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Cytogenetic and molecular characterization of A2BP1FOX1 as a candidate gene for autism.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:869 –876 (2007)
Cytogenetic and Molecular Characterization of
A2BP1/FOX1 as a Candidate Gene for Autism
Christa Lese Martin,1* Jacqueline A. Duvall,2 Yesim Ilkin,3 Jason S. Simon,4 M. Gladys Arreaza,4
Kristin Wilkes,2 Ana Alvarez-Retuerto,2 Amy Whichello,5 Cynthia M. Powell,6 Kathleen Rao,6
Edwin Cook,7 and Daniel H. Geschwind2
11
Department of Human Genetics, Emory University, Atlanta, Georgia
Departments of Neurology and Human Genetics, and Center for Autism Research, Semel Insitute, UCLA, Los Angeles, California
3
Department of Human Genetics, University of Chicago, Chicago, Illinois
4
Schering-Plough Research Institute, Kenilworth, New Jersey
5
Duke University School of Nursing, Durham, North Carolina
6
Division of Pediatric Genetics and Metabolism, University of North Carolina, Chapel Hill, North Carolina
7
Institute for Juvenile Research, Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois
2
Cytogenetic imbalances are increasingly being
realized as causes of autism. Here, we report a de
novo translocation between the short arms of
chromosomes 15 and 16 in a female with autism,
epilepsy, and global developmental delay. FISH
analysis identified a cryptic deletion of approximately 160 kb at the boundary of the first exon and
first intron of the 1.7 Mb ataxin-2 binding protein-1
(A2BP1) gene, also called FOX1. Quantitative real
time PCR (Q-PCR) analysis verified a deletion of
exon 1 in the 50 promoter region of the A2BP1 gene.
Reverse transcription PCR (qRT-PCR) showed
reduced mRNA expression in the individual’s
lymphocytes, demonstrating the functional consequence of the deletion. A2BP1 codes for a brainexpressed RNA binding or splicing factor.
Because of emerging evidence in the role of RNA
processing and gene regulation in pervasive
developmental disorders, we performed further
screening of A2BP1 in additional individuals
with autism from the Autism Genetics Resource
Exchange (AGRE) collection. Twenty-seven SNPs
were genotyped across A2BP1 in 206 parent-child
trios and two regions showed association at
P 0.008 level. No additional deletions or clear
mutations were identified in 88 probands by resequencing of all exons and surrounding intronic
regions or quantitative PCR (Q-PCR) of exon 1.
Although only nominal association was observed,
and no obvious causal mutations were identified,
these results suggest that A2BP1 may affect
susceptibility or cause autism in a subset of
patients. Further investigations in a larger sample may provide additional information regarding
the involvement of this gene in the autistic
phenotype.
ß 2007 Wiley-Liss, Inc.
This article contains supplementary material, which may be viewed
at the American Journal of Medical Genetics website at http://www.
interscience.wiley.com/jpages/1552-4841/suppmat/index.html.
Grant sponsor: National Institute of Mental Health; Grant
numbers: R01-MH64547.
*Correspondence to: Christa Lese Martin, Ph.D., Department
of Human Genetics, Emory University, Atlanta, Georgia 30322.
E-mail: clmartin@genetics.emory.edu
Received 6 November 2006; Accepted 23 February 2007
DOI 10.1002/ajmg.b.30530
ß 2007 Wiley-Liss, Inc.
KEY WORDS: cytogenetic imbalance; copy number variation; association analysis; splicing factor; RNA binding
Please cite this article as follows: Martin CL, Duvall JA,
Ilkin Y, Simon JS, Arreaza MG, Wilkes K, AlvarezRetuerto A, Whichello A, Powell CM, Rao K, Cook E,
Geschwind DH. 2007. Cytogenetic and Molecular Characterization of A2BP1/FOX1 as a Candidate Gene for
Autism. Am J Med Genet Part B 144B: 869–876.
INTRODUCTION
Autism is a neurodevelopmental disorder characterized by
the clinical triad of social deficits, impaired communication and
repetitive restrictive behavior patterns. The prevalence of
Autism Spectrum Disorders (ASD) has been estimated to be as
high as 1/166, with males being affected at least four times
more frequently than females [Yeargin-Allsopp et al., 2003;
Muhle et al., 2004]. Many different biological causes have
been implicated in the etiology of autism, but genetic factors
appear to be the most important: family studies have revealed a
recurrence risk of 4% among siblings of affected probands
and twin studies have shown a significant difference in the
concordance rates of autism between monozygotic and dizygotic twins [Veenstra-VanderWeele and Cook, 2004]. The genetic
mechanism underlying autism has been studied using various
approaches, including linkage, candidate gene, and chromosome analyses, however, there is still a paucity of information
regarding causative genetic mechanisms of the autistic
phenotype.
