A genetic variant that disrupts MET transcription
is associated with autism
Daniel B. Campbell*, James S. Sutcliffe†‡, Philip J. Ebert*, Roberto Militerni§, Carmela Bravaccio§, Simona Trillo¶,
Maurizio Elia?, Cindy Schneider**, Raun Melmed††, Roberto Sacco‡‡§§, Antonio M. Persico‡‡§§, and Pat Levitt*‡¶¶
Departments of *Pharmacology and†Molecular Physiology and Biophysics and‡Vanderbilt Kennedy Center for Research on Human Development,
Vanderbilt University, Nashville, TN 37203;§Department of Child Neuropsychiatry, Il University of Naples, I-80131 Naples, Italy;¶Associazione Anni Verdi
ONLUS, 00148 Rome, Italy;?Unit of Neurology and Clinical Neurophysiopathology, Scientific Institutes for Research, Hospitalization and Health Care (IRCCS)
Oasi Maria SS, 94018 Troina, EN, Italy; **Center for Autism Research and Education, Phoenix, AZ 85012;††Southwest Autism Research and Resource Center,
Phoenix, AZ 85006;‡‡Laboratory of Molecular Psychiatry and Neurogenetics, University Campus Bio-Medico, I-00155 Rome, Italy; and§§IRCCS
Fondazione Santa Lucia, 00179 Rome, Italy
Edited by Mary-Claire King, University of Washington, Seattle, WA, and approved September 1, 2006 (received for review June 23, 2006)
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, con-
sistent 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% confi-
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,
basis for this behaviorally and medically complex disorder.
autism spectrum disorder ? association ? candidate gene ? hepatocyte
growth factor ? hepatocyte growth factor receptor
pairments, and repetitive behaviors with restricted interests.
The population prevalence of autism is debated; recent reports
indicate that ?1 in 500 individuals have autism and as many as
1 in 166 individuals have an autism spectrum disorder (ASD) (1,
2). Here, we broadly use the term ‘‘autism’’ to refer to any
use a binary code to designate as ‘‘affected’’ any individual
diagnosed with autism or ASD and ‘‘unaffected’’ any individual
lacking such a diagnosis. The etiology of this complex disease
likely involves environmental factors, but autism is highly heri-
table. Twin studies demonstrate concordance rates of 82–92% in
monozygotic twins and 1–10% concordance rate in dizygotic
twins (3). Sibling recurrence risk (6–8%) is 35 times the popu-
of all neuropsychiatric disorders.
The most widely accepted hypotheses regarding autism etiology
include oligogenic inheritance and epistatic interactions among
common vulnerability-conferring genetic variants and, possibly,
gene–environment interactions. Genomewide linkage scans, an
unbiased approach to localize genetic factors, have identified
utism is a complex, behaviorally defined neurodevelopmen-
tal disorder characterized by social deficits, language im-
several chromosomal regions as promising locations for autism
vulnerability genes, including peaks on chromosomes 2q, 7q, 15q,
is very powerful, but the heterogeneity present within autism
families has led thus far to mixed success in identifying candidate
genes (9, 10). In pursuing specific candidates, most studies have
own evaluation of linkage peaks extended beyond genes with
selective brain expression to consider the complex medical condi-
tions seen in autism patients. In addition to the well known
behavioral core features, some individuals with autism exhibit
gastrointestinal, immunological, or nonspecific neurological symp-
exhibit more medical complications compared with typical individ-
uals is debated, it is possible that autism vulnerability could include
genes involved more broadly in multiple biological processes that
impact the development and function of the brain and other organ
systems in parallel.
We applied the convergence of developmental biological and
genetic information to analyze the gene encoding the MET recep-
tor tyrosine kinase (OMIM 164860; GenBank accession
(16), due to somatic gain-of-function mutations, and in mediating
hepatocyte growth factor (HGF)?scatter factor signaling in periph-
eral organ development and repair (17–19). MET signaling con-
23, 24). Recent studies by our group and others revealed that MET
also contributes to development of the cerebral cortex (25, 26) and
autism (28, 29). Hypomorphic MET?HGF signaling in the cerebral
cortex results in abnormal interneuron migration from the gangli-
regions of cortex (25, 26). Hypomorphic MET?HGF signaling in
the cerebellum causes decreased proliferation of granule cells and
a concomitant reduction in the size of the cerebellum, particularly
consistent with those observed in brains of individuals with autism
(28, 29). We therefore pursued MET as an autism candidate gene.
