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Morrow, et. al. 2008

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http://www.sciencemag.org/content/321/5886/218.full.pdf
[1] To find inherited causes of autism-spectrum disorders, we studied families in which parents
share ancestors, enhancing the role of inherited factors. We mapped several loci, some containing
large, inherited, homozygous deletions that are likely mutations. The largest deletions implicated
genes, including
PCDH10
(
protocadherin 10
)and
DIA1
(
deleted in autism1
,or
c3orf58)
,whose
level of expression changes in response to neuronal activity, a marker of genes involved in
synaptic changes that underlie learning. [2] A subset of genes, including
NHE9
(
Na
+
/H
+
exchanger 9
),
showed additional potential mutations in patients with unrelated parents. [3]Our findings highlight
the utility of
homozygosity mapping
in heterogeneous disorders like autism but also suggest that
defective regulation of gene expression after neural activity may be a mechanism common to
seemingly diverse autism mutations.
A
utism is a severe neuropsychiatric dis-
order characterized by impaired social
interaction and communication and by
repetitive and stereotyped interests and behav-
ior. Autism includes mental retardation in up
to 70% (
1
) and seizures in 20 to 25% of cases
(
2
). [4]Although autism spectrum disorders (ASDs)
are highly heritable, they exhibit wide clinical
variability and heterogeneous genetic architec-
ture, which have hindered gene identification
(
3
,
4
).
[5]The great majority of identified ASD genes
have high rates of de novo mutation. [6]Large,
de novo, microscopically evident chromosomal
anomalies have been reported in 1 to 2% of cases
of autism (
5
), and recent work has iden
tified
submicroscopic deletions and duplications, col-
lectively called copy number variants (CNVs),
affecting many loci, in 10% or more of sporadic
cases (
4
,
6
9
). [7]Mutations in
FMR1
,
TSC1
,
TSC2
,
NF1
,
UBE3A
,and
MECP2
that cause mono-
genic neurological disorders also cause syn-
dromic autism and are all associated with high
rates of de novo or recent mutation. The ex-
treme genetic heterogeneity of autism, and the
high de novo mutation rate, have hindered link-
age studies of inherited autism susceptibility
loci (
3
,
4
). [8]The accumulating number of dis-
tinct, individually rare genetic causes in autism
(
5
,
10
,
11
) suggests that the genetic architecture
of autism resembles that of mental retardation
and epilepsy, with many syndromes, each indi-
vidually rare, as well as other cases potentially
reflecting complex interactions between inherited
changes (
12
).
Homozygosity mapping
(
13
,
14
)[9]inpedi-
grees with shared ancestry has been a success-
ful methodology to discover autosomal recessive
disease genes for many genetically heterogeneous
neurodevelopmental conditions, such as brain mal-
formations and mental retardation (
15
17
). Be-
cause of the large amount of genetic information
that can be obtained from pedigrees in which
parents share a recent common ancestor, the need
to pool information from multiple families is re-
duced. Homozygosity mapping has been sug-
gested as potentially useful for mapping complex
traits as well (
18
), but this hypothesis has not
been tested, other than one study of patent
ductus arteriosus (generally considered to be a
multifactorial condition) (
19
). Although segre-
gation analyses have supported a role for auto-
somal recessive genes in ASD (
20
), homozygosity
mapping has not been applied to autism to date.
Here, we show that homozygosity mapping can
be useful for identifying loci and genes in ASDs
in consanguineous populations.
Ascertainment of pedigrees with autism and
recent shared ancestry.
The Homozygosity Map-
ping Collaborative for Autism (HMCA) (
21
)
has recruited 104 families (79 simplex and 25
multiplex) from the Arabic Middle East, Turkey,
and Pakistan (table S1 and fig. S1), of which 88
pedigrees (69 simplex and 19 multiplex) have
cousin marriages (i.e., parental consanguinity).
To establish thorough research diagnoses, inter-
national participating clinicians received training
in accepted autism research scales. When re-
search scales were not available in the language
of their country, these clinicians enrolled patients
and family members based on DSM-IV-TR di-
agnoses that were informed by these clinicians
experience with validated research scales. Ad-
ditional direct assessments of patients were con-
ducted by clinical members of the Boston team,
which included developmental psychologists
(J.W., E.L., R.M.J.), pediatric neurologists (G.M.,
A.P.), a clinical geneticist (W.H.T.), and a neuro-
psychiatrist (E.M.M.). Reliability between clini-
cian assessments was high; a description of
clinical methods is available in (
22
).