Multiple lines of evidence support the location of an
autism gene in the chromosomal region 16p13. First, several
genome-wide linkage scans and association studies have
obtained peaks at 16p13 [IMGSAC, 1998; Philippe et al.,
1999; IMGSAC, 2001; Lucarelli et al., 2003; Barnby et al.,
2005]. One region, located between D16S407 (10 Mb from
pter) and D16S3075 (12 Mb from pter) has been implicated by
various genome-wide linkage studies with an MLS ranging
from 0.74 [Philippe et al., 1999] to 2.19–2.52 [IMGSAC, 2001].
In addition, an association study focusing on the 16p region
also identified a marginal association between D16S502
(7.8 Mb from pter) and autism [Lucarelli et al., 2003].
Interestingly, this region on 16p is close to the 1.7 Mb ataxin2 binding protein-1 (A2BP1) gene which binds to the ataxin-2
protein, implicated in Spinocerebellar Ataxia type 2 (SCA2).
A2BP1 has been previously characterized and shown to code
for an RNA-binding protein which is expressed in muscle
and brain. The gene is highly conserved in C. elegans,
870
Martin et al.
D. melanogaster, mouse and human. A previous cytogenetic
study reported two unrelated individuals with abnormal
phenotypes who carry de novo chromosome rearrangements
that disrupt A2BP1 [Bhalla et al., 2004]. One individual was
reported to have severe intellectual and developmental
retardation and had a seizure when 5 days old. The second
individual presented with mild mental retardation, a history of
seizures and short absences, delayed milestones, behavioral
problems, mild dysmorphism, clinodactyly of the fifth finger
and mild cutaneous bilateral syndactyly of the second and
third toes.
Here, we report the detailed clinical, cytogenetic and
molecular findings in a female with autism, epilepsy, mental
retardation and other clinical features, who carries a translocation between the short arms of chromosomes 15 and 16 that
disrupts the A2BP1 gene, affecting its expression level.
Further, we present data from quantitative PCR, sequence
and SNP association analyses that were utilized to characterize the A2BP1 gene in a large cohort of individuals from the
Autism Genetics Resource Exchange (AGRE).
CASE REPORT AND METHODS
Clinical Report
The proband was enrolled in AGRE (sample number
AU077504) following approved Institutional Review Board
protocols. A lymphoblastoid cell line from the proband and
peripheral blood samples from the proband and her parents
were utilized for the analyses in this study. Although most
families in the AGRE collection are multiplex, the proband is
the only affected individual in this AGRE family.
The proband was a 3,500 g term infant of an uneventful
pregnancy delivered by repeat C-section. At 6 weeks, liver
enzymes (AST 119, ALT 173, GGT 650) were elevated and an
abdominal ultrasound revealed a gallstone. She underwent a
cholecystectomy at 9 months due to failure to thrive and
irritability after feedings. Liver enzymes were within normal
limits post-operatively.
At 17 months, she had myringotomy and tube placement due
to a history of chronic otitis media. Elevated liver enzymes
(AST 132, ALT 165, GGT 370) were again detected by routine
pre-operative laboratory testing. Over the next several
months, close monitoring of liver enzymes showed fluctuations
of AST, ALT, and GGT with no apparent clinical signs or
symptoms. The highest levels were noted at 20 months (AST
1041, ALT 1564, GGT 668). Over the next 2 years she had three
liver biopsies that were consistent with mild portal fibrosis and
active cholangiolitis of uncertain etiology, with no evidence of
chronic active hepatitis. Liver enzymes improved with the
long-term administration of ursodeoxycholic acid.
At 2 years 8 months she was referred for a genetic evaluation
due to liver disease of unknown etiology, developmental delay,
hypotonia, uneven gait and mild dysmorphic features, including slight frontal bossing, wide-set eyes, epicanthal folds and
a flat nasal bridge. She weighed 25.5 pounds (just below
5th centile), her height was 34 inches (5th centile) and head
circumference was 49 cm (50th centile). Routine and highresolution chromosome analyses at this time were reported as
normal.
Complex partial seizures were first observed at 3 years
3 months and an EEG showed frequent sharp waves and spikes
originating in the right frontal and right anterior temporal
region. These seizures persisted until age 6. After 4 years of no
known clinical seizure activity, the patient was weaned off
anti-epileptic medication, however, at 12 years, she began
exhibiting frequent rapid eye blinking episodes and had early
morning myoclonic seizures; an EEG showed multifocal sharp
waves in the bilateral temporal, posterior and frontal head
regions. A Magnetic Resonance Imaging (MRI) study was
reported to be normal, but upon more detailed review was
consistent with mild cerebellar atrophy.
