Supplementary Figure 1. Families with autosomal recessive variants. (A) P60-ASNS variants. (A1, A2, A3)
Proband’s MRI sections (performed 8 days after birth) showing the gyral simplification and delay on white matter
maturation. (A3) Arrow showing petechial lesions in basal ganglia. (B) P180-VPS13B variants. (B1) Patient’s MRI
sections showing dysmorphic and thick corpus callosum and (B2, B3) the lateral ventricular asymmetry. (c) P231RELN variants. (C1,C2) Patient’s MRI sections showing extended pachygyria, pons hypoplasia and severe cerebellar
hypoplasia. (D) P286-WDR62 homozygous variant. (D1, D2) Proband’s MRI sections (performed at 11 days) showing
the severe microcephaly, the lissencephaly and posterior bilateral schizencephaly. (D3, D4) Mother prenatal MRI at
30 GW showing reoccurrence of microcephaly, lissencephaly and bilateral schizencephaly in the fœtus. (E) P533WDR62 variants. (E1, E2, E3) Patient’s MRI sections (performed at 6 years old) showing extensive perisylvian
polymicrogyria affecting also both occiptal and frontal lobes.
Supplementary Figure 2. STX7 expression levels in human tissues. Quantitative RT-PCR using RNA extracted
from different tissues shows that STX7 transcripts are ubiquitous with a predominant level in central nervous system.
Supplementary Figure 3. Parental origin of de novo variants. (A) P5 family: intronic Single Nucleotid
Polymorphism (SNP: G>A heterozygous variation at 102 451 981 genomic position) near the c.1738G>A variant in
DYNC1H1, shared by P5 proband and his father. Allele-specific PCR from proband DNA, with both c.1738G>A
mutant and wild-type primers, containing the SNP of interest is shown. The variant is associated with the G (wildtype) allele, consistent with mother’s origin of the mutation. (B) P474 family: exonic SNP (C>G heterozygous
transition at position 49 579 696), 695 bp distant from c.1148C>T variant in TUBA1A gene, present in the proband
and his mother. Chromatograms of PCR fragment from proband DNA, containing both variant and SNP, are shown.
The 2 different haplotypes were obtained in independent clones by cloning PCR products into a plasmid vector using
the TOPO®TA Cloning®Kit (Invitrogen, Life technologies, Carlsbad, CA, USA). The variant (allele mut) is
associated with the SNP allele consistent with mother’s origin of the mutation. (C) P374 family: microsatellite near
the c.8159G>A variant in DYNC1H1 gene showing that the 3 affected fetuses share the same haplotype. We did not
find any informative SNP in the same region. Abbreviations: wt = wild type; mut = mutant
Family
Proband
MDC
Microcephaly
Corps
Brainstem
Callosum
Hypoplastic
Hypoplastic
Cerebellum Associated Features
IUGR, akinesia, severe
amyotrophy, hydrops fetalis,
multiple pterygiums, facial
dysmorphy
Severe epileptic
encephalopathy
P5
(2 affected)
Male fetus
Microlissencephaly
Yes
Posterior
agenesis
P60
(2 affected)
9 month
Gyral simplification
dead female
Yes
N
N
N
P64
Female
(2 affected + 1N)
fetus
N
Severe
hypoplasia +
mesencephalosynapsis
Severe
hypoplasia +
Vermian
agenesis
Club feet
Dyslpastic
Dysplastic
IUGR, facial dysmorphy,
enlarged germinative zones
(neuropathology)
P150 (2 affected + 1N)
P180 (2 affected)
P213 (2 affected)
Lissencephaly
32 wg
Delayed gyration
female fetus with pachygyria
11 yo
female
Female
fetus
P229 (2 affected + 1N) Male fetus
Microcephaly
Microlissencephaly
Microlissencephaly
+ nodular
periventricular
heterotopia
No
Severe
Yes
Yes
Yes
N
Thick and
N
Dysmorphic
N
N
N
Hypoplastic
Other Members
Affected female fetus
Affected 9 month
dead male
Affected female fetus
Normal female
Male fetus
