Tbx1 haploinsufficiency is linked to behavioral
disorders in mice and humans: Implications
for 22q11 deletion syndrome
Richard Paylora,b, Beate Glaserc,d, Annalisa Mupod,e,f,g, Paris Ataliotisd,h, Corinne Spencera,b, Angela Sobotkae,
Chelsey Sparkse, Chul-Hee Choii, John Oghalaii, Sarah Curranj, Kieran C. Murphyk, Stephen Monksk, Nigel Williamsc,
Michael C. O’Donovanc, Michael J. Owenc,l, Peter J. Scamblerh, and Elizabeth Lindsaye,f,m
Departments ofaMolecular and Human Genetics,bNeuroscience,ePediatrics (Cardiology), andiOtolaryngology, Baylor College of Medicine,
Houston, TX 77030;cDepartment of Psychological Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom;fCEINGE Biotecnologie Avanzate
andgEuropean School of Molecular Medicine (SEMM), 80145 Naples, Italy;hMolecular Medicine Unit, Institute of Child Health, 30 Guilford Street,
London WC1N 1EH, United Kingdom;kDepartment of Psychiatry, Royal College of Surgeons in Ireland, Dublin 9, Ireland; andjDepartment of
Psychological Medicine, Institute of Psychiatry, London SE4 8AF, United Kingdom
Edited by Edward M. Scolnick, The Broad Institute, Cambridge, MA, and approved March 10, 2006 (received for review January 9, 2006)
About 35% of patients with 22q11 deletion syndrome (22q11DS),
which includes DiGeorge and velocardiofacial syndromes, devel-
ops psychiatric disorders, mainly schizophrenia and bipolar disor-
der. We previously reported that mice carrying a multigene dele-
tion (Df1) that models 22q11DS have reduced prepulse inhibition
(PPI), a behavioral abnormality and schizophrenia endophenotype.
Impaired PPI is associated with several psychiatric disorders, in-
cluding those that occur in 22q11DS, and recently, reduced PPI was
reported in children with 22q11DS. Here, we have mapped PPI
deficits in a panel of mouse mutants that carry deletions that
partially overlap with Df1 and have defined a PPI critical region
encompassing