At a follow-up genetics evaluation at 10 years 3 months,
additional dysmorphic features were noted, including slight
upslanting palpebral fissures, slight confluence of eyebrows,
full lips and a broad nasal tip. Long, thin fingers, a wide space
between the first and second toes and a sacral dimple were also
observed. Due to the constellation of clinical features suggestive of a chromosomal imbalance, repeat chromosome analysis
was ordered; the findings from these studies are reported in
the Results.
Developmentally, the proband sat up at 6–7 months,
crawled at 10 months, and began walking at 17 months. Her
walking was broad based and slightly unstable due to lower
extremity hypotonia. Evaluation by a developmental pediatrician demonstrated delays in both receptive and expressive
language skills at 2 years 6 months. By 3 years, some hand
biting and posturing of arms would occur with excitement or
frustration. Distractibility, sensory defensiveness, perseveration, and occasional pinching of other children upon frustration
during group therapy were also concerns.
At 4 years 6 months, the proband began having drastic mood
and behavior changes. Extreme irritability and aggression
toward herself and others occurred frequently and she could
sustain tantrums/rages for up to 2 or more hours. She also
experienced loss of spontaneous language (e.g., no longer called
family members by name) and increased sensory defensiveness. An initial diagnosis of autism was made at 5 years
6 months by pediatric psychiatry. At 6 years 2 months, a
developmental evaluation reported a Vineland Adaptive
Behavior Composite Standard Score of 46 and a MerrillPalmer ratio I.Q. of 30. She had a score of 39.5 on the Childhood
Autism Rating Scale (CARS). Autism Diagnostic Interview—
Revised (ADI—R) summary scores at age 12 years 2 months
were consistent with autism (Social ¼ 29; Communication ¼
29; Restricted and repetitive behaviors ¼ 10). Developmentally, the proband remains severely impaired and requires
constant supervision due to symptoms of autistic disorder and
she continues to have frequent treatment refractory myoclonic
seizures which generalize up to10 times per month.
Cytogenetic and FISH Analyses
G-banded chromosome analysis was performed on peripheral blood samples from the proband and her parents following
routine procedures. FISH analysis was carried out on metaphase cells from the proband with a probe specific to the 16p
subtelomere region [Knight et al., 2000] (D16S3400, Abbott
Molecular, Inc., Des Plaines, IL) and with probes for the
chromosome 15 alpha satellite (D15Z4) and classical satellite
regions (D15Z1).
Further FISH characterization was pursued to identify the
breakpoint of the rearrangement. BAC clones were identified
from the telomeric region of the short arm of chromosome
16 based on publicly available genome resources (http://
genome.ucsc.edu/, www.ensembl.org). As shown in Figure 1,
genomic clones were selected at approximately 1 Mb intervals
covering the most distal 10 Mb of 16p. When the breakpoint
was narrowed to a region of 2 Mb, additional clone coverage
was identified for fine mapping. All clones are from the RPCI11 library, unless otherwise specified. All clones were PCRverified using STS primers and FISH was used to verify each
clone’s cytogenetic position and unique localization (i.e., no
cross-hybridization signals to other chromosomes).
DNA was isolated using an automated DNA isolation system
(AutoGen 740, Integrated Separation Systems, Natick, MA)
and directly-labeled with either Spectrum Orange-dUTP
(Abbott Molecular, Inc.), Spectrum Green-dUTP (Vysis, Inc.)
A2BP1/FOX1 as a Candidate Gene for Autism
871
Fig. 1. Schematic diagram showing genomic clone coverage for the most distal 10 Mb of 16p with results from FISH mapping in the proband with a
15p;16p translocation that disrupts the A2BP1 gene.
or Diethylaminocoumarin-5-dUTP (DEAC/aqua, PerkinElmer
Life Sciences, Inc., Boston, MA) using a standard nicktranslation reaction. Slide preparation, probe preparation,
hybridization and post-hybridization washing were completed
using previously described methods [Chong et al., 1997;
Martin et al., 2002]. At least 10 cells were analyzed using
direct microscopic visualization and digital-imaging analysis
(ViewPoint software, Abbott Molecular Inc.). Chromosome
identification was achieved by inverted DAPI staining.
Quantitative PCR Analysis
Real-time quantitative PCR (Q-PCR) was performed on
genomic DNA from the proband with the 15p;16p translocation, her parents, and 88 additional autistic probands from the
AGRE collection to examine exon 1 of the A2BP1 gene for
heterozygous deletions. Each sample was tested a total of eight
times and normalized to the control gene MPZ. Primers
utilized are listed in Supplementary Table I. The primer
concentration for each gene was 100 nm. The samples were run
on an ABI Prism 7700 using the Bio-Rad iTaq SYBR Green
Supermix with ROX under the following conditions: an initial
958C denaturation (3 min) was followed by 45 cycles of 958C
(15 sec) and 608C (45 sec). A heat dissociation curve was run at
the end of the PCR to confirm the presence of a single product.