Marriage between first cousins increases
the prevalence of neurological birth defects by
about 100%, with this excess attributable to
increased autosomal recessive causes (
23
,
24
),
and with de novo chromosome anomalies repre-
senting a correspondingly reduced portion of the
total (
24
). Although comparable epidemiological
data for autism are not available, we reasoned
that a prominent involvement of autosomal re-
cessive genes in autism would be signaled by
differences in the male-to-female (M/F) ratio of
affected children in consanguineous (related) ver-
sus nonconsanguineous marriages (although re-
cessive causes of autism may still retain some
gender-specific difference in penetrance). Across
the HMCA, the M/F ratio of affected individu-
als was typical, at 4.8:1 (115 males: 24 females).
However, in consanguineous, multiplex pedigrees,
the M/F ratio was 2.6:1 (34 males: 13 females)
(fig. S1), compared to 7.4:1 (81 males: 11 fe-
males) for the other categories of families (i.e.,
nonconsanguineous and consanguineous sim-
plex) (chi-square = 5.37, df = 1,
P
= 0.02). The
M/F ratio of 2.6:1 is close to what would be
predicted if the prevalence of autism were doubled
in these families, with the excess attributable to
recessive causes (
23
,
24
).
An increased role for inherited factors in autism
families with shared ancestry was also suggested
by a low rate of de novo CNVs that segregated
with disease, despite the use of two sensitive meth-
ods for detecting them: the Affymetrix Gene-
Chip Human Mapping 500K single-nucleotide
polymorphism (SNP) array, as well as bacterial
artificial chromosome (BAC) comparative ge-
nomic hybridization (CGH) microarrays [princi-
pally an extensively validated, commercial micro-
array from Signature Genomics (see
22
)]. Whereas
rates of inherited CNVs (some potentially causa-
tive) were high in both the SNP and BAC arrays,
ranging in size from 1.4 kb to 3.9 Mb (tables S2
and S3), overall rates of de novo CNVs that
segregated with ASD were 0% in consanguineous
multiplex (0 of 42 patients) and 1.9% in consan-
guineous simplex families (1 of 52 patients), which
were considerably lower than reported for non-
consanguineous families: 1.28% in the HMCA
overall versus 7.1% using representational oligo-
nucleotide microarray analysis in autism (
6
)(chi-
square = 4.438, df = 1,
P
<0.02),orversus
27.5% (
7
) (chi-square = 17.733, df = 1,
P
< 0.01)
using another BAC array in syndromic autism. A
large study, using identical BAC arrays run in the
same lab as our study, found 5.6% (84 of 1500)
of patients referred to Signature Genomics with
de novo or pathogenic CNVs (chi-square = 3.052,
df = 1,
P
<0.05)(
25
).TheHMCArateofdenovo
CNVs was similar to previously reported rates in
multiplex pedigrees with autism [1.28% in the
HMCA versus 2.6%, or 2 of 77, in multiplex
autism (
6
), chi-square = 0.557, df = 1,
P
= 0.22]
and in controls [1.28% HMCAversus 1.0%, or 2
of 196, in control subjects (
6
), chi-square = 0.001,
df = 1,
P
= 0.49], despite the fact that the 500K
platform used here has significantly higher cov-
erage. The single large de novo CNV discovered
was a 3-Mb deletion at 22q11.21, the velocardio-
facial syndrome (VCFS) locus (encompassing all
SNPs between rs432770 and rs1014626), which
has been previously reported in autistic patients
(
26
). The relatively reduced M/F ratio of affected
children and the reduced rate of linked de novo
CNVs in the consanguineous sample (not signifi-
cantly different from rates in control) both suggest
that consanguineous pedigrees with autism are
enriched for autosomal recessive causes similar
to other congenital neurological disorders in con-
sanguineous populations (
23
,
24
).
Homozygosity mapping implicates hetero-
geneous loci and genes.
Homozygosity map-
ping in consanguineous autism pedigrees suggested
considerable genetic heterogeneity, implicating
several genetic loci, with limited overlap between
pedigrees. Using the Affymetrix Gene-Chip Human
Mapping 500K SNP arrays, a locus-exclusionary
approach was taken assuming a model of auto-
somal recessive inheritance and high penetrance.
