Supplementary material: Rare familial 16q21 microdeletions under a linkage peak
implicate cadherin 8 (CDH8) in susceptibility to autism and learning disability
Alistair T Pagnamenta1, Hameed Khan2, Susan Walker2, Dianne Gerrelli3, Kirsty Wing1,
Maria Clara Bonaglia4, Roberto Giorda5, Tom Berney6, Elisa Mani7, Massimo Molteni7,
Dalila Pinto2, Ann Le Couteur6, Joachim Hallmayer8, James S Sutcliffe9, Peter
Szatmari10, Andrew D Paterson2, Stephen W Scherer2, Veronica J Vieland11, Anthony P
Monaco1
In situ hybridization
Human fetal tissue, at 9 weeks of gestation, was dissected and fixed in 4%
paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C. Following
fixation, tissues were dehydrated and embedded in paraffin wax. Sagittal sections of the
head were cut at 8 µm using a standard microtome and attached to Superfrost Plus
microscopic slides (VWR). Before hybridization, tissue sections were de-waxed,
hydrated, fixed in 4% PFA/PBS and rinsed twice with PBS. Proteins were removed by
incubation with proteinase K (20 mg/ml) in PBS. After washing with PBS, the sections
were re-fixed in the same PFA solution, and treated with 0.1 M triethanolamine
containing 0.25% acetic anhydride. Slides were dehydrated through an alcohol series and
air-dried.
Probe regions comprising the 3’-UTR region of the short and long CDH8 isoforms
were amplified from whole brain cDNA using primers described in the main text. The
resulting amplicon was ligated into pGEM-T (Promega). Antisense and sense probes
were prepared by linearising plasmids with SphI and NotI respectively. DigoxigeninUTP was incorporated into riboprobes during in vitro transcription using the DIG RNA
labeling mix (Roche) according to the manufacturer’s instructions. Antisense and sense
probes were generated using SP6 and T7 polymerase respectively.
Hybridization solution contained riboprobe (300ng DIG labeled RNA probe),
RNAguard (1 ml/ml) and tRNA (0.5 mg/ml) in hybridization buffer (50% formamide, 0.3
M NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8.0, 10% dextran sulphate and 1x
Denhardt's solution). A 100 µl aliquot of hybridization probe was added to each slide,
which was incubated in a sealed chamber moistened with 50% formamide/1x standard
saline citrate (SSC) overnight at 65°C. Stringency washes were performed in the
following order: 2x SSC (twice at 65°C); 50% formamide/2x SSC (twice at 65°C); 2x
SSC (twice at 65°C); 0.2x SSC (65°C) and 0.2x SSC (65°C cooled to room temperature).
Slides were then incubated for 1 hour in 150 mM NaCl and 100 mM Tris-HCl pH 7.5
containing 10% fetal calf serum (FCS). For antibody detection, slides were incubated in
anti-digoxigenin antibody conjugated with alkaline phosphatase (anti-Dig antibody
diluted 1:1000, containing 2% FCS) overnight at 4°C. Expression patterns were
visualized using the NBT/BCIP system (Roche). Sections were mounted in VectaMount
(Vector Labs) and analyzed using the Axioplan 2 imaging system (Zeiss).
Quantitative PCR testing of sample NA18852
Quantitative PCR (qPCR) was used to determine whether a duplication involving CDH8
in HapMap sample NA18852, detected by Wang et al [1] and reported in the Database of
Genomic Variants (DGV) [2], is real. qPCR primers were designed for CDH8 coding
exons 5-7 and a non-repetitive region of the intron immediately following exon 7, using
Primer 3 (Supplementary Table 2 and Supplementary Figure 1). Exon 14 of DOCK4 was
used as the control PCR, as genome imbalances in this gene are rare and primers were
already available [3].
PCRs were carried out in triplicate in volumes of 25 μl, using 50 ng of DNA, primers
at 200 nM and iQ SYBR Green Supermix (BioRad, Hercules, CA), according to the
manufacturer’s instructions. Thermocycling and data acquisition was carried out using
the iQ5 iCycler (BioRad). Ct outliers were removed in rare cases where the SD of the
triplicates was >0.5 cycles.
DNA was obtained for HapMap sample NA18852 (Lot B2, 6/7/2006) from Coriell
Cell Repositories (Camden, NJ) and we also used family 3099 as these DNA samples had
also been derived from cell lines. 3099_009 was chosen as the reference sample, whilst
3099_006 (eldest affected son) was used as a deletion control, as these two samples had
similar absorbance profiles to NA18852, as determined by the NanoDrop machine
(Thermo Scientific, Waltham, MA).
For the five qPCRs reactions, all samples gave mean Ct values between 20.76 and
24.24 and so we deemed it unnecessary to test relative amplification efficiencies.
Relative CDH8 dosage was therefore calculated using the 2–ΔΔCt method [4].
We
considered that normal copy number results would lie between a threshold of 0.7 and 1.3.
The experiment was carried out three times.
The whole gene deletion of CDH8 was validated successfully in sample 3099_006. In
contrast, the relative CDH8 copy number for sample NA18852 did not increase above 1.3
for any of the CDH8 qPCR probes, in any of the three replicate experiments
(Supplementary Figure 1B). There was also no increase between exons 6 and 7 of
CDH8, as would be predicted by the CNV call for sample NA18852 listed in the DGV
(Supplementary Figure 1).
