the midbrain (1, black

arrows) and a single lesion area in the pons below the aqueduct (2, black arrow), also visible under UV-

light (2’). Shown images are representative of analyses made on 4 animals per genotype.


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Supplementary Figure 3. Calcified nodules are surrounded by astrocytes and microglia. (A) Numerous,

strongly GFAP- positive astrocytes surround calcified nodules in the midbrain area in Pdgfbret/ret mice. In

control animals, only few vessel-associated GFAP-positive astrocytes are visible. (B) 3D-reconstruction of

confocal z-stacks images shows green auto-fluorescent laminated nodule in Pdgfbret/ret mice in close

vicinity to the blood vessel (visualized by podocalyxin staining, in green), which is surrounded by strongly

CD45-positive microglia (in red). Shown images are representative of analyses made on 3 animals per

genotype.


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Supplementary Table 1: Statistics of next generation sequencing in Families S and B A. Genome sequencing in Family S 4 (III-5) 9 (IV-3) Paternal cousin Phenotypic status affected affected unaffected Coverage and quality measures Mapped sequence (Gb) % of the genome covered (≥ 5x) % of the exome covered (≥ 5x) Average sequencing depth

152,584 99.2 99.7 52.4

149,244 99.1 99.7 52.3

147,525 99.0 99.7 51.6

Sequence variation measures Single nucleotide variants Exonic Non-synonymous Synonymous Stop-gain Stop-loss Splicing Untranslated regions (UTRs) Intronic and intergenic Indels Exonic Frameshift In-frame Splicing Untranslated regions (UTRs) Intronic and intergenic

3,380,052 20,829 9,709 11,037 71 12 471 28,583 3,339,169 476,694 622 464 158 114 5,137 470,821

3,367,052 20,487 9,604 10,806 65 12 460 28,647 3,317,458 475,485 649 490 159 128 5,165 469,543

3,338,031 20,418 9,441 10,905 61 11 448 28,864 3,288,301 454,076 652 471 181 119 5,013 448,292

Shared variants (present in both affected, but not in the unaffected) Genome-wide Protein-changing Novel Nonsense Frameshift In-frame deletion Missense Splicing

317,473

998 28 1 1 1

25 0

Variants tested for segregation Novel nonsense Novel frameshift

PDGFB; Chr. 22: 39627650- 39627650; NM_002608:c.433C>T;p.Q145X CELA1; Chr.12: 51740414- 51740416; NM_001971:exon1:c.7_9GAC>CG


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B. Exome sequencing in Family B 4 (I.6) 7 (II.2) Phenotypic status affected affected Coverage and quality measures Mapped sequence (Gb) % Reads in targets % of the exome covered (≥ 5x) Average sequencing depth

8.2 75.16% 93.53% 29

4.9 74.81% 91.53% 45

Sequence variation measures Single nucleotide variants Exonic Non-synonymous Synonymous Stop-gain Stop-loss Splicing Untranslated regions (UTRs) Other Indels Exonic Frameshift In-frame Splicing Untranslated regions (UTRs) Other

45,162 22,263 11,692 10,288 253 28 127 1,794 20.978 1,144 72 25 45 4 65 1,003

50,134 26,938 15,306 11,037 551 41 201 1,797 21,198 905 61 25 35 3 48 793

Shared variants among both affected Total Protein-changing Novel (absent from dbSNP135) Nonsense Frameshift In-frame deletion or insertion Missense Splicing

27,551 6,756 1,400

67 6 2

1,290 35

Variants tested for segregation

PDGFB; Chr. 22: 39627650- 39627650; NM_002608:c.T356C:p.L119P


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Supplementary Table 2: Genetic features of the families with PDGFB mutations. Annotation of variants was based on transcript ENST00000331163. Family F10 F13 B S F8 F Mutation (cDNA) c.3G>A c.26T>G c.356T>C c.433C>T c.445C>T c.726G>C Mutation (protein) p.Met1? p.Leu9Arg p.Leu119Pr

o p.Gln145* p.Arg149* p.*242Yext*89

Exon 1 1 4 4 4 6 Total number of tested individuals/ mutation carriers/ individuals with calcifications/ symptomatic individuals

6/4/3/3 (no CT done for 1 individual with mutation)

6/4/4/4

10/3/3/3 9/6/6/4 10/5/5/5 19/9/9/5

SIFT N/A damaging (0)

damaging (0)

N/A N/A N/A

PolyPhen N/A benign (0.132)

probably damaging (1)

N/A N/A N/A

Mutation Taster disease causing

polymor phism

disease causing

disease causing

disease causing

polymorphism

Controls screened (ethnicity)

173 (French)

173 (French)

100 (Brazilian) 278 (German) 173 (French)

300 (Serbian) 173 (French) 278 (German)

278 (German) 173 (French)

173 (French)

Electropherogram

Reference 5 5 32 13 33 34


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Supplementary Table 3: Clinical and radiologic findings of mutation carriers.