Several single families showed one or two loci
with strong support for linkage [multipoint loga-
rithm of the odds ratio for linkage (lod) scores
ranging from 2.4 to 2.96] (table S4), corroborated
by microsatellite analysis. Potentially linked loci
were generally nonoverlapping between families,
consistent with genetic heterogeneity, although
two families shared linkage to an overlapping re-
gion of chromosome 2q (AU-4500, lod = 2.41,
and AU-4200, lod = 1.81) that has been pre-
viously implicated in other autism linkage studies
(
27
). The higher lod scores from single families,
although not achieving genome-wide signifi-
cance, are comparable to the highest lod scores
achieved by pooling hundreds of nonconsanguin-
eous pedigrees (
3
,
4
,
27
).
Although the large size of linked loci pre-
cluded systematic gene sequencing in most cases,
we were surprised to see that several consan-
guineous pedigrees showed large, rare, inherited
homozygous deletions within linked regions,
some of which are very likely causative muta-
tions (Figs. 1 and 2 and table S5). Such deletions
were present in 5 of 78 consanguineous pedi-
grees (6.4%) and ranged in size from 18 thou-
sand base pairs (kbp) to > 880 kbp. For example,
patient AU-3101, a boy diagnosed with autistic
disorder and seizures, demonstrated a 74-cM seg-
ment of identity by descent (IBD) on chromo-
some 3q (lod score = 1.45) (Fig. 1), with an
~886-kbp homozygous deletion within 3q24.
The deletion is hemizygous in both parents and
an unaffected sibling (hence inherited from a
common grandparent or more distant ancestor)
but was not present in any of the other 393 sam-
ples from our autism pedigrees, nor in 184 Mid-
dle Eastern control chromosomes, nor in 2200
samples from the Autism Genetic Resource Ex-
change (AGRE) repository (www.agre.org). This
deletion was confirmed using Agilent oligo ar-
rays (fig. S2) and polymerase chain reaction (PCR)
(fig. S3). The deletion completely removes
c3orf58
,
which encodes an uncharacterized protein with a
signal peptide that localizes to the Golgi (
28
).
Moreover, the deletion is near the 5
region of
NHE9
, such that only 60 to 85 kbp upstream of
the transcription initiation site is spared.
NHE9
(also known as
SLC9A9
)encodesa(Na
+
,K
+
)/H
+
exchanger previously reported to have been dis-
rupted in a pedigree with a developmental neuro-
psychiatric disorder and mild mental retardation
(
29
). Of note, SNPs within
NHE9
and less than
100 kb from
c3orf58
were among the top 21 re-
gions (
P
< 0.00001) in the human genome show-
ing adaptive selection (i.e., evidence for recent
evolutionary selection) in a recent genome-wide
analysis (
30
). A second >300 kbp, linked, homo-
zygous deletion (again not present in >2000
individuals other than this family) is closest to
PCDH10
on 4q28 (Fig. 2 and table S5), which
encodes a cadherin superfamily protein essen-
tial for normal forebrain axon outgrowth (
31
).
Smaller deletions (also unique to the individ-
ual family) (table S5) were closest to
CNTN3
,
encoding BIG-1, an immunogloglobulin super-
family protein that stimulates axon outgrowth
(
32
);
RNF8
, encoding a RING finger protein that
acts as a ubiquitin ligase and transcriptional co-
activator (
33
); and
SCN7A
(amid a cluster of
voltage-gated sodium channels that also in-
cludes
SCN1A
,
SCN2A
,
SCN3A
, and
SCN9A
)
on 2q. Homozygous deletions were confirmed
by PCR (
22
). Of note, all of the implicated
genes have high levels of expression in brain.
Although without further data it is not known
that all of these
private
homozygous deletions
are causative, some are very likely to be, with
larger deletions also more commonly pathogenic
than smaller ones (
4
,
6
).
Autism-associated genes are regulated by
neuronal activity.
Unexpectedly, the three genes
within or closest to the two largest deletions
(
c3orf58
,
NHE9
,and
PCDH10
) were all inde-
pendently identified by unbiased screens look-
ing for genes regulated by neuronal activity or
for targets of transcription factors induced by
activity. Neuronal activity induces a set of tran-
scription factors (including
MEF2
,
NPAS4
,
CREB
,
EGR
,
SRF
, and others) with time courses of mi-
nutes to hours, and these transcription factors
induce or repress specific target genes that me-
diate synaptic development and plasticity (
34
).