Author contributions: D.B.C., P.J.E., A.M.P., and P.L. designed research; D.B.C. performed
research; J.S.S., R. Militerni, C.B., S.T., M.E., C.S., and R. Melmed contributed new reagents?
analytic tools; D.B.C., J.S.S., R.S., and A.M.P. analyzed data; and D.B.C., J.S.S., P.J.E., A.M.P.,
and P.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: FBAT, family based association test; HBAT, haplotype-based association
See Commentary on page 16621.
¶¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
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Screen of the MET Gene for Variants in Autism. To identify variants
in the MET gene, we screened the 21 exons and key regulatory
regions of the gene in 86 individuals with autism by using
temperature gradient capillary electrophoresis and direct rese-
quencing. Primers used to amplify the exonic regions of the MET
gene are listed in Table 2, which is published as supporting
information on the PNAS web site. Two rare nonsynonymous
variants were identified in exon 14: C3095T, a nonconserved
arginine-to-cysteine substitution at amino acid 988 (R988C), and
C3162T, a threonine-to-isoleucine substitution at amino acid
1010 (T1010I). These same variants were reported in small cell
lung cancer cell lines as functional somatic mutations (30). We
3, which is published as supporting information on the PNAS
web site) to determine the frequencies of the two nonsynony-
mous exon 14 variants. For each of the variants, R988C and
T1010I, the rare allele was present in five cases (1.8%) and two
controls (0.6%). These differences are not significant either for
genotypic (?2? 1.773; df ? 2; P ? 0.412) or for allelic
frequencies (?2? 1.762; df ? 1; P ? 0.184). Thus, there is no
genetic evidence to support autism association for these rare
alleles. Synonymous SNPs were identified in exon 2
(rs11762213), exon 7 (rs13223756), exon 20 (rs41736), and exon
21 (rs2023748 and rs41737). The initial screen also identified
variants in the promoter region (rs184953 and rs1858830) and a
variant (rs41739) in the 3? untranslated region of the MET gene.
Family Based Association Analyses. To determine a possible associ-
ation between MET and autism, we tested for transmission dis-
equilibrium in a two-stage study design by using nine markers that
span the entire MET locus (Fig. 1). SNPs were genotyped in an
original sample consisting of 204 families (178 simplex and 26
multiplex), followed by a replication sample of 539 families (87
simplex and 452 multiplex) (Table 3). Analysis of intermarker
linkage disequilibrium (LD) revealed that MET contains two
distinct LD blocks (Fig. 1): a 17-kb block at the 5? end of the gene
and an expansive 110-kb block that includes exons 2–21, the entire
test (HBAT) (31). HBAT analysis revealed significant transmission
the presence of an autism-associated variant in the MET promoter
The LD block 1 haplotype association supported the possibility
of identifying a variant that disrupts MET gene regulation. We thus
examined transmissions of single markers by using the family based
association test (FBAT) (32). Parent-to-affected offspring trans-
missions observed (TOBS) were compared with transmissions ex-
pected (TEXP), generating a P value representing the probability of
observing the transmission disequilibrium by chance. We observed
a significant overtransmission of the rs1858830 C allele to affected
individuals (Fig. 2; see Tables 4–6, which are published as sup-
porting information on the PNAS web site). Transmission disequi-
librium for rs1858830 under a dominant model was significant in
both the original 204-family sample (TOBS? 81, TEXP? 65, P ?
0.0005) and in the replication sample of 539 families (TOBS? 225,
TEXP? 198, P ? 0.001); combined analysis of these samples was
The rs1858830 variant is a common G?C SNP, situated just 20 bp
function as dominant mutations (33). However, FBAT analyses
using an additive model yielded similar results: the original sample
a trend in the replication sample (TOBS? 490, TEXP? 464, P ?
0.072) and again a significant association in the combined samples
in LD with rs1858830, only the most informative based on allele
was significantly associated with autism; less informative markers
(rs184953 and rs40238; minor allele frequency ?0.178; pairwise r2
to rs1858830 ? 0.164; Table 1) failed to show an association. No
other marker consistently reached significant transmission disequi-
librium after corrections for multiple comparisons (Fig. 2; Tables
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
r2values are provided in Table 1, which is published as supporting information on the PNAS web site.
MET locus genomic structure, genotyping markers, and definition of haplotype blocks. The MET locus consists of 21 exons spanning 125-kb on
Campbell et al.
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vol. 103 ?
no. 45 ?