Family (Ethnicity)

Patient (labels from original publication when applicable)

Ag e

Sex

Radiologic features (calcifications on CT scan)

Clinical features Onset (years) and initial symptom

S (Serbian)

2 (II-5)

83 F ++ basal ganglia, ++ cerebellum

mild chorea, dysarthria n/a

4 (III-5)

56 F ++ basal ganglia, ++ thalamus, ++ cerebellum, + cerebral white matter

mild to moderate generalized chorea, prominent orobuccal dyskinesia, mild dysexecutive syndrome, psychiatric symptoms (anxiety, depression, social withdrawal)

55, anxiety

5 (III-7)

58 M +++ basal ganglia, +++ thalamus, ++ cerebellum, +++ cerebral white matter

progressive motor disturbances (mainly gait problems), parkinsonism, lingual dyskinesia, dysexecutive syndrome, psychiatric symptoms (depression with suicidal ideation)

47, gait disturbance

7 (IV-1)

32 M ++ basal ganglia, + thalamus

orobuccal dyskinesia, mild hand choreoathetoid movements, anxiety disorder

21, mild orobuccal dyskinesia

8 (IV-2)

26 M + basal ganglia, + subcortical white matter

asymptomatic, normal examination

n/a

9 (IV-3)

23 M ++ basal ganglia, + subcortical white matter

lingual dyskinesia, hand tremor

22, lingual dyskinesia

B (Brazilian)

4 (I-6)

57 M ++ basal ganglia, +++ cerebellum, + cerebral white matter

severe motor and language impairment

46

6 (II-1)

28 M + basal ganglia, + thalamus, + cerebellum, + cerebral white matter

migraine 10, migraine

7 (II-2)

28 M + basal ganglia, + thalamus, + cerebellum, + cerebral white matter

migraine 10, migraine

F (French) 2 (II-3)

67 F +++ basal ganglia, ++ cerebellum, ++ cerebral white matter

dystonia, parkinsonism, orofacial dyskinesia, dementia (including memory, dysexecutive syndrome), psychosis

38, psychosis

5 (III-2)

57 M ++ basal ganglia asymptomatic, normal examination

n/a

7 41 F ++ basal ganglia, asymptomatic, normal n/a


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(III-6) + cerebral white matter examination 8

(III-8) 37 M +++ basal ganglia,

++ cerebral white matter dysexecutive syndrome, memory impairment, psychosis

33, psychosis

10 (III-10)

39 M +++ basal ganglia, +++ cerebellum

parkinsonism, dysexecutive syndrome, memory impairment psychosis

31, psychosis

11 (IV-6)

19 M ++ basal ganglia apathy childhood

12 (IV-7)

16 F ++ basal ganglia, + cerebral white matter

one generalized tonic-clonic seizure at age 7, normal examination

n/a

13 (IV-8)

23 M ++ basal ganglia, + cerebral white matter

asymptomatic, normal examination

n/a

14 (IV-9)

11 M ++ basal ganglia, + cerebral white matter

moderate intellectual disability

childhood

F10 (Unknown)

1 34 F + basal ganglia cramping, headaches, neck twitches, dyslexia

n/a

2 15 F + basal ganglia short term memory problems, migraines, muscle tightening

n/a

4 17 F + basal ganglia no clinical information n/a ? 38 F no CT scan ticks, headaches n/a F8 (German)

2 (II-2)

38 F +++ basal ganglia mild headaches, vertigo and dizziness

n/a

3 (II-3)

38 F +++ basal ganglia mild headaches, vertigo and dizziness

n/a

4 (III-1)

13 M +++ basal ganglia mild headaches, vertigo and dizziness

n/a

5 (III-2)

17 M +++ basal ganglia mild headaches, vertigo and dizziness

n/a

6 (III-3)

11 F +++ basal ganglia no clinical symptoms n/a

F13 (Finnish-Swedish)

1 56 M ++ basal ganglia, ++ white matter

migraines with aura, postural tremor, depression

13, migraine

3 30 F ++ basal ganglia, ++ thalamus, ++ white matter (frontal and parietal), + cerebellum dentate nucleus

severe migraines with aura, speech and concentration difficulties, depression

16, migraine

4 26 M ++ basal ganglia, ++ thalamus, + white matter, + cerebellum dentate nucleus

migraines with aura, suspected attention deficit hyperactivity disorder

14, migraine

5 21 F ++ basal ganglia, ++ white matter, + cerebellar peduncles

migraines with aura, mild postural tremor

12, migraine

CT scan – computed tomography scan; ‘+’ – mild calcification; ‘++’ – moderate calcification; ‘+++’ –

severe calcification.