Normal female
N
Lateral ventricular
asymmetry, neutropenia,
facial dysmorphy, joint
hypermobility, severe
developmental delay
Affected 5yo male
N
IUGR, cranio-facial
dysmorphy, digital
anomalies, anterior
displacement of the anus,
club feet
Affected female fetus
Hypoplastic
Affected male fetus
Interhemispheric cyst,
microphthalmia, IUGR, facial
Normal female
dysmorphy
Severe
hypoplasia
Severe epileptic
encephalopathy, ataxia,
bilateral transverse palmar
crease, growth retardation
Affected 29yo female
P231 (2 affected + 3N)
17 yo
female
Pachygyria
No
N
Pons
hypoplasia
Normal 32yo female
Normal 26yo male
Normal male
P248 (2 affected)
3 yo male
Posteriorly
predominant
Subcortical band
heterotopia
No
N
N
N
Autistic features, moderate
developmental delay,
infantile spams, facial
dysmorphy, hypotonia
36gw affected female
fetus
Family
P255 (3 affected)
P286 (2 affected)
Proband
P405 (2 affected)
P474 (2 affected)
P533 (2 affected)
Corps
Brainstem
Callosum
Cerebellum Associated Features
Moderate developmental
delay, joint hypermobility,
multiple dysmorphic
features, groth retardation
11 yo
female
Periventricular
No
nodular heterotopia
Dysmorphic N
9 yo female
Posteriorly
predominant
lissencephaly,
Schizencephaly
Severe
Short
N
N
Severe hypotonia,severe
developmental delay,
pyramidal syndrome
Posterior and
asymmetric
Polymicrogyria
Yes
N
N
N
N/A
Male
N
14mo dead
male
11yo male
4yo male
Perisylvian
Polymicrogyria
Sevelry affected dead
newborn
Affected fetus
(Schizencephaly)
No
Severe
hypoplasia
enlarged germinative zones,
(neuropathology)
N
N
N
N/A
Affected female
Complete
Agenesis
Severe
brainstem and Hypoplastic,
Medulla
neuronal
Hypoplasia,
ectopias
kinking junction
Prenatal diagnosis 14mo Severe encephalopathy,
Blepharophimosis, fused
thalami
Affected female
Affected 32wg female
fetus
Agenesis
Polymicrogyria
Yes
N
N
N
Mild Microcephaly
Mild
N
N
mild
Hypoplastic
Arachnoid cyst, Severe
Intellectual disability
Vermian
hypoplasia
Fetuses: Cleft palate,
urogenital anomalies,
ventricular septal defect,
digital anomalies
No
Affected 22gw female
fetus
Abnormal
cortico-spinal
tract
Severe epileptic
encephalopathy, severe
develomental delay, axial
hypotonia, limb spasticity
Pachygyria
Affected 7yo dead
male
Normal female
No
Lissencephaly
Other Members
Affected 5 yo male
Pseudo-PMG with
24 wg
multiples
female fetus heterotopia (band
and nodules)
P612 (2 affected + 1N) 3yo male
P625 (3 affected)
Microcephaly
Severe
cerebellar
dysplasia
P370 (2 affected + 1N) 14 yo Male
P374 (3 affected)
MDC
Dysmorphic N
Afected male fetus
13yo male
Normal female
Affected 25gw male
fetus
Affected 27gw male
fetus
Supplementary Table 1. Summary of characteristics of families included in project. For each family information regarding the pedigrees, clinical and brain imaging
phenotype are indicated. Proband is the affected individual selected for exome sequencing. Abbreviations: MDC= malformation of cortical development; IUGR= intrauterine
growth retardation; N= normal; yo= years old; wg= weak of gestation; N/A= not available.
Patient
Mean depth
>5X (%)
>15X (%)
P5
122
99
98
P60
94
99
97
P64
156
99
98
P150
117
99
98
P180
91
99
97
P213
120
99
98
P229
119
99
98
P231
167
99
98
P248
161
99
98
P255
141
99
98
P286
127
99
98
P370
123
99
98
P405
128
99
98
P474
128
91
81
P533
164
99
99
P612
114
99
98
P625
153
99
98
Mean
141
98.2
96.4
Supplementary Table 2. Exome sequencing quality data. Mean depth, percentage of nucleotides with a coverage
depth of at least 5X and 15X for each proband. We identified approximately 7 000 variants in each proband and an
average of 245 variations per subject after filtering.