4–6). Examination of transmissions of all nine markers to unaf-
fected siblings showed no significant transmission distortion, indi-
to autism and not due to altered viability.
To further understand the heritability of the MET promoter
allele in our large sample (1,231 individuals with autism from 743
families), we examined the 265 simplex (one affected child, irre-
spective of number of siblings) and 478 multiplex (more than one
affected child) families independently (Fig. 2c; Tables 7 and 8,
which are published as supporting information on the PNAS web
site). Within the framework of a genetically complex, heteroge-
neous, polygenic disorder like autism, common genetic predispos-
ing factors are likely to be enriched in multiplex families, whereas
a fraction of the simplex family cases are more likely to be caused
Association of the rs1858830 C allele was restricted to multiplex
families, with simplex families displaying no association under
model (multiplex: TOBS? 494, TEXP? 447, P ? 0.001; simplex:
TOBS? 135, TEXP? 136, P ? 0.886). In addition, we used the
Autism Genetic Resource Exchange database to identify 299
individuals with autism from 182 families who are positive for a
narrow diagnosis, based on Autism Diagnostic Interview-Revised,
Autism Diagnostic Observational Schedule, and a clinical diagno-
sis. The rs1858830 C allele was significantly overtransmitted to
individuals with narrowly defined autism (TOBS? 94, TEXP? 75,
P ? 0.0002) (Table 9, which is published as supporting information
on the PNAS web site). For comparison, exclusion of the 182
families with narrow diagnosis from the entire 743-family sample
resulted in a significant but somewhat weaker association (TOBS?
212, TEXP? 188, P ? 0.003). Thus, both subpopulations contribute
to the association of the rs1858830 C allele in the combined sample
(TOBS? 306, TEXP? 263, P ? 0.000005).
Case-Control Association of MET Promoter Variant rs1858830. Geno-
type at the rs1858830 locus was determined in a group of 189
unrelated Italian and American control individuals. A single indi-
vidual with autism was randomly selected from each of the pedi-
grees genotyped in the combined family based association sample.
Significant differences in genotypic and allelic frequencies were
detected between the individuals with autism and controls (geno-
typic: ?2? 12.150; df ? 2; P ? 0.002; allelic: ?2? 10.440; df ? 1;
P ? 0.001; Table 10, which is published as supporting information
on the PNAS web site). Compared with the GG genotype, the
autism diagnosis relative risk was 2.27 [95% confidence interval
(CI): 1.41, 3.65] for the CC genotype and 1.67 (95% CI: 1.11, 2.49)
for the CG genotype when analyzing cases and unrelated controls
(Table 10). This relative risk may be biologically relevant in a
polygenic disease such as autism. Thus, the rs1858830 C allele is
common and overrepresented in individuals with autism.
Transcription Assays. Given the location of the rs1858830 G?C
variant (20-bp 5? to the transcription start site), we hypothesized
that the associated allele would affect transcription of the MET
gene. To test this hypothesis, we generated two reporter con-
structs containing 726 bp of the human MET promoter, differing
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.
The autism-associated MET promoter variant rs1858830 allele C
overtransmitted alleles (points) and global haplotype analyses (lines). Signif-
icance 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 ?
(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 indi-
viduals 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).
Plots of FBAT and HBAT P values. Plotted are log10 P values for
www.pnas.org?cgi?doi?10.1073?pnas.0605296103Campbell et al.
only at the rs1858830 nucleotide, and transfected them into
mouse neural cell lines N2A and SN56 and the HEK cell line.
The reporter construct containing the C allele produced less
than half the luciferase activity than the construct containing the
G allele (P ? 0.05 for each of the three cell lines; Fig. 3). The
2-fold reduction in promoter activity indicates that the autism-
associated rs1858830 C allele is less efficient in driving tran-
scription than the G allele, demonstrating that the rs1858830
variant is a functional regulatory element of MET transcription.
Identification of Transcription Factors That Differentially Bind the
rs1858830 Variant. We next attempted to identify the mechanisms
through which the rs1858830 MET variant might influence tran-
sequences. The transcription factor database TRANSFAC (35)
predicted that the G and C alleles would differentially bind the
transcription factors SP1 and AP2 (Fig. 4a). Indeed, in EMSA, a G
allele-containing oligonucleotide probe robustly bound a single
protein complex in HeLa nuclear extracts, whereas an oligonucle-
otide probe containing the C allele weakly bound at least two
protein complexes, one similar in size to that bound by the G allele
oligonucleotide probe and another that migrated more slowly (Fig.
that the G allele oligonucleotide probe bound a single transcription
is published as supporting information on the PNAS web site). To
determine the specific transcription factors involved in the DNA–
protein complexes, we performed supershift assays with antibodies
as the SP1-family member SP3 and a transcription factor identified
in a preliminary screen, PC4. Incubation of the DNA–protein
complexes with specific antibodies revealed that the predominant
protein in the complex is the SP1 transcription factor: Addition of
SP1 antibody created a visibly supershifted band, representing a
oligonucleotide probe in HeLa nuclear extracts (Fig. 4c). Addition
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
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.