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Supplementary Table 4. Mouse Serum levels of electrolytes and albumin Genotype Ca 2+

(mmol/l) PO4

3+ (mmol/l)

Mg2+ (mmol/l)

Albumin (g/l)

Control (n= 6) 2.57±0.06 1.61±0.24 0.99±0.04 36.00±2.90 Pdgfb ret/ret (n=7) 2.62±0.06 1.90±0.25 0.98±0.07 31.14±1.95* Control (n=6) 2.55±0.13 1.50±0.47 0.92±0.06 35.83±2.32 Pdgfb-‐/-‐; R26P+/0 (n=7) 2.64±0.11 1.59±0.22 0.92±0.05 34.42±1.51 Results are means ±SD, *p= 0.004 (Student t-test, two-tailed) Supplementary Table 5: Primer pairs used for screening the three genes linked to familial brain calcifications. Different primer sets for the same exons were used to double check for candidate SNPs and to rule out possible PCR artifacts. The exons numbers indicated refer to coding exons Gene/Primer pairs Exons Oligo sequence 5´-3´

SLC20A2 Forward Exon 2 CATGCCAAAGTTAGATCCCA Reverse

AGAAAATAAATGGTTGCCTGA

Forward

CATTGTCCAGCACTTTCCAG Reverse GCAAAGTACTGCAGGGAAGC Forward Exon3 CGCTTTGTAAAGAAACAATTCACA Reverse

GCTCACGCCTATAATCCTG

Reverse

TCACGCCTATAATCCTGGCTC

Forward

ATTCACAGAAAATTAAACTCTGG Reverse

GTAACTTGTAATAAAACTTAC

Forward

GGCTGTAGCGCTTTGTAAAGAA Reverse CAGCCTGGCAACAGAGTG Forward Exon 4 GTCAGCTCTGCCAAGTCA Forward

AAATGGAAGTTGGCCCAGC

Reverse

ACAATTATTCCTCTAACCCCTC

Forward

GAAGGTCAGCTCTGCCAAGT Reverse GGTGCTGTGCCTTGACAAT


11

Forward Exon 5 CAACAGTGGGCTCTTTGACA Forward

TGTTTCCAAGTCTGACATCAG

Reverse

TTACTATCAGCCAACAACTCC

Forward

GATCACAGGCAGTGACAACG Reverse CAACTCCAGCACCTATTCCA Forward Exon 6 TTTAAGCACATATTCGCCAGA Reverse

CTTCCAGTTACTCATGGCAAC

Forward

TGAACATACAGCTTTCTACTCCAAA Reverse CACCCACACTTCCATTTCCT Forward Exon 7 CCTGGCCTCAACTTCATTTTCTC Reverse

CCCCAGTGCCTCCGGTTAG

Forward

AAAATCAGTGCTAAAGCATAAAG Forward

TCCAAAATCAGTGCTAAAGCATA

Reverse TGAGCCTGCTCTGCTGACTA Forward Exon 8 CCTCCTAGCTTGTCCTGGAG Reverse

TTCGAGCTGCGGACTCAT

Forward

CTGCCACCGCGTTACAGT

Reverse

ACATGTCCTTTATGGCCAACTT

Forward

AAGACTGGAAAATGAGACC Reverse

CTTCCATCGGTGCCGTTCACT

Forward

GACTGGAAAATGAGACCTTCTC Reverse

TCGGTGCCGTTCACTGCTG

Forward

TGAGGGAGAATCGCTTGAAC Reverse

CCCACCAGCTTCTCACTGTC

Forward

CAGCCCAGGAAAGCAACTAC Reverse GCTGACTTATGGGTCTGTCCA Forward Exon 9 CCGCGGCTGTAGTCTCAATTA Reverse

GGGGCCTGTTTAAGTCTGTGC

Reverse

GCTGCCAACTGCCAGTGCTG

Forward

AAAACATCATATCTGTGGAGTGTGA Reverse ACACAGCCGGGAAGCTCTAC


12

Forward Exon 10 CTGCATTTTGCACAACCATC Reverse

TGGGAATTGCTGTTGTTGAG

Forward

GTTCTGTTAAAATTGATCTTGCCC

Reverse

CCCGGAGACCTGGAGAACC

Forward

GGAATTGCTGTTGTTGAGCA Reverse GAGACCTGGAGAACCTGGAG Forward Exon 11 GCTGAAGAGAAGAATCCCCAAAC Forward

