Autism is a common complex neurological disorder with a high heritability 90% 1'

1
Linkage and Candidate Gene Studies of Autism in
EU Populations
R. Holt1, G. Barnby1, E. Maestrini2, E.
Bacchelli2, D. Brocklebank1, A. J. Bailey3, A. P.
Monaco1 EU Autism MOLGEN Consortium
1 Wellcome Trust Centre for Human Genetics,
Roosevelt Drive, Oxford, OX3 7BN, UK, 2
Dipartimento di Biologia, Università di Bologna,
Via Selmi 3, 40126 Bologna, Italy, 3 University
Department of Psychiatry, Park Hospital for
Children, Oxford, UK
Results
Introduction

• Linkage
• 379 SNPs and 1209 samples passed quality
  control with a mean genotyping success rate of
  gt0.99.
• Singlepoint analysis (CHECK!) was performed on
  affacted sib pairs from a total of 397 families
  when EU and AGP results were combined.
• Results were analysed both with and without the
  Finnish samples (32 families), as they are from
  an isolated population and may, therefore, have
  different susceptibility loci.
• Analysis of all results found weak evidence of
  linkage on chromosomes 3 and 6 (LOD scores of
  1.64 and 1.57, respectively).
• With Finnish samples excluded, evidence for
  linkage increased on chromosome 2 (LOD 1.87),
  but decreased on chromosomes 3 and 6 (LOD 1.08
  and 1.17, respectively).
• Figures 1 A C show the linkage pattern found
  for these three loci.
• LOD scores were not significant when linkage
  disequilibrium was taken into account (r2 of 0.5
  and 0.3).
• Parent-of-origin analysis showed that the
  linkage on chromosomes 2 and 3, in particular, is
  due to maternal alleles.
• The analysis of haplotypes is in progress.

• Autism is a common complex neurological disorder
  with a high heritability (90) 1.
• Attempts to identify the underlying causal
  variants have met with limited success.
• Linkage studies have identified possible
  susceptibility loci on the majority of
  chromosomes 2.
• Association has been reported for a large number
  of candidate genes, although replication has
  proved difficult.
• We have utilised three approaches to attempt to
  find or replicate autism susceptibility loci
  using European Union (EU) populations
• Linkage meta-analysis of seven previously
  implicated regions by combining data from an
  Autism Genome Project (AGP) study 3 with that of
  newly generated genotypes for additional samples.
  
• A search for extended haplotypes in regions of
  linkage using trios from two isolated European
  populations.
• Association analysis of seven previously reported
  candidate genes.

Materials and Methods

• SNP Selection
• Separate Illumina GoldenGate 384 SNP arrays
  were designed for both the linkage/haplotype and
  association studies.
• SNPs with a MAF gt0.05 were chosen from the
  HapMap CEU to tag variation within the regions
  investigated.
• SNPs included on the linkage array were chosen
  from those on the Affymetrix 10K SNP array, used
  by the AGP, to allow the combination of our data
  and that of the AGP.
• Additional SNPs of interest from published
  literature were also included on the association
  array.
• Table 1 shows the distribution of SNPs for the
  various loci covered by each array.

A
Figure 1 Pairwise linkage results for chromosomes
2 (A), 3 (B) and 6 (C). Graphs plot lod score
against position along the chromosome in cM.
Green lines indicate the results for all
families, blue lines are the result when the
Finnish families have been excluded.
C
B
Table 1 Distribution of SNPs on the two
genotyping arrays

• Association
• The FST test showed there is no significant
  population structure in the combined data set.
• 357 SNPs and 1127 samples passed quality
  control with a mean genotyping success rate
  gt0.99.
• The two strongest associations were for rs362780
  (RELN) and rs2518261 (GRIK2) (Table 3) (Figure 2)
  (382 families).
• Weak association found in PRKCB1 for the IMGSAC
  population (279 families) (rs11074601, P
  0.00596).
• No associations were significant after Bon
  Ferroni correction for multiple testing.
• Analysis of the results for ASMT is ongoing.