These results are inconsistent with the CDH8 duplication listed for sample NA18852
in the DGV. This duplication was originally detected by only 4 probes from the 550k
SNP array in the original study (Supplementary Figure 1A) and was not detected by a
subsequent higher-resolution genome-wide CNV scan that used 42 M probes [5].
Therefore we conclude that the CNV call in NA18852 by Wang et al [1] is highly likely
to be a false positive result.
Supplementary References
1.
Wang K, Li M, Hadley D, Liu R, Glessner J, Grant SF, Hakonarson H, Bucan M.
PennCNV: an integrated hidden Markov model designed for high-resolution copy
number variation detection in whole-genome SNP genotyping data. Genome Res
2007;17(11):1665-74.
2.
Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, Scherer SW,
Lee C. Detection of large-scale variation in the human genome. Nat Genet
2004;36(9):949-51.
3.
Maestrini E, Pagnamenta AT, Lamb JA, Bacchelli E, Sykes NH, Sousa I, Toma
C, Barnby G, Butler H, Winchester L, Scerri TS, Minopoli F, Reichert J, Cai G,
Buxbaum JD, Korvatska O, Schellenberg GD, Dawson G, Bildt AD, Minderaa
RB, Mulder EJ, Morris AP, Bailey AJ, Monaco AP. High-density SNP
association study and copy number variation analysis of the AUTS1 and AUTS5
loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility. Mol
Psychiatry 2009.
4.
Pfaffl MW. A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Res 2001;29(9):e45.
5.
Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, Zhang Y, Aerts J, Andrews
TD, Barnes C, Campbell P, Fitzgerald T, Hu M, Ihm CH, Kristiansson K,
Macarthur DG, Macdonald JR, Onyiah I, Pang AW, Robson S, Stirrups K,
Valsesia A, Walter K, Wei J, Tyler-Smith C, Carter NP, Lee C, Scherer SW,
Hurles ME. Origins and functional impact of copy number variation in the human
genome. Nature 2009;464(7289):704-12.
Supplementary Table 1: CDH8 sequencing primers. Exons are numbered starting from
the first coding exon that is common to both short and long CDH8 isoforms. * Due to
amplicon size, internal primers were also used (available upon request).
Sequence
Direction
GAGCCTATGCTAAGAACCCTCA
GGGTCTCACAATCACCCTATTC
CGCAAACCAGTTTCTGATAGG
CCTTGGGATTAATCGTATTCCT
GCAGAAATTGATGATGTTATGTAAAAA
ATCCTTGATGCCAGGAGCTA
TCCTTTTATTCCCTACACATGGA
ATTGATGCAGGCAGTGAAGT
TAGTACCAAAATCATGCCATACC
TCATTGATGTAACAGGCACACA
GGTGCCCAAAAGATATTTGC
CACAAGTCCCTCAACTTTAAAACTC
GACTCTGAAGTGAAATTTTGATGG
TCTTATTGTTCCTGGCTCTGG
TCTTCATCAGCATCCATTTTTG
TTCCTCTTACTTTAAAATGCAAAGC
GGAGAAATGCCCACTGTTTTA
ACTCAGCAAATGGTGTTTGAA
ACTGATAAGCTGAATTTCAAGTGC
GGTAATGCTCTTAATTTTCATCAAC
GACGAGTCAGGGAAAAAGCC
GGCACTGAAAGTCCTCTTCATTTGG
TTTTGTAAAAATACCCCTCTCTCC
TTCCTTGCAGGTCCGTATTT
Product
Size (bp)
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
1042*
Exon
amplified
Noncoding
Region
5'-UTR (long
isoform)
5'-UTR and
coding
851
Exon 1
478
Exon 2
Coding
344
Exon 3
Coding
480
Exon 4
Coding
343
Exon 5
Coding
781
Exon 6
Coding
688
Exon 7
Coding
1449*
Exon 8
Coding
471
Exon 9
Coding
607
Exon 10
Coding
966
Exon 11
Coding and
3'-UTR
Supplementary Table 2: qPCR primers, conditions and amplicon details.
CDH8
exon 5
CDH8
exon 6
CDH8
exon 7
CDH8
Sequence (5’-3’)
AAAAGTGCTGTTCCATCTCCAT
CTCAGTACCGGAAGATGTGGTT
ATCACGGAGTTTAGAGCAGCAT
CAGTCAAAATCGTGGTTGAAGA
GAAATGAACAAATCCAGGCACT
TAATGCAGACGATGGGAAGATA
CCTATGGGGTTCTGCTATTCTG
Annealing
temperature
Amplicon
Tm
Amplicon
size
56°C
80.9°C
123 bp
56°C
81.0°C
100 bp
56°C
80.0°C
117 bp
56°C
76.8°C
111 bp
Genome position
(NCBI b26)
Chr16:6041238360412505
Chr16:6040893260409031
Chr16:6038072660380842
Chr16:60372010-
intronic
DOCK4
exon 14
TTATTTTCCAAAGCCACAGACC
AACCTGTGTGTTCTTCCCTTTG
GACCACCTGGGACTGTTGTTAT
59°C
82.5°C
112 bp
60372120
Chr7:111327743111327854
Supplementary Figure 1: Quantitative PCR of the CDH8 gene. A) Customised UCSC
plot for chr16:60,350,000-60,420,000 showing qPCR probe positions in relation to the
putative duplication #9760 in sample NA18852 (red). B) qPCR results showing CDH8
dosage relative to 3099_009 and results normalised to DOCK4. Mean ±SEM from three
experiments are plotted.