TGGAAGGGAGGCAGAGAGCTG

Reverse

GGTGAACAGTGTGGGATGGAG

Forward

AACAAATGTAGGGGACTGGGTA Reverse TTAGCTCGGGAAGGTGAGTC

PDGFB

Forward Exon 1 AGGCCTGAGCGCCTGATCGC Reverse

CGCTGTTGCCTTCCCTTAGA

Forward

AGGCCTGAGCGCCTGAT Forward

TTCTTCCGGGAAAGAGTGAA

Reverse

TGGGGTTGACACTCCAGTAG

Forward

TGAGCGCCTGATCGCCGCG Reverse CCCAGCCCGAAGAGGTCAC Forward Exon 2 GAGGCCTTTGTGCTCCTGAT Reverse

CAAGTCCCAGGTACCAACCC

Forward

TCCGTCTCCCTGTGACCTTG Reverse GATGGGTGTGGCCACGCTCT Forward Exon 3 CTGGAAGGAGGGACTGTTCT Reverse

AGTTCGCTCAGTCCTGAATGT

Forward

CAGGACCCGTGCATTTATGG Reverse TTACTCTCTCAGTTCGCTCAGTC Forward Exon 4 TAATGACAGCCAGGACTTGAAAC Reverse

TGCCCAGTCAAGGAAGCCTGGTCA

Reverse

CAGGAATGTCCTTCGACACA

Forward

ATGACAGCCAGGACTTGAAAC Reverse AAGAGTGGGCCTCTCTGG Forward Exon 5 CCGGGCTTTCGAGGAAAGAT Reverse

CTTGTGTCTCAGCAAGATG

Forward

CGGGCTTTCGAGGAAAGAT


13

Reverse

AAAGAAAGCCTCCCGTGAAT

Forward

TTCGAGGAAAGATTGCTGCAGG Reverse AACCTGGCTTGTGTCTCAGC Forward Exon 6 AGAAGGTCCATGGCAGGCCTTGGT Reverse

CACAGGATTCTGGGCCTCAGTTG

Forward

AGCTGGTGAGCAGTGGAGTT Reverse

GGAAAAATCCTTTCCCCTGA

Forward

CCTTGGTCAGTGGGGAGAG Reverse

CCATCCACAGACCACAGAGA

Forward

TGAGCAGTGGAGTTGGGATC Reverse GGCTGGATTTTCTATGGAAAAATC

PDGFRB Forward Exon 2 ACTTCAGCCCCTCGGACAT Reverse

GAAGGGCAGGGCAGTGTG

Forward

AGGCTTGGGACGTGGTGG Reverse TGGAAATCCTGAAAACTAAAGCTG Forward Exon 3 GACCTGAGGGCTGTGCATAC Reverse

GAACACACTCCCCGACTGAG

Forward

GTCCTCAGAGCCATGGCTG Reverse TCTGCTTTTCTAGGATGGCTGC Forward Exon 4 GAGACAGAGGGCCAGAGATG Reverse

CTGAGCATCAGGCCAGAAAG

Forward

GAGACAGAGGGCCAGAGATG Reverse CGAAGGCTGTGGTGAGAATCC Forward Exon 5,6 TTTCCCTCTCTAGCCCCCTA Reverse

CTCTGCACCCAGCAAAGTG

Forward

GAATCTTCTACATCAGTCATCC Reverse CAGCCCTGATCACTGTATC Forward Exon 7 GGCCCCAGCATCCATATTAG Reverse

GCCTAGGTTTGTGGCTGAAA

Forward

CCTCAGCTCCGGAAACTCCATG Reverse CTCTGGTCGCTGCAGCATC


14

Forward Exon 8 CCTGTGGGTGAGGATTTACG Reverse

GACAGTGCAGGAAGGGAGAG

Forward

GCCGAGATGACCTTGGCAAGC Reverse GCAGTGGGTCAGTGGCCTAG Forward Exon 9 GAACCCATCGGTTTTCACAC Reverse

CTATGCCCGGAACAATATGC

Forward

CACACTGATTCCTGGTATTTAATG Reverse TCCTAGGATGCAACTCTATGC Forward Exon 10 TGACCAGTAGAGGGAGCACA Reverse

TTCACAATGCTCCTGTTTGC

Forward

CAGTGGGACATAGGAACTGG Reverse ACACAACAGGGTTTCACAATGC Forward Exon 11 CCTAGAGCGAGATGCTGAGG Reverse

