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. 2006 Nov 7;103(45):16834-9.
doi: 10.1073/pnas.0605296103. Epub 2006 Oct 19.

A genetic variant that disrupts MET transcription is associated with autism

Daniel B Campbell  1 , James S Sutcliffe, Philip J Ebert, Roberto Militerni, Carmela Bravaccio, Simona Trillo, Maurizio Elia, Cindy Schneider, Raun Melmed, Roberto Sacco, Antonio M Persico, Pat Levitt
Affiliations

A genetic variant that disrupts MET transcription is associated with autism

Daniel B Campbell et al. Proc Natl Acad Sci U S A. .

Abstract

There is strong evidence for a genetic predisposition to autism and an intense interest in discovering heritable risk factors that disrupt gene function. Based on neurobiological findings and location within a chromosome 7q31 autism candidate gene region, we analyzed the gene encoding the pleiotropic MET receptor tyrosine kinase in a family based study of autism including 1,231 cases. MET signaling participates in neocortical and cerebellar growth and maturation, immune function, and gastrointestinal repair, consistent with reported medical complications in some children with autism. Here, we show genetic association (P = 0.0005) of a common C allele in the promoter region of the MET gene in 204 autism families. The allelic association at this MET variant was confirmed in a replication sample of 539 autism families (P = 0.001) and in the combined sample (P = 0.000005). Multiplex families, in which more than one child has autism, exhibited the strongest allelic association (P = 0.000007). In case-control analyses, the autism diagnosis relative risk was 2.27 (95% confidence interval: 1.41-3.65; P = 0.0006) for the CC genotype and 1.67 (95% confidence interval: 1.11-2.49; P = 0.012) for the CG genotype compared with the GG genotype. Functional assays showed that the C allele results in a 2-fold decrease in MET promoter activity and altered binding of specific transcription factor complexes. These data implicate reduced MET gene expression in autism susceptibility, providing evidence of a previously undescribed pathophysiological basis for this behaviorally and medically complex disorder.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MET locus genomic structure, genotyping markers, and definition of haplotype blocks. The MET locus consists of 21 exons spanning 125-kb on chromosome 7q31. Nine SNPs spanning the MET locus were chosen to perform association studies and Taqman Assays-on-Demand were used to determine genotype. The nine genotyping markers defined two distinct linkage disequilibrium blocks. Pairwise linkage disequilibrium (D′) values are indicated. Pairwise r2 values are provided in Table 1, which is published as supporting information on the PNAS web site.
Fig. 2.
Fig. 2.
Plots of FBAT and HBAT P values. Plotted are log10 P values for overtransmitted alleles (points) and global haplotype analyses (lines). Significance thresholds for Bonferroni corrected P values (P = 0.025) are indicated. (a) FBAT dominant model: MET promoter variant rs1858830 (marker 3) allele C was overtransmitted to individuals with autism in the original sample (P = 0.00005), replication sample (P = 0.001), and combined sample (P = 0.000005). (b) FBAT and HBAT additive model: MET promoter variant rs1858830 (marker 3) allele C was overtransmitted to individuals with autism in the original sample (P = 0.006) and combined sample (P = 0.005). Global haplotype analyses indicated significant transmission disequilibrium (P = 0.008) in LD block 1, which includes rs1858830. (c) FBAT and HBAT additive model: MET promoter variant rs1858830 (marker 3) allele C was overtransmitted to individuals with autism in multiplex families (P = 0.001) but not simplex families (P = 0.886). A marker in linkage disequilbrium with rs1858830 (rs437; marker 1) exhibited significant transmission disequilibrium in multiplex families (P = 0.009) but not in simplex families (P = 0.377). Global haplotype analyses indicated transmission disequilibrium in LD block 1 in multiplex families (P = 0.007) and in simplex families (P = 0.022).
Fig. 3.
Fig. 3.
The autism-associated MET promoter variant rs1858830 allele C produced a 2-fold decrease in transcript. Two independent mouse neural cell lines, SN56 and N2A, and the human embryonic kidney (HEK) cell line were transfected with firefly luciferase reporter constructs carrying 762-bp of the MET promoter with either the G allele or the C allele at rs1858830. Data are presented as fold-induction compared with promoterless vector. Error bars represent SEM (n = 4). ∗, P < 0.05 compared with G allele construct by two-tailed unpaired t test.
Fig. 4.
Fig. 4.
The MET promoter variant rs1858830 alleles G and C differentially bind transcription factor complexes. (a) The double-stranded rs1858830 oligonucleotide probes used in EMSAs and supershift assays. The probes correspond to MET promoter nucleotides −35 to −6 (with zero defined as the transcription start site) and differ only at the rs1858830 locus. Predicted transcription factor binding sites are indicated (35). The rs1858830-G oligonucleotide probe is predicted to contain a single SP1-binding site, whereas the rs1858830-C probe is predicted to have two different SP1-binding sites. (b) HeLa nuclear extract EMSA revealed that rs1858830 G allele probe binds robustly a single transcription factor complex, whereas the rs1858830 C allele probe binds two transcription factor complexes. (c) HeLa cell nuclear extract supershift assays by using antibodies directed to specific transcription factors. To test the hypothesis that a DNA–nuclear protein complex contains a specific transcription factor, an antibody to the transcription factor is incubated with the complex. Observation of a slower migrating (supershifted) band representing a DNA–protein–antibody complex confirms the presence of the transcription factor. Alternatively, a reduction in the amount of the DNA–protein complex indicates that the specific transcription factor-directed antibody decreases stability of the DNA–protein complex. A supershifted band was observed upon incubation of the G allele probe–protein complex with antibody directed to the SP1 transcription factor (compare lane 1 to lane 3). Reduced DNA–protein complex was observed upon incubation of the C allele probe–protein complex with antibodies directed to transcription factors SP1 and PC4 (compare lane 8 to lanes 10 and 13). Antibodies directed to transcription factors SP3 and AP2 had moderate effects on DNA–nuclear protein complex stability (lanes 4, 5, 11, and 12). For comparison, the reduction in probe-complex formation caused by competition with 100× molar excess unlabeled probes is provided (lanes 2 and 9). Thus transcription factors SP1 and PC4 are likely regulators of MET transcription with differential binding of the rs1858830 variant alleles.
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