Supplementary Figures and Tables

Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral

cortical development

Silvia Cappello, Mary J. Gray, Caroline Badouel, Simona Lange, Melanie Einsiedler, Myriam Srour,

David Chitayat, Fadi F. Hamdan, Zandra A. Jenkins, Tim Morgan, Nadia Preitner, Tami Uster,

Jackie Thomas , Patrick Shannon, Victoria Morrison, Nataliya Di Donato, Lionel Van Maldergem,

Teresa Neuhann, Ruth Newbury-Ecob, Marielle Swinkells, Paulien Terhal, Louise C. Wilson, Petra

J.G. Zwijnenburg, Andrew J. Sutherland-Smith, Michael A. Black, David Markie, Jacques L.

Michaud, Michael A. Simpson, Sahar Mansour, Helen McNeill, Magdalena Götz, Stephen P.

Robertson

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Nature Genetics: doi:10.1038/ng.2765

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Nature Genetics: doi:10.1038/ng.2765

Nature Genetics: doi:10.1038/ng.2765

Supplementary Tables

Supplementary Table 1: 454 sequencing coverage statistics for individuals D1-D3.

Supplementary Table 2: Variants within the target region identified in 454 data from subjects D1, D2 and D3.

D1 D2 D3

Annotated Homozygous variants 2322 2970 3227

Annotated Heterozygous variants 1353 181 132

Novel Heterozygous variants 140 46 43

Novel Homozygous variants 68 154 124

Novel Non-Synomous variants 6 3 4

Galaxy [http://main.g2.bx.psu.edu/root] was used to initially filter datafiles of sequence alignable to the reference genome (454AllDiffsReg.primary_target_region.txt). Off target region and all heterozygous variants (defined as those varients seen in 15-85% of reads) were removed. The resulting variants were then sorted in Microsoft Excel, filtered for representation in dbSNP (those not listed are termed novel) and examined for sharing across all three sequenced samples. Supplementary Table 3: Homozygous variant sharing in 454 data from subjects D1, D2 and D3.

Total Variants (all individuals) 10185

Total homozygous variants (all individuals) 8638

Genes with homozygous variants shared by at least 2 individuals 51

Genes with homozygous variants shared by 3 individuals 23

Genes with novel homozygous variants in 2 individuals 1

Genes with novel homozygous variants in 3 individuals 1

Sample Mapped Bases (Mb) Reads on Target Mean Depth (target region)

D1 126.0 64.9% 26.3X

D2 133.7 64.9% 27.0X

D3 166.2 64.5% 33.1X

Nature Genetics: doi:10.1038/ng.2765

Supplementary Table 4. Identification of FAT4 mutations in family F1 by exome sequencing. Under a recessive model and after filtering for novel variants as previously described1, loci were identified in individual F1 with compound heterozygous, homozygous or hemizygous variants.

Zygosity Chr Start (hg18) Stop (hg18) Ref/variant allele

Effect/found in dbSNP130

Gene Exon

Compound heterozygous variants

Het 4 126620347 126620348 G/T Non-synon; Novel FAT4

14

Het 4 126630619 126630620 G/A Non-synon; Novel 17

Het 1 144007780 144007781 G/T Non-synon; Novel

NBPF10

3

Het 1 144011299 144011300 A/C Acc. splice site; Novel 6+3

Het 1 144076725 144076726 T/G Non-synon; Novel 80

Het 14 63527497 63527498 C/G Non-synon; Novel SYNE2

21

Het 14 63704113 63704114 A/C Acc splice site; Novel 92+3

Het 9 35221122 35221123 T/A Non-synon; Novel UNC13B

3

Het 9 35393553 35393554 A/G Non-synon; Novel 38

Homozygous/hemizygous variants

Homo 10 75083201 75083202 C/T Non-synon; Novel SYNPO2L 3

Homo 9 138859797 138859798 G/T Non-synon; Novel C9orf172 1

Hemi X 134513702 134513703 T/A Non-synon Novel DDX26B 8

Supplementary Table 5. Sequences of constructs encoding shRNAs, miRNAs and primers for qRT-PCR amplication of Fat4 and Dchs1 transcripts.

Dchs1- sh#1 CGATAGCCAGTAGCTCTTTACAGAATTCAAGAGATTCTGTAAAGAGCTACTGGCTATCG

Dchs1- sh#2 GCGGATGGATTGAGTGACACGTCTT TTCAAGAGAAAGACGTGTCACTCAATCCATCCGC

Fat4- sh#1 CATGATTCTTTCACGTGCTCAGAAATTCAAGAGATTTCTGAGCACGTGAAAGAATCATG

Fat4- sh#2 CCGATGCAGATGACGAAGATAATTATTCAAGAGATAATTATCTTCGTCATCTGCATCGG

Yap-mi#1 TGCTGTTAAGAAAGGGATCGGAACTAGTTTTGGCCACTGACTGACTAGTTCCGCCCTTTCTTAA

Yap-mi#2 TGCTGAAGAATGGAAAGTTCACCAGTGTTTTGGCCACTGACTGACACTGGTGATTTCCATTCTT

Yap-mi#3 TGCTGTTACCTATGACCTCGCAGCATGTTTTGGCCACTGACTGACATGCTGCGGTCATAGGTAA

Primer Fat4-1 ATCAGAAGATGCACTGTTGGG

Primer Fat4-2 GAGACACGTCCTCCATACCG

Primer Dchs1-1 ACTGTGCCAGCTATGCCTTT

Primer Dchs1-2 AGCCAGGGTTGGTTTGTTGA

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Supplementary Table 6. Sources and working dilutions of antibodies used for Immunohistochemistry (IHC) and western blotting (WB).

Antigen Company Species IHC WB

Actin Millipore mouse 1:5000

-Catenin Sigma rabbit 1:500

Activated Caspase 3 Millipore rabbit 1:50

CyclinD1 Labvision rabbit 1:10

Flag Sigma mouse 1:200

GFP Aves Labs chicken 1:1000

Ki67 Novocastra rabbit 1:200

NeuN Millipore mouse 1:100

Pax6 Millipore mouse 1:200

Ph3 Biomol rabbit 1:200

Phospho-Yap Cell Signaling rabbit 1:200 1:1000

Tbr1 Abcam rabbit 1:100

Tbr2 Abcam rabbit 1:1000

Tuj1 Sigma mouse 1:100

Yap Santa Cruz rabbit 1:200

Yap Cell Signaling rabbit 1:1000

References 1. Simpson, M.A. et al. Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of

severe and progressive bone loss. Nat Genet 43, 303-5 (2011).

Nature Genetics: doi:10.1038/ng.2765