CACAAGTCCCCACCTGAGTT

Forward

GGAGGTGGGCGCTTCTATTCTG Reverse CAGGTTGGCCCAGACTGCTG Forward Exon 12 ATATGCCCCTGCCCCTCT Reverse

CTGGGACCAGACCTCAGAGA

Forward

CATGTGTCCTAGACGGACGA Reverse

CTCACAGGGTCACAGGATCA

Forward

CTCAGTTGAGCACTTGCTGTG Reverse TAGGTCTCATAGCTAGTCATGGC Forward Exon 13,14 AGTGGCTCTTTCCCCTCACT Reverse

CCCAAACCAGACCCAGACT

Forward

GAGTGGCTCTTTCCCCTCACT Reverse TAAACCCCACCGCCCTCTGG Forward Exon 14 CCTGGGTCTTGCAGCACA Reverse TAAACCCCACCGCCCTCT Forward Exon 15 ACCACAGAGGCAGGAAAAGA Reverse

GACAAAAGGAGGGGAAGGAG

Forward

AGAATCCAGATTTGCAAGGCAC Reverse ATCTTGTGGAACAGCCCTAGC


15

Forward Exon 16 TGGGATAGACTGAAGGAAGATGA Reverse

CATCTATGAAATGGGCACGA

Forward

CTTTCCTACTGGGATAGACTG Reverse CACTACAGATCAGTTTTGGCC Forward Exon 17,18 AAGCAGGAGAACTTTGAGTTTGA Reverse

ACTGCCCATGTGCGAGAG

Forward

ACGATCAGTAGCCACCTGTC Reverse CAGCCCATCACTGCTGTCAG Forward Exon 18 ATCCTCCAAGAGCACACCAG Reverse AGCCCCACACAGATTTCCTA Forward Exon 19,20 ATGGACATGGGAACTGTGCT Reverse

TATCAGGGCTCGTCCCATAG

Forward

GGAACTGTGCTGCGCAGAG Reverse CTGATGCCATCCTTTGAATGGC Forward Exon 20 AGCATGGCTCTCCTTGTCCT Reverse TGGTTTAAGCCAGTAGAGTTGGA Forward Exon 21 ATACACCCAGCCACCACTTC Reverse

AAAGGGTGGTCCCCTAAATG

Forward

TCCCTCCAGCTCCTTTCCATG Reverse TAGAACCAGCAGGGAAACTGAG Forward Exon 22 GCAGCGGGGGTCTTATTTAT Reverse

CTTACCCTACGTAACTTACCTCTGA

Forward

AGATTCTATGTGAGGTAGGAGG Reverse TGCACAATTTCCTTGGCCCC Forward Exon 23 GCAGGCCTTGCATAGTTTTC Reverse

CACAACACGTCAGGAGCAGA

Forward

GGACACACCGAAGGCTCTTC Reverse AGGGTTTGGGGCACAACACG


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Supplementary Note Medical records were reviewed for all patients from the six families with the PDGFB mutations and a

detailed history was obtained on motor and non-motor features typical of IBGC in a semi-structured

interview. All living participants underwent a clinical neurological examination by a neurologist

experienced in movement disorders. The diagnosis of IBGC was established based on the neuroimaging

findings on standard computed tomography that was performed in 48 participating individuals

(independent of the clinical status). Apart from the six families in which the PDGFB mutations were

identified, we investigated 26 additional families and 22 sporadic patients of Canadian, Finnish-Swedish,

French, German, Iranian, Portuguese, Serbian, and Spanish origin. Sixteen out of the 25 families had

autosomal dominant inheritance pattern. The diagnosis of IBGC was established based on the

neuroimaging findings on standard computed tomography. The study was approved by the ethics boards

of each of the participating clinical centers and all patients and healthy family members gave written

informed consent. Ethics boards included: Ethics committee for Human subjects-Federal University of

Pernambuco, Recife, Brazil; Comité de Ética de la Fundación Pública Galega de Medicina, Xenómica-

Servicio Galego de Saude-Xunta de Galicia, Santiago de Compostela, Spain; Ethikkommission der

Universität zu Lübeck, Lübeck, Germany; Etički komitet Medicinskog Fakulteta Univerziteta u Beogradu,

Belgrade, Serbia; Office of the Human Research Protection Program (OHRPP), Los Angeles, CA, USA;

Comité de Protection des Personnes- Ile de France II- 45 rue des Saints-Pères, 75006 Paris, France;

Regionala etikprövningsnämnden i Stockholm; Ethics Committee of the Avicenna Research Institute,

Tehran, Iran.


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