Semi-Quantitative RT-PCR Analysis
Total RNA was extracted from lymphoblasts from the
proband with the 15p;16p translocation, the proband’s normal
family members, and an unrelated normal control individual
using the RNeasy1 Mini Kit (Qiagen, Valencia, CA) according
to the manufacturer’s instructions. First-strand cDNA was
synthesized from purified RNA using the SuperscriptTM RT
First-Strand cDNA Synthesis kit following the manufacturer’s
protocol (Invitrogen, Carlsbad, CA).
PCR primers for isoform 4 and isoforms 1, 2 and 3 were
designed based on the human A2BP1 sequence (NCBI accession numbers: NM_145891; NM_145892; NM_145893; see
Supplementary Table I for primer sequences). PCR components for each 50 ml reaction were: 2.2 ml First-Strand cDNA,
2.5 U Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA),
5 ml 10 PCR buffer A (Fisher Scientific), 1.1 ml 10 mM A2BP1
forward and reverse primers, 2.2 ml 1 mM 18S rRNA forward
and reverse primers, 1.5 ml 300 mM dNTPs. Initial PCR was
performed and 8 ml was removed at 24, 26, 28, 30, 32, and
34 cycles to identify the plateau phase of PCR and final PCR
was done prior to the plateau. Thermocycling conditions were:
2 min at 948C, followed by 26 thermal cycles of 30 sec at 948C,
30 sec at 598C, 30 sec at 728C and 5 min at 728C. Forty
microliters of each PCR reaction were run on a 1.2% agarose gel
and stained with 1 SYBR1 Green I nucleic acid gel stain
(BMA, Rockland, ME) for 30 min. Stained gels were imaged
using a TyphoonTM scanner and analyzed using ImageQuantTM software (Amersham Biosciences, Piscataway, NJ).
Each band image volume was individually corrected for
background. A2BP1 band volumes were normalized to the
18S rRNA load control band volumes. A two-sample t-test
(assuming equal variances) was performed on the normalized
A2BP1 volumes using three independent replicates per
sample.
SNP Genotyping and Association Analysis
Twenty-seven SNPs from dbSNP and Celera databases were
genotyped by Illumina, Inc. (San Diego, CA; www.illumina.
com) in 206 case-parent trios from AGRE. SNPs spanned both
exons and introns between exon 5 (isoform 4) to the 30 end of the
gene with an average density of 63 kb/SNP (Table I).
The Haploview program was used to assess the genotyping
success (%geno), Mendelian Errors (ME), Hardy–Weinberg
equilibrium (HWE) and minor allele frequencies (MAF)
[Barrett et al., 2005]. Transmission Disequilibrium tests
(TDT) were performed for individual SNPs as well as all
possible groupings of 2, 3, and 4 consecutive SNPs using a
sliding window TDT analysis with Genehunter 2.0 [Daly et al.,
1998].
DNA Sequence Analysis
All 16 exons of the A2BP1 gene, including approximately
200 bases of intronic flanking regions, were sequenced in
88 autistic probands. Supplementary Table II lists the primers
utilized for sequence analysis. Following DNA amplification,
PCR reactions were diluted to 50 ml in PCR buffer containing
0.5 ml of ExoSAP-IT (USB Corporation, Cleveland, OH) and
incubated 15 min at 378C followed by inactivation of the
872
Martin et al.
TABLE I. SNPs Covering the A2BP1 Gene
NCBI ID
rs1395579
rs1911492
rs1395585
rs1507030
rs1507031
rs7501006
rs2063087
rs1003614
rs1003615
rs1034977
rs8060733
rs1034980
rs1019190
rs1848173
rs1507008
rs4787048
rs7204945
rs3785228
rs1024650
rs740677
rs3785214
rs917544
rs763649
rs12149686
rs2191133
rs3785189
rs874584
Position (Build 36)
Major allele
Minor allele
Minor allele Frequency
7527735
7534508
7534959
7551157
7551379
7560547
7564832
7570144
7570219
7572495
7572881
7573264
7574873
7576229
7588603
7604103
7608397
7619581
7646994
7651762
7657122
7663573
7669146
7672064
7684601
7696446
7700167
A
A
A
C
A
A
A
A
A
A
A
A
A
C
A
A
A
A
C
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
C
T
C
G
G
G
G
G
G
G
G
C
G
C
G
G
G
G
0.34
0.09
0.23
0.38
0.48
0.41
0.03
0.41
0.41
0.12
0.15
0.11
0.04
0.17
0.16
0.30
0.29
0.16
0.33
0.42
0.45
0.32
0.50
0.18
0.33
0.24
0.34
enzymes at 808C for 15 min. Cycle sequencing in the forward
and reverse directions was performed using ABI PRISM
BigDye terminator v3.1 Cycle Sequencing DNA Sequencing
Kit (Applied Biosystems, Foster City, CA) according to the
manufacturer’s instructions. Briefly, 1 ml of each PCR product
was used as template and combined with 4 ml sequencing
reaction mix containing 5 pmol M13 sequencing primer
(21M13 or M13Rev), 0.5 Sequencing buffer and 0.25 ml
BDTv3.1 mix. Sequencing reactions were denatured for 1 min
at 968C followed by 25 cycles at 968C for 10 sec, 508C for 5 sec
and 608C for 4 min. Sequencing reactions were purified by
filtration using Montage SEQ384 plates (Millipore Corp.