The MET promoter variant rs1858830 alleles G and C differentially bind transcription factor complexes. (a) The double-stranded rs1858830
Campbell et al.
November 7, 2006 ?
vol. 103 ?
no. 45 ?
of SP1 antibody in supershift assays with the C allele oligonucle-
otide probe caused markedly decreased DNA–protein complex
formation, indicating a specific interaction of the antibody with the
DNA–protein complex. Similar results were observed in SP1-
antibody supershift assays with human fetal brain nuclear protein
effectively with the C allele probe–protein complex than with the
G allele probe–protein complex (Figs. 4c and 5b), indicating a
differential interaction of the PC4 transcription factor with the
The genetic and molecular data reported here indicate genetic
association of a common, functional variant of MET with autism
attractive aspects of these findings. First, neuropathological
findings in autism indicate altered organization of both the
cerebral cortex and cerebellum, both of which are disrupted in
mice with decreased MET signaling activity. There is co-
occurrence of autism with a number of neurological and cogni-
tive disorders, including epilepsy, atypical sleep patterns, and
mental retardation (36). Together with well known dysfunction
of cortical information processing, the role of MET signaling in
interneuron development is relevant as a central component of
the hypothesized GABAergic pathophysiological changes in
autism (37). Second, the rise in autism diagnosis likely represents
changing diagnostic criteria, increased awareness and an in-
creased incidence (1, 4). Although yet to be identified environ-
mental factors likely contribute to the development of autism,
heritability studies suggest that the impact of those factors must
be imposed upon individuals genetically predisposed to the
disorder. Only a limited number of disease-related functional
alleles have been identified to date in autism cases, and they only
account for a small fraction of cases (38). We hypothesize that
the common, functionally disruptive rs1858830 C allele can,
together with other vulnerability genes and epigenetic and
environmental factors, precipitate the onset of autism. The
existence of epistatic interactions among common genetic vari-
ants at several different loci is further supported by the associ-
ation between the rs1858830 C allele and autism in multiplex
families and not in simplex families. Third, although admittedly
still debated in terms of prevalence, individuals with autism can
present complex medical profiles, such as gastrointestinal, im-
mune, and nonspecific neurological dysfunctions (14, 15). In
addition to brain development, the pleiotropic MET receptor
tyrosine kinase has specific roles in digestive system develop-
ment and repair (18, 23, 24) and modulation of T cell-activated
peripheral monocytes and dendritic antigen-presenting cells (20,
22). We raise the possibility, still to be tested, that increased risk
for autism, due to a functional polymorphism in the MET gene,
may impart in certain individuals shared etiology of a parallel,
although independent, disruption of brain and peripheral organ
development and function. Further investigations in clinical
populations will be needed to determine the contribution of the
functional promoter variant of MET reported here to specific
characteristics of the complex phenotype in autism.
Subjects. Families recruited by the centers listed in Table 2 were
used for this study. Clinical characterization has been described
in detail in refs. 11 and 12. All research was approved by the
Vanderbilt University Institutional Review Board.
40 individuals with autism from the Italian sample and 46 individ-
uals with autism from the Autism Genetic Resource Exchange
Consortium were screened for exonic variants. Primers and ampli-
fication conditions used to amplify the 21 exons of the MET gene
are listed in Table 2. Reveal temperature gradient capillary elec-
trophoresis (SpectruMedix, State College, PA) was used to screen
for variants in the exons of the MET gene. Amplicons identified as
variant-positive then were directly resequenced to identify the
SNP Genotyping. Genotyping was performed by using TaqMan
SNP Genotyping Assays on the ABI Prism 7900HT and analyzed
with SDS software. Assays-On-Demand SNP Genotyping were
obtained from Applied Biosystems (Foster City, CA). Eight of
the nine assays provided reproducible results; the Assay-on-
Demand for rs1858830 consistently failed to give reliable geno-
types from genomic DNA template. Neither a TaqMan Assay-
by-Design nor an Epoch Eclipse Quencher assay (Nanogen, San
Diego, CA) was able to reliably provide rs1858830 genotype
from genomic DNA, probably because of an inability to generate
a specific amplicon within this ?85% GC region. We therefore
generated a 652-bp amplicon, including rs1858830, from
genomic DNA for each sample and used separately generated
652-bp amplicons as templates for Taqman Assay-on-Demand
and Epoch Eclipse Quencher genotyping assays. To ensure
proper genotype calls, we also genotyped rs1858830 in each
sample by using Reveal temperature gradient capillary electro-
phoresis (SpectruMedix). If inconsistency in any of the three
indirect genotyping assays was detected, then the genotype at
rs1858830 was determined by direct resequencing.