Patient
P5
P150
P180
P213
P229
P231
P248
P370
P474
P612
Gene
Refseq
cDNA Variation
Protein
Polyphen
Sift
ZSCAN2
DYNC1H1
DHTKD1
PTPRT
COL3A1
AIPL1
TLR6
PRR12
PLA2G4E
STX7
LACTB
ADAM19
TUBA1A
LRRC66
DROSHA
NM_181877.3
NM_001376.4
NM_018706.5
NM_133170.3
NM_000090.3
NM_001033055.1
NM_006068.4
NM_020719.1
NM_001206670.1
NM_003569.2
NM_032857.3
NM_033274.3
NM_001270399.1
NM_001024611.1
NM_013235.4
c.152_153delCT
c.1738G>A
c.2519+1G>C
c.4306C>T
c.2110G>A
c.824C>A
c.1729_1730delAT
c.4397_4398insCCCTCA
c.1717A>G
c.159A>C
c.46G>C
c.2563C>T
c.1148C>T
c.1823delG
c.755G>A
p.(Pro51fs)
p.(Glu580Lys)
splice effect
p.(Arg1436Cys)
p.(Glu704Lys)
p.(Ala275Glu)
p.(Met577fs)
p.(Thr1466_Pro1467insThrPro)
p.(Met573Val)
p.(Gln53His)
p.(Gly16Arg)
p.(Pro855Ser)
p.(Ala383Val)
p.(Gly608fs)
p.(Arg252Gln)
0
0.991
/
1
0.994
0
0
0.26
0.996
0.957
0.511
0.62
0
0.266
0
/
0.02
0
0
0.73
0.13
0
0.02
Present in
affected sibs
No
Yes
No
No
No
No
No
No
No
Yes
Yes
No
Yes
No
No
Supplementary Table 3. Summary of de novo variants identified by exome sequencing and validated by Sanger sequencing. Polyphen-2 score >0.85 predicts that the
variant is probably damaging, scores 0.15–0.85 are interpreted as potentially damaging and scores <0.15 as benign. SIFT score below 0.05 suggest that the amino acid change
affects protein function, higher than 0.05 is tolerated. Variants in bold text involve known MCD-related genes.
Patient
Gene
Refseq
cDNA Variation Protein
Polyphen
Sift
P5
ZNF41
GDPD2
FLNA
GYG2
CXorf30
SLC38A5
HDAC6
NAP1L2
FAM47A
PHKA2
ARMCX4
ZNF75D
MBTPS2
DMD
CACNA1F
DACH2
ZMAT1
BEX2
RBMX2
TREX2
PNPLA4
FRMPD3
FAM47A
TBC1D25
NAP1L3
NM_007130.2
NM_001171192.1
c.707A>T
c.395C>T
c.7903G>A
c.433G>A
c.649A>G
c.806T>C
c.1538G>A
c.508T>C
c.127A>G
c.2951T>C
c.574A>T
c.1100G>A
c.1013A>T
c.8296G>A
c.1568G>A
c.1261C>T
c.1813C>T
c.32G>A
c.860G>A
c.259C>T
c.224G>A
c.2447G>T
c.1310C>T
c.1145T>G
c.1310C>G
0.795
0.194
0.368
1
0.001
1
0.021
0.135
0.005
0.86
0.21
0.985
0.079
0.718
0.35
0.276
0.002
0.045
0.001
0.135
0.912
0.196
0.032
0.2
1
0.08
0.2
0.29
0.28
0.31
0.01
0.16
0.36
0.12
0.03
0.97
0.06
0.23
1
0.01
0.06
0.1
0.08
P248
P370
P405
P474
P533
P612
P625
NM_001110556.1
NM_003918.2
NM_001098843.4
NM_033518.2
NM_006044.2
NM_021963.3
NM_203408.3
NM_000292.2
NM_001256155.1
NM_007131.3
NM_015884.3
NM_004011.3
NM_005183.2
NM_053281.3
NM_001011657.3
NM_001168399.1
NM_016024.2
NM_080701.3
NM_004650.2
XM_042978.7
NM_203408.3
NM_002536.2
NM_004538.5
p.(His236Leu)
p.(Ala132Val)
p.(Glu2635Lys)
p.(Asp145Asn)
p.(Met217Val)
p.(Phe269Ser)
p.(Arg513His)
p.(Ser170Pro)
p.(Met43Val)