This activity-based gene expression results in pro-
tein changes that selectively enhance or repress
synapses, likely forming a part of learning para-
digms (
35
). In microarray screens using cul-
tured rat hippocampal neurons (performed blind
to the genetic study), 1005 complementary DNAs
(cDNAs) (of 22,407 nonre
dundant genes tested,
i.e.,
5% of the transcriptome) were identified as
altered in expression after neuronal membrane
depolarization by elevated KCl (
21
). Among
these
neural activity
regulated
genes,
c3orf58
(deleted in patient AU-3101) was robustly increased
within 6 hours of membrane depolarization (Fig.
3A).
c3orf58
contained several evolutionarily con-
served binding sites for
MEF2
,
CREB
,and
SRF
(Fig. 3B), and depolarization-dependent transcrip-
tion of
c3orf58
was strongly inhibited by RNA
interference (RNAi) knock-down of the
MEF2
transcription factor (Fig. 3A), which suggests that
c3orf58
may be a direct or indirect MEF2 target.
We propose renaming this gene
DIA1
(
deleted in
autism-1
). In the same forward screen, transcription
of
PCDH10
(the gene closest to the second-
largest, >300-kbp, homozygous deletion in patient
AU-7001) (Fig. 2A) was strongly up-regulated in
hippocampal neurons in response to membrane
depolarization (Fig. 3C). Although
PCDH10
was
not greatly affected by MEF2 RNAi,
PCDH10
was robustly induced by a MEF2-VP16 fusion
protein, reaching 1.31 ± 0.09 fold-induction of
transcription and 1.94 ± 0.23 fold-induction at
1 hour and 2.5 hours, respectively. This tran-
scriptional activation also suggests strongly that
PCDH10
is a transcriptional target of MEF2.
Because the KCl depolarization assay identified
fewer than 5% of the transcriptome as altered in
expression, the identification in this assay of two of
three genes associated with the two largest dele-
tions is quite unlikely by chance alone (binomial
test,
P
< 0.006 for two or more genes in the
altered
expression
category). Enrichment of genes in-
duced by membrane depolarization remains sig-
nificant even when genes closest to all of the five
homozygous deletions are considered (two of six
transcripts found on the array, binomial test,
P
<
0.027). Moreover, a separate screen using RNAi
knock-down of
NPAS4,
a transcription factor acti-
vated in response to depolarization, showed that
NHE9
was one of 292 out of 22,407 cDNAs
(1.3%) whose transcription was significantly altered
(in this case, increased) (Fig. 3D) (
22
), although
NHE9
expression was not detectably altered by
membrane depolarization alone. These transcrip-
tional effects suggest that the homozygous dele-
tions in autism patients may preferentially involve
activity-regulated genes, either mutating their cod-
ing sequence (e.g.,
DIA1
) or potentially affecting
conserved DNA sequences (
NHE9
,
PCDH10
)
that may be critical for proper transcriptional
regulation.
A subset of genes identified in HMCA sam-
ples shows potential mutations in nonconsan-
guineous pedigrees.
Further analysis of
NHE9
demonstrated deleterious sequence variants asso-
ciated with similiar autistic phenotypes in patients
whose parents were not related. Because the pro-
band AU-3101 with the deletion juxtaposed to
NHE9
showed autism as well as epilepsy, we
sequenced
NHE9
in other patients with autism
and epilepsy. A heterozygous CGA to TGA tran-
sition, changing arginine 423 residue to a stop
codon, was found, and it creates a predicted pro-
tein truncation in the final extracellular loop of
this multispanning transmembrane protein (Fig.
4, A to C). This nonsense change occurs within
two amino acids of a similar nonsense mutation in
Nhe1
that causes slow-wave epilepsy in mice (
36
)
(Fig.4B).The
swe
mouse mutation results in a
gene dosage-dependent reduction of protein
levels and loss of function in brain (
36
). A simi-
lar nonsense mutation in the final extracellular
loop has recently been found in the related
NHE6
gene in a patient with an Angelman-like syndrome,
which involves both autism symptoms and epilepsy
(
37
). The
NHE9
nonsense change here is carried by
two male siblings with autistic disorder as well as
their mother, who was reported to have had child-
hood language delay based on a parental language
questionnaire (blind to genotype). One autistic
son has electroencephalogram-confirmed epilepsy,
and the second autistic son had two probable
seizures but does not have known epilepsy. This
nonsense change was not found in greater than
3800 control chromosomes. Complete resequencing
of all exons and exon-intron boundaries in a greater
than fivefold excess (480) of controls revealed no
nonsense changes (table S6). Rare, nonconserva-
tive coding changes were more common in pa-
tients with autism with epilepsy compared with
control subjects (5.95% versus 0.63%, Fisher
s
exact test,
P
= 0.005 for total changes), although
autistic patients without seizures did not differ
significantly from controls in the rate of noncon-
servative changes (1.14% autism without sei-
zures versus 0.63% controls). The heterozygous
changes, in particular the nonsense mutation,
likely affect gene dosage and suggest that the
study of consanguineous pedigrees may identify
genes of importance in nonconsanguineous pop-
ulations as well.