TABLE II. Locations and Frequencies of SNPs in A2BP1 in Autistic Individuals
SNP namea
SNP4020 (G662A)
SNP3bpDel298040
SNP4027 (C298121G)
SNP4028 (G635524A)
SNP4029 (A635528T)
SNP4030 (G635530C)
SNP4031 (C635605A)
SNP4032 (T635606A)
1bpDel31195
SNP4100 (T31272C)
4 bpDel311820
SNP4022 (A311931G)
SNP4024 (G497405A)
SNP4025 (C497570T)
SNP4102 (C558948T)
SNP4103 (C559111T)
rs1507010 (A586531G)
rs4616299 (A586590G)
rs321705(2bp632944)
SNP4088 (C632950G)
SNP4089 (C633048T)
SNP4090 (G688277A)
SNP4091 (G688294A)
SNP4092 (G688364T)
SNP3570 (G690344T)
a
Chr. 16 locationb
Gene location
Bases from nearest exonc
Minor allele frequencyd
6069906
6367284
6367365
6704768
6704772
6704774
6704849
6704850
7101836
7101913
7382461
7382572
7568046
7568211
7629589
7629752
7657172
7657231
7703585
7703591
7703689
7758918
7758935
7759005
7760985
Exon 1 (50 UTR)
Intron 1
Intron 2
Intron 2
Intron 2
Intron 2
Intron 2
Intron 2
Intron 3
Intron 4
Intron 4
Exon 4b (50 UTR)
Exon 5
Intron 5
Exon 6
Intron 6
Intron 10
Intron 10
Intron 11
Intron 11
Exon 12
Exon 15
Intron 15
Intron 15
Exon 16 (30 UTR)
482 (UTR)
4
þ12
130
126
124
49
48
20
þ15
85
22 (UTR)
100
þ21
12
þ31
þ33
þ92
31
25
74
62
þ3
þ73
562 (UTR)
0.60% (1/166)
0.56% (1/180)
0.56% (1/178)
31.88%(51/160)
17.07%(28/164)
31.71%(52/164)
23.26%(40/172)
25.00%(43/172)
38.10% (64/168)
0.59% (1/170)
19.32%(34/176)
0.81% (1/124)
0.57% (1/176)
9.20% (16/174)
0.57% (1/174)
1.69% (2/118)
48.85% (85/174)
33.91% (59/174)
43.60%(75/172)
0.58% (1/172)
0.57% (1/174)
1.14% (2/176)
5.68% (10/176)
25.00% (44/176)
1.28% (2/156)
Bold ¼ reported in NCBI dbSNP Build 120; Italics ¼ indel.
Chr. 16 location from UCSC Genome Browser, July 2003.
Bold ¼ in coding sequence; ¼ upstream of 1st base of exon; þ ¼ downstream of last base of exon.
d
Number of chromosomes carrying the minor allele out of total number of chromosomes is shown in parentheses; bold ¼ >10%.
b
c
A2BP1/FOX1 as a Candidate Gene for Autism
873
Bedford, MA), dissolved in 25 ml deionized water and resolved
by capillary electrophoresis on an Applied Biosystems 3730XL
DNA Analyzer. Chromatograms were transferred to a Unix
workstation (DEC alpha, Compaq Corp), base called with
Phred (version 0.990722.g), assembled with Phrap (version
3.01) and scanned with Polyphred (version 3.5) [Nickerson
et al., 1997]. The final results were viewed with Consed
(version 9.0) (Phred, Phrap, and Consed available at http://
www.genome.washington.edu, PolyPhred is available at
http://droog.mbt.washington.edu). Analysis parameters were
all maintained at the individual software’s default settings.
RESULTS
Cytogenetic and FISH Characterization of the t(15;16)
G-banding analysis revealed a possible abnormality involving the short arms of chromosomes 15 and 16. FISH analysis
using a probe for the 16p telomere region verified an apparently balanced translocation between the short arms of
chromosomes 15 and 16, with one hybridization signal on the
normal 16p and one signal on 15p. FISH analyses with a
chromosome 15 alpha-satellite probe and a probe from the
classical satellite region on 15p both showed signals only on the
normal 15 and derivative 15; there was no reciprocal signal on
16p. Parental cytogenetic analysis was normal, indicating the
rearrangement occurred de novo in the proband. Microsatellite
analysis of the proband and her parents demonstrated a
paternal origin of the deletion. The karyotype was therefore designated: 46,XX,?t(15;16)(p11.2;p13.3)dn.ish t(15;16)
(D16S3400þ,D15Z1þ,D15Z4þ;D16S3400,D15Z1,D15Z4).