p.(Ile984Thr)
p.(Thr192Ser)
p.(Cys367Tyr)
p.(His338Leu)
p.(Asp2766Asn)
p.(Arg523His)
p.(Pro421Ser)
p.(Pro605Ser)
p.(Cys11Tyr)
p.(Arg287His)
p.(Arg87Ter)
p.(Arg75Lys)
p.(Arg816Leu)
p.(Thr437Met)
p.(Leu382Arg)
p.(Ala437Gly)
Present in
affected sibs
N/A
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Yes
No
No
No
No
Supplementary Table 4. Summary of X-linked variants identified by exome sequencing and validated by Sanger sequencing. Polyphen-2 score >0.85 predicts that the
variant is probably damaging, scores 0.15–0.85 are interpreted as potentially damaging and scores <0.15 as benign. SIFT score below 0.05 suggest that the amino acid change
affects protein function, higher than 0.05 is tolerated. Abbreviations: N/A = not available.
Patient
Gene
Refseq
cDNA Variation
Protein
Polyphen
Sift
P150
METTL1
RFX1
WDR62
CLC
DYRK1B
MEIS3
NM_005371.5
NM_002918.4
NM_001083961.1
NM_001828.5
NM_006484.1
NM_020160.2
c.542T>C
c.2695G>A
c.2030T>C
c.389T>G
c.625G>A
c.202C>A
p.(Leu181Pro)
p.(Gly899Ser)
p.(Leu677Pro)
p.(Ile130Ser)
p.(Val209Ile)
p.(Leu68Ile)
0.998
0.777
0.994
0.998
0.931
0.993
0.02
0.02
0.12
-
P286
Present in
affected sibs
Yes
N/A
Yes
N/A
N/A
N/A
Supplementary Table 5. Summary of homozygous variants identified by exome sequencing and validated by Sanger sequencing. Polyphen-2 score >0.85 predicts that
the variant is probably damaging, scores 0.15–0.85 are interpreted as potentially damaging and scores <0.15 as benign. SIFT score below 0.05 suggest that the amino acid
change affects protein function, higher than 0.05 is tolerated. Variants in bold text involve known MCD-related genes. Abbreviations: N/A = not available.
Patient Gene
P5
P60
P64
P150
P180
P213
P231
P248
P255
MUC16
MUC16
MUC16
MUC16
ASNS
ASNS
NXPE3
NXPE3
NXPE3
GRID1
GRID1
KANK1
KANK1
PI4K2B
PI4K2B
PARP14
PARP14
ACSM2A
ACSM2A
VPS13B
VPS13B
PLEKHG2
PLEKHG2
CELSR1
CELSR1
HLA-A
HLA-A
RELN
RELN
MROH2A
MROH2A
MUC16
MUC16
EVPL
EVPL
TTN
TTN
Refseq
cDNA Variation
Protein
Polyphen Sift
NM_024690.2
NM_024690.2
NM_024690.2
NM_024690.2
c.26180C>T
c.10300T>C
c.6979delT
c.6955A>G
c.1648C>T
c.1439C>T
c.452_459delTCAAGCTG
c.462delG
c.956G>C
c.442C>T
c.437C>T
c.5delC
c.3992C>G
c.10C>A
c.861G>T
c.1455T>A
c.2083C>A
c.72C>G
c.1414C>T
c.3582delT
c.6370_6371delAT
c.1230C>A
c.4073T>C
c.7268C>T
c.6224C>T
c.383G>A
c.658G>C
c.9427T>G
c.2213G>A
c.1952G>A
c.2834_2835insGG
c.26366C>T
c.2500_2501delAG
c.4126G>T
c.314G>A
c.88972A>G
c.1051G>A
p.(Ala8727Val)
p.(Ser3434Pro)
p.(Ser2327fs)
p.(Ile2319Val)
p.(Arg550Cys)
p.(Ser480Phe)
p.(Leu151fs)
p.(Gln154fs)
p.(Gly319Ala)
p.(Arg148Cys)