Discussion.
Our copy number analysis, link-
age, and resequencing together support other re-
cent studies (
3
,
4
,
6
) suggesting that autism is
highly heterogeneous genetically, but our data
further suggest that homozygosity mapping pro-
vides an important approach to dissect this hetero-
geneity. We show that individuals with related
parents are more likely to have inherited causes
of disease, likely autosomal and recessive, and
that these pedigrees allow mapping of loci from
small numbers of families. Such families can also
provide linkage evidence to support the identifi-
cation of mutations, both coding and noncoding.
Genes that act in a recessive manner may make
good candidates for future analysis in association
or resequencing studies, because they may show
interactions with other nonlinked mutations (
38
).
Our data implicating noncoding elements in pa-
tients with shared ancestry, as well as the hetero-
zygous nonsense changes in patients without
shared ancestry, suggest that loss of proper reg-
ulation of gene dosage may be an important
genetic mechanism in autism. This possibility is
also strongly supported by the numerous hetero-
zygous CNVs found in patients from noncon-
sanguineous pedigrees (
4
,
6
).
Our data add to accumulating evidence for
numerous individually rare loci in autism. These
include
FMR1
,
MECP2
,
NLGN3
,
NLGN4
(
9
),
SHANK3
(
10
),
CNTNAP2
(
39
41
),
A2BP1
(
41
),
NRXN1
(
4
) (also implicated by inherited CNVs
in our study, table S2), and now candidate genes
such as
PCDH10
,
DIA1
(
c3orf58
)
,NHE9,CNTN3
,
SCN7A
,and
RNF8
, in addition to chromosomal
and CNVanomalies (
4
,
6
8
,
26
). Whereas genes
involved in glutamatergic transmission seem to
be important in autism (
4
), data from our study
and others (
6
,
42
) implicate other biological
mechanisms as well. Potential disease mecha-
nisms include failures in neuronal cell adhesion
molecules such as
NLGN3
,
NLGN4
,and
NRXN1
;
PCDH10
and
CNTN3,
identified as potential can-
didates here, may have similar roles. Endosomal
trafficking and protein turnover is another poten-
tial mechanism implicated by
NHE9
,whichitselfis
localized to endosomes (
43
). Further, mutations
in
NHE6
(which encodes a protein highly related
to that encoded by
NHE9
) were found in a series
of patients with an Angelman syndrome
like
phenotype, with epilepsy and autism-like symp-
toms in some patients (
37
).
DIA1
(
c3orf58
)
appears to encode a protein localized to the Golgi
apparatus (
28
), and so may also relate to protein
trafficking.
[10]The regulation of expression of some autism
candidate genes by neuronal membrane depolar-
ization suggests the appealing hypothesis that
neural activity
dependent regulation of synapse
development may be a mechanism common to
several autism mutations. Early brain develop-
ment is driven largely by intrinsic patterns of gene
expression that do not depend on experience-
driven synaptic activity (
44
). [11]Mutations in the
genes active in early development can lead to
brain malformations or severe mental retardation.
In contrast, postnatal brain development requires
input from the environment that triggers the re-
lease of neurotransmitter and promotes critical
aspects of synaptic maturation. During this pro-
cess, neural activity alters the expression of hun-
dreds of genes, each with a defined temporal
course that may be particularly vulnerable to
gene dosage changes. The connection between
experience-dependent neural activity and gene
expression in the postnatal period forms the basis
of learning and memory, and autism symptoms
typically emerge during these later stages of de-
velopment. Our finding that deletions of genes
regulated by neuronal activity or regions poten-
tially involved in regulation of gene expression in
autism suggests that defects in activity-dependent
gene expression may be a cause of cognitive def-
icits in patients with autism. Therefore, disruption
of activity-regulated synaptic development may
be one mechanism common to at least a subset
of seemingly heterogeneous autism-associated
mutations.

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