The short arms of acrocentric chromosomes contain multiple
copies of the ribosomal RNA genes, which can be lost without
phenotypic consequences. Therefore, the breakpoint on chromosome 16 was targeted with additional FISH studies to
determine if the rearrangement disrupted a gene that could be
involved in causing the proband’s phenotype.
As shown in Figure 1, BAC clones for FISH analysis were
selected at approximately 1 Mb intervals for the most distal
10 Mb of the short arm of chromosome 16. The clone located
approximately 5 Mb from the 16p telomere (RP11-35P16) was
translocated to 15p, but the clone at 7 Mb (RP11-545E8)
remained on 16p. Interestingly, the 6 Mb clone (RP11-509E10)
showed an apparent partial deletion, with the hybridization
signal on the derivative 16 chromosome showing a reduced
intensity compared to the signal on the normal chromosome 16.
Also shown in Figure 1, is additional clone coverage that was
selected in this region for fine mapping, including clones distal
to and proximal to the 6 Mb clone. Figure 2 shows representative images from FISH analysis using these clones. RP11-19H6
was shown to be translocated to 15p while RP11-167B4
remained on 16p. Only a single hybridization signal was
observed on the normal chromosome 16 for RP11-578P21,
demonstrating a deletion at the translocation breakpoint.
Parental FISH analysis using clone RP11-578P21 was normal,
ruling out an inherited deletion polymorphism of this clone and
no copy number polymorphisms for this region have been
reported.
The gene content at the translocation breakpoint and deleted
region was examined and the only known gene contained in
this interval is the ataxin-2 binding protein (A2BP1) gene.
From FISH analysis, the deletion was estimated to include
exon 1 of the A2BP1 gene. Further confirmation of this result
was pursued using molecular methods.
Q-PCR Analysis for Deletion
Detection of Exon 1 in A2BP1
To precisely determine if exon 1 of the A2BP1 gene is deleted
in the proband, real-time Q-PCR using genomic DNA was
Fig. 2. Representative FISH analysis showing results from breakpoint
mapping of the 15p;16p translocation. The color coding used in the schematic
above the FISH images is the same as that used in Figure 1. BAC 19H6 is
distal to the translocation breakpoint and shows a hybridization signal (red)
on the short arm of the derivative 15 and the normal 16p. BAC 578P21 is
deleted and only shows a single hybridization signal (red) on the normal 16;
the derivative 15 and normal 15 are marked by a centromere probe for
chromosome 15 (aqua).
performed in the proband, her parents, an unaffected brother,
and an unrelated control individual. As shown in Figure 3, the
genomic dose of A2BP1 in the proband is approximately 50% of
that in the four other individuals tested, demonstrating a
heterozygous deletion of exon 1 in the proband. Deletions of
exon 1 in A2BP1 were also tested for by Q-PCR in an additional
88 individuals (that also had complete gene sequencing). No
evidence for a deletion in exon 1 was observed in any of the
individuals tested, consistent with the presumed rarity of this
deletion.
A2BP1 Expression Analysis of Isoforms 1–4
The A2BP1 gene has 4 known isoforms and the total gene
is among the longest in the genome, 1.7 Mb (spanning from
6.0–7.7 Mb on 16p). Isoform 4 spans the entire 1.7 Mb, but
isoforms 1, 2, and 3 cover only 380 kb at the 30 end of the gene.
The first 3 exons and part of the 4th exon of isoform 4 consist of
the untranslated region of the gene. Interestingly, these
4 exons cover 1 Mb total genomic sequence. Since the deletion
in the proband occurs in an untranslated region that is more
than a megabase away from the coding sequence, but was
predicted to potentially contain regulatory sequences, we
hypothesized that the deletion might alter A2BP1 levels. To
assess whether the deletion had such functional consequences,
expression of A2BP1 was examined by semi-quantitative RTPCR in lymphoblasts. As shown in Figure 4a, qRT-PCR for
isoform 4 showed the expression was significantly decreased
in the proband compared to controls (P < 0.0001). The same
experiment was performed using primers specific for isoforms
1, 2, and 3. Expression of isoforms 1, 2, and 3 (collectively) was
also significantly decreased, although to a lesser extent than
isoform 4 (Fig. 4b). These results demonstrate that the deletion
has a significant effect on A2BP1 expression.