p.(Pro146Leu)
p.(Ala2fs)
p.(Ser1331Cys)
p.(Pro4Thr)
p.(Gln287His)
p.(Ser485Arg)
p.(Pro965Thr)
p.(Tyr24Ter)
p.(Arg492Trp)
p.(Ala1194fs)
p.(Met2124fs)
p.(Phe410Leu)
p.(Leu1358Ser)
p.(Thr2423Met)
p.(Ala2075Val)
p.(Gly128Glu)
p.(Asp220His)
p.(Tyr3143Asp)
p.(Cys738Tyr)
p.(Arg651Gln)
p.(Lys945fs)
p.(Pro8789Leu)
p.(Ser834fs)
p.(Val1376Leu)
p.(Arg105Gln)
p.(Ile29658Val)
p.(Val351Met)
0
1
0.991
0
0
0.023
0.966
0.857
0
0.999
0.731
0.777
0.077
0.026
0.998
0
0
0.115
0.994
0.93
0.839
1
0.999
0.997
0.995
0.021
0
0
0.143
0.01
-
NM_133436.3
NM_133436.3
NM_001134456.1
NM_001134456.1
NM_001134456.1
NM_017551.2
NM_017551.2
NM_001256876.1
NM_001256876.1
NM_018323.3
NM_018323.3
NM_017554.2
NM_017554.2
NM_001010845.2
NM_001010845.2
NM_017890.4
NM_017890.4
NM_022835.2
NM_022835.2
NM_014246.1
NM_014246.1
NM_002116.7
NM_002116.7
NM_005045.3
NM_005045.3
NM_001287395
NM_001287395
NM_024690.2
NM_024690.2
NM_001988.2
NM_001988.2
NM_001267550.1
NM_001267550.1
0
0
0
0.82
0.01
0.01
0
0.17
0.01
0.54
0
0
0.24
0.22
0.3
0.06
0
0
0.13
0.1
-
Present in
affected sibs
N/A
N/A
N/A
N/A
Yes
No
No
No
No
No
No
Yes
No
N/A
N/A
N/A
N/A
Yes
No
No
No
No
Patient Gene
P370
P405
P474
P533
P612
URI1
URI1
THADA
THADA
TTN-AS1
TTN-AS1
TMX2
TMX2
ATXN7
ATXN7
FILIP1
FILIP1
FILIP1
TNK2
TNK2
WDR62
WDR62
FAM205A
FAM205A
FAM205A
FAM205A
FAM205A
PLEKHG4B
PLEKHG4B
SCN10A
SCN10A
SLC13A1
SLC13A1
WDR73
WDR73
AHNAK2
AHNAK2
FAT2
FAT2
Refseq
cDNA Variation
Protein
Polyphen Sift
NM_001252641.1
NM_001252641.1
NM_001271643.1
NM_001271643.1
NM_001267550.1
NM_001267550.1
NM_015959.3
NM_015959.3
NM_001177387.1
NM_001177387.1
NM_015687.2
NM_015687.2
NM_015687.2
NM_001010938.1
NM_001010938.1
NM_001083961.1
NM_001083961.1
NM_001141917.1
NM_001141917.1
NM_001141917.1
NM_001141917.1
NM_001141917.1
NM_052909.3
NM_052909.3
NM_006514.2
NM_006514.2
NM_022444.3
NM_022444.3
NM_032856.2
NM_032856.2
NM_138420.2
NM_138420.2
NM_001447.2
NM_001447.2
c.33C>A
c.644C>T
c.2468C>T
c.362G>A
c.32462C>T
c.7961G>A
c.326A>G
c.691C>T
c.211T>G
c.844G>A
c.3316G>C
c.1483A>G
c.923C>T
c.1966G>A
c.955C>T
c.2515C>T
c.3304C>T
c.3986A>C
c.3619A>C
c.2939A>T
c.2554G>A
c.2452G>T
c.1567G>A
c.2762G>A
c.2530C>T
c.597G>T
c.838A>G
c.277A>C
c.333delG
c.293T>C
c.5025G>C
c.2695G>T
c.2773T>C
c.930T>G
p.(His11Gln)
p.(Thr215Ile)
p.(Ser823Leu)
p.(Arg121His)
p.(Pro10821Leu)
p.(Arg2654Lys)
p.(Asp109Gly)
p.(Arg231Trp)
p.(Ser71Ala)
p.(Val282Met)
p.(Gly1106Arg)
p.(Lys495Glu)
p.(Ser308Leu)
p.(Val656Met)
p.(Arg319Cys)
p.(Ser839Trp)
p.(Gln1102Ter)
p.(Asn1329Thr)
p.(Thr1207Pro)
p.(Asn980Ile)
p.(Val852Ile)
p.(Val818Leu)
p.(Glu523Lys)
p.(Arg921Gln)