Association Analyses. All single and haplotype association analyses
were performed by using the FBAT (32) and HBAT (31) (FBAT
version 1.5.5). HBAT and FBAT analyses were performed by using
the empirical variance (‘‘-e’’ option; Fig. 2 and Tables 4–8) because
gene and because the empirical variance provides a more conser-
vative estimate of association. However, little evidence for linkage
at the MET locus has been reported in the samples tested here for
association (Supporting Text, which is published as supporting
information on the PNAS web site). Therefore, HBAT and FBAT
analyses were repeated without the -e option of FBAT (Tables
web site). The conclusions with and without the assumption of the
presence of linkage remain the same.
Corrections for Multiple Comparisons. Appropriate corrections for
multiple comparisons are an ongoing debate in human genetics.
The presence of two distinct LD blocks indicates that a Bonferroni
correction for multiple comparisons of two is appropriate; we
consider significant only those associations with P ? 0.025
(? 0.05?2). More stringent corrections for multiple comparisons
are possible, but do not change the conclusions. An a priori design
hoc decision to analyze the data by using two models of association
could be argued to bring the appropriate Bonferroni correction
factor to 8 (23). Thus, a very stringent correction for multiple
comparisons would lead to a significance threshold of P ? 0.006
(0.05?8). All associations at rs1858830 exceed this more stringent
Transcription Assays. A 762-bp fragment of the MET promoter was
cloned into the pGL4.10[luc2] luciferase reporter vector (Promega,
Madison, WI). Luciferase assays were conducted by using the
Dual-Glo Luciferase Assay kit (Promega) according to the manu-
Electrophoretic Mobility Shift and Antibody Supershift Assays. All
reactions included double-stranded,
probe at 50,000 cpm. EMSAs were performed by using the Pro-
mega Gel Shift Assay System, according to the manufacturer’s
www.pnas.org?cgi?doi?10.1073?pnas.0605296103 Campbell et al.
protocol. HeLa nuclear extract was purchased from Promega.
Human fetal brain nuclear protein, obtained from a spontaneously
aborted 22-week female fetus, was purchased from BioChain
Institute, Inc. (Hayward, CA; catalog no. P2244035; lot no.
A304059). Nuclear protein (5 ?g) was incubated at room temper-
ature either alone or with 100? molar excess unlabeled competitor
probe for 20 min before addition of
on a 4% nondenaturing acrylamide gel. Supershift assays were
at 4°C with 2 ?g of antibody before loading on the gel.
32P-labeled probe, then
Supporting Information. See Supporting Text for detailed methods
information on the PNAS web site, for additional data.
We thank the patients and families participating in this study for their
valuable and generous contributions. Drs. Randy Blakely, Kathleen
Dennis, Bernie Devlin, Kathie Eagleson, Chun Li, Laura Lillien, Wendy
Stone, and Barbara Thompson provided comments, and Shaine Jones,
Cara Ballard-Sutcliffe, Denise Malone, Stefania Salamena, and Ping
Mayo provided technical assistance. The Autism Genetic Resource
Exchange is a program of Cure Autism Now and is supported in part by
H. Geschwind). This work was supported in part by NIMH Grant
MH65299 (to P.L.), National Institute of Child Health and Human
Development Core Grant HD15052 (to P.L.), the Marino Autism
Research Institute (P.L.), Telethon-Italy Grant GGP02019 (to A.M.P.),
Cure Autism Now (A.M.P.), the National Alliance for Autism Research
(A.M.P.), the Fondation Jerome Lejeune ( A.M.P.), a National Alliance
for Research on Schizophrenia and Depression Young Investigator
fellowship (P.J.E.), and NIMH Grant MH61009 (to J.S.S.).
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Campbell et al.
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no. 45 ?