SNP Analysis and Sequencing
Rare chromosomal abnormalities, such as the deletion
described here, in people with disease symptoms may indicate
that the interrupted gene contributes to the disease. Motivated
by the identification of this rare disease-causing variant, we
reasoned that common variants in this gene may contribute to
autism. To test this in a preliminary manner we conducted
a pilot association analysis using AGRE autism families.
874
Martin et al.
Fig. 3. Results from Q-PCR analysis of the A2BP1 gene in the proband, the proband’s unaffected family members and an unrelated normal control
showing reduced dosage for A2BP1 in the proband. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Twenty-seven SNPs, in both introns and exons of the A2BP1
gene (Fig. 5), from dbSNP and the Celera database (Table I)
were genotyped in 206 parent child trios from AGRE. All SNPs
had a %geno 99%, ME < 2, MAF 0.03, and HWE P 0.03.
No single SNP showed significant association when tested
for transmission disequilibrium (TD). However, nominally
significant multipoint TD was observed between SNPs
rs1395579, rs1911492, and rs1395585, which span a 7 kb
region, and between SNPs rs917544, rs763649, rs12149686,
and rs2191133, spanning 21 kb (TDT P ¼ 0.004 and P ¼ 0.008,
respectively; Fig. 5). Linkage disequilibrium was assessed
using the Haploview program. SNPs within each of the two
regions did not appear to be in strong LD, with r2 < 0.54 among
SNPs in each of the regions.
Re-sequencing of A2BP1 was also carried out to identify
potential mutations in this gene within the AGRE sample.
A2BP1 was sequenced for all 16 exons and surrounding
intronic regions in one proband chosen from 88 affected sibspairs who demonstrated the most allele sharing across the
region between markers D16S2622 and D16S407, which flank
the A2BP1 gene. No non-synonymous amino acid changes were
identified in this cohort. Twenty-five single nucleotide variants
were identified by sequencing the 88 autistic individuals
(Table II). Seven of the 25 SNPs were located in exons.
However, none caused an amino acid change and were not
predicted to be deleterious.
DISCUSSION
Various genome-wide genetic approaches, including linkage
scans, association studies, cytogenetic and FISH analyses
have been pursued to identify chromosomal regions harboring
Fig. 4. qRT-PCR results demonstrating reduced expression of (A)
isoform 4 and (B) isoforms 1, 2, and 3 (collectively) of the A2BP1 gene in
the proband compared to normal family members and a normal unrelated
control.
causative or susceptibility genes for autism. Using these
strategies, several studies have implicated the chromosomal
region 16p13 [IMGSAC, 1998; Philippe et al., 1999; IMGSAC,
2001; Lucarelli et al., 2003; Bhalla et al., 2004; Barnby et al.,
2005; Vorstman et al., 2006].
We identified a translocation involving the short arms of
chromosomes 15 and 16 in a female with autism, severe
developmental disability, epilepsy, delayed walking with mild
residual ataxia, behavioral regression, fluctuating liver function tests and mild cerebellar atrophy. Using a combination of
FISH and Q-PCR analyses, we demonstrated that a deletion of
approximately 160 kb is present in exon 1 of the A2BP1 gene at
the breakpoint of this chromosomal rearrangement. Furthermore, rtPCR showed significantly reduced expression, consistent with the supposition that the A2BP1 deletion in this
individual is functional.
A2BP1 is one of the largest genes in the human genome,
covering 1.7 Mb and encoding a 377 amino acid protein. It is
highly conserved in C. elegans, D. melanogaster, mouse and
human [Kiehl et al., 2001] and has been previously characterized in human [Shibata et al., 2000], C. elegans [Kiehl et al.,
2000] and mouse [Kiehl et al., 2001]. In humans, A2BP1 codes
for an RNA-binding protein that binds to the C-terminus of
ataxin-2, which has been implicated in spinocerebellar ataxia
type 2 (SCA2) [Shibata et al., 2000]. Recently, the A2BP1
protein was shown to regulate alternative splicing of tissuespecific exons by binding to the hexanucleotide UGCAUG
through its RNA recognition motif and is considered a neuronspecific splicing factor [Nakahata and Kawamoto, 2005;
Underwood et al., 2005; Auweter et al., 2006].
The A2BP1 protein is predominantly expressed in muscle
and brain. Specifically in the brain, A2BP1 is found in the cytoplasm of Purkinje cells and dentate neurons in a punctate
pattern [Shibata et al., 2000; Underwood et al., 2005].
Postmortem studies of brains from individuals with autism
have shown neuroanatomic abnormalities of the cerebellum
and limbic system, including the hippocampus [Bauman and
Kemper, 2005]. Therefore, A2BP1 is a plausible candidate gene
for ASD given its expression pattern. Fine mapping studies of
the proband with the 15p;16p translocation revealed a deletion
involving exon 1, an untranslated region of the A2BP1 gene.