p.(Arg844Cys)
p.(Leu199Leu)
p.(Asn280Asp)
p.(Ile93Leu)
p.(Trp111fs)
p.(Leu98Pro)
p.(Lys1675Asn)
p.(Val899Leu)
p.(Cys925Arg)
p.(Asn310Lys)
0.006
0.313
0.001
0.987
0.856
0.596
0.053
0.911
0.998
0.197
0.721
1
0.996
0.002
0.108
0.004
0.034
1
0.998
0.944
0
0.999
0.686
0.012
0.96
0.03
0.22
0.1
0.16
0.14
0.07
0.4
0.14
0.07
0.14
0.01
1
1
0.02
0.1
0.01
0.09
1
0.03
1
0
0.13
0.42
0.01
0.7
Present in
affected sibs
No
No
No
Yes
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Yes
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Yes
No
Yes
No
Yes
Supplementary Table 6. Summary of compound heterozygous variants identified by exome sequencing and validated by Sanger sequencing. Polyphen-2 score >0.85
predicts that the variant is probably damaging, scores 0.15–0.85 are interpreted as potentially damaging and scores <0.15 as benign. SIFT score below 0.05 suggest that the
amino acid change affects protein function, higher than 0.05 is tolerated. Variants in bold text involve known MCD-related genes. Abbreviations: N/A = not available.
Positive signals
Accepted droplets (Nb)
Positive droplets for target "mutant allele"
Positive droplets for reference "WT allele"
Double positive droplets (Fam+ Hex+)
Number of "mutant allele" molecules per well
(poisson corrected)
Number of "WT allele" molecules per well
(poisson corrected)
Mutant Allele Ratio (%)
Mean (%)
SD (Mutant Allele Ratio)
Father well 1
Father well 2
Father well 3
Proband
15 306
152
3 821
61
15 251
153
3 734
54
15 106
183
3 682
56
14 637
1 669
1 740
313
214.50
208.42
240.91
2 129.69
4 477.44
4 354.47
4 294.48
2 212.04
4.57
4.57
5.31
49.05
4.82
0.43
Supplementary Table 7. Droplets digital PCR results for P248 Father and Proband. As highlighted in the table
and Figure 1, variant allele is present in 213 out of 3 882 positive droplets in first father well; 207 out of 3 788 in
second well; 239 out of 3 738 in third well; absent in the mother and present in 1,982 out of 2,053 droplets in the
proband. Calculations were possible while we obtained more than 10 000 droplets per well (good quality score).
Absolute quantification results were normalized according to the Poisson distribution (Poisson modeling = (LN(accepted droplets-positive droplets-double positive droplets)/accepted droplets) x accepted droplets). The overall
results are confirm that the proband carries a heterozygous variant (ratio about 50%); and indicate that the father
carries a somatic mosaicism with variant alleles in about 4.8% of blood cells. Abbreviations: WT= wild type; SD=
Standard deviation.