• Samples
• Samples used were either
• Genomic DNA with a concentration of
  60-100ng/µl.
• Whole genome amplified (WGA) DNA.
• The sample populations used in this study were
  IMGSAC (European and North American), PARIS
  (French and Swedish samples), Finnish and Dutch.
• Table 2 details the distribution of samples
  genotyped for each array.
• Replication of three SNPs from the candidate
  gene array was performed in 215 trios from the
  Dutch population using WGA DNA.

Figure 2 TDT meta-analysis of candidate gene
genotyping. 1 rs362780 (RELN) P 0.0016, 2
rs2518261 (GRIK2) P 0.0017, 3 rs11074601
(PRKCB1) P 0.017.
Table 2 Number of families run per
array (Multiplex MPX)
SLC6A4
NOSTRIN
GRIK2
RELN
PRKCB1
SHANK3
2
1
3

• Genotyping and Analysis
• Genotyping was performed using standard
  Illumina protocols.
• Replication of three association array SNPs was
  performed using Sequenom iPlex genotyping
  (standard protocols).
• Genotypes were called in BeadStudio, with some
  manual adjustment of clusters. Poor clustering
  SNPs were excluded.
• Mendelian errors were identified and removed
  using PedCheck. Samples with a genotype call rate
  of lt80 or with gt10 Mendelian inheritance errors
  were also excluded.
• Prior to analysis, the genotypes from the
  linkage array where combined with those for an
  additional 362 IMGSAC and 49 PARIS families
  genotyped for the same SNPs by the AGP.
• Linkage analysis was performed using Merlin.
  Parent-of-origin analysis was performed with the
  sex_split option of ASPEX.
• Association analysis was performed using the
  transmission disequilibrium test with the STATA
  package. The data was analysed both by combining
  the results across all populations genotyped and
  by performing a meta-analysis using weighted odds
  ratios.
• Association to autism and the broader phenotype
  of autism spectrum disorders (ASDs) was tested.
• Stratification was analysed using an FST test
  on 105 SNPs (including 30 specifically genotyped
  for the purpose table 1) from the association
  array.

Table 3 Top association results for combined and
meta-analyses.
Conclusions

• We were unable to identify significant regions
  of linkage, either when our results were combined
  with those of samples from the same populations
  previously genotyped by the AGP, nor when they
  were analysed separately (data not shown). Weak
  evidence for linkage was found on chromosomes 2,
  3 and 6.
• Haplotype analysis in two founder populations
  is ongoing.
• We found weak evidence for association for
  single SNPs in RELN, GRIK2 and PRKCB1. This weak
  association failed to reach significance when
  accounting for multiple testing.
• Replication of the three most significant
  association results is currently being performed
  in a Dutch population.
• We have, therefore, been unable to narrow the
  known autism linkage regions. Our results do,
  however, support the plausibility of RELN
  containing autism susceptibility variants.
• Increasingly, the importance of rare variants
  of large effect 4, including copy number variants
  5, are being recognised in autism susceptibility.
  Therefore, it is perhaps not surprising that
  there is continued difficulty in identifying
  susceptibility alleles using methods designed to
  find common alleles of moderate effect, such as
  association studies.

References

• Rutter, M. Genetic studies of autism from the
  1970s into the millennium. J Abnorm Child
  Psychol. 28(1), 3-14 (2000).
• Bacchelli, E., Maestrini, E. Autism spectrum
  disorder molecular genetic advances. Am J Med
  Genet C Semin Med Genet. 142C(1), 13-23 (2006).
• Szatmari, P. et al. Mapping autism risk loci
  using genetic linkage and chromosomal
  rearrangements. Nat Genet. 39(3), 319-28 (2007).
• Durand, C. M. Mutations in the gene encoding the
  synaptic scaffolding protein SHANK3 are
  associated with autism spectrum disorders. Nat
  Genet. 39(1), 25-7 (2007).
• Jacquemont, M. L., et al. Array-based comparative
  genomic hybridisation identifies high frequency
  of cryptic chromosomal rearrangements in patients
  with syndromic autism spectrum disorders. J Med
  Genet. 43(11) 843-9 (2006).

Acknowledgments
Our thanks go to Chris Allan for performing the
genotyping. This work has been funded by the EU
Sixth Framework Programme.