However, qRT-PCR analysis of lymphoblasts showed reduced
expression of A2BP1 for all 4 isoforms compared to other
normal family members and an unrelated normal control.
Therefore, the chromosomal region 1.5 Mb up from the coding
sequence is likely involved in regulating gene expression, as its
deletion affects the expression of all known A2BP1 isoforms in
this individual.
Given the causative deletion of A2BP1 and because of
emerging evidence in the role of RNA processing and gene
regulation in pervasive developmental disorders, we performed further screening of this gene in additional individuals
with autism from the AGRE collection to identify other rare
A2BP1/FOX1 as a Candidate Gene for Autism
875
Fig. 5. A2BP1 gene structure and SNP association results. The A2BP1 gene is shown schematically at the top, spanning from 6,009 kb on chr16 to
7,702 kb based on NCBI Genome Build 36. The coding region, which was the focus of our association analysis is depicted by the brackets. Exons are
represented by short vertical lines. The region of interest is magnified below with relative locations of genotyped SNPs. The LD plot generated in Haploview
shows r2 values between genes with darker color indicating higher LD. The two haplotype blocks that showed significant association are boxed in red and
SNPs in those blocks are shown under the magnified gene structure.
variants. We did not detect any additional deletions of exon 1 or
mutations in the 88 probands screened. However, we cannot
rule out whole gene deletions or deletions of other parts of the
gene since we only examined exon 1 of A2BP1 by Q-PCR.
Association analysis of SNPs across A2BP1 identified two
potential regions harboring risk alleles. Although only nominal association was observed, which is not significant if one
applies a strict Bonferroni correction, these results remain
suggestive, since the SNPs are not totally independent, and a
Bonferroni correction may be overly strict. Similarly, while no
obvious causal mutations were identified in this study, these
results do not rule out the possibility that A2BP1 may affect
susceptibility or cause autism in a subset of patients. Multiple
lines of evidence, including linkage studies and cytogenetic
deletions support A2BP1 as a good candidate gene for
involvement in ASD. Therefore, further investigations of the
potential risk variants identified here in a larger sample of
multiplex families or in sporadic autism cases, both with and
without phenotypic features similar to the case reported, may
provide additional information regarding the involvement of
this gene in the autistic phenotype.
ACKNOWLEDGMENTS
This work was supported by NIMH grant R01-MH64547, A
Genome Wide Search for Autism Susceptibility Loci (to CLM
and DHG). We gratefully acknowledge the resources provided by the Autism Genetic Resource Exchange (AGRE)
Consortium1 and the participating AGRE families. AGRE is a
1
Dan Geschwind, M.D., Ph.D., UCLA, Los Angeles, CA; Maja
Bucan, Ph.D., Univ. of Pennsylvania, Philadelphia, PA; W.Ted
Brown, M.D., Ph.D., F.A.C.M.G., N.Y.S. Inst. for Basic Research in
Developmental Disabilities, Staten Island, NY; Rita M. Cantor,
Ph.D., UCLA, Los Angeles, CA; John N. Constantino, M.D.,
Washington Univ., St. Louis, MO; T.Conrad Gilliam, Ph.D., Univ.
of Chicago, Chicago, IL; Martha Herbert, M.D., Ph.D., Harvard
Medical School, Boston, MA; Clara Lajonchere, Ph.D, Cure Autism
Now, Los Angeles, CA; David H. Ledbetter, Ph.D., Emory Univ.,
Atlanta, GA; Christa Lese Martin, Ph.D., Emory Univ., Atlanta,
GA; Janet Miller, J.D., Ph.D., Cure Autism Now, Los Angeles, CA;
Stanley F. Nelson, M.D., UCLA, Los Angeles, CA; Gerard D.
Schellenberg, Ph.D., Univ. of Washington, Seattle, WA; Carol A.
Samango-Sprouse, Ed.D., George Washington Univ., Washington,
D.C.; Sarah Spence, M.D., Ph.D., UCLA, Los Angeles, CA;
Matthew State, M.D., Ph.D., Yale Univ., New Haven, CT; Rudolph
E. Tanzi, Ph.D., Massachusetts General Hospital, Boston, MA.
program of Cure Autism Now and is supported, in part, by
grant MH64547 from the National Institute of Mental Health
to Daniel H. Geschwind (PI). The goal of the program is to
facilitate more rapid progress in the identification of the
genetic causes of autism and autism spectrum disorders by
promoting sharing and collaboration. A full description of this
resource has been published (Geschwind et al. (2001) Am J
Hum Genet, 69:2) and is available on the web at www.agre.org.
The authors would also like to thank Susan Christian, PhD, for
microsatellite analysis to determine the parental origin of the
deletion, and William Dobyns, M.D., for review of the MRI
scans.
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