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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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
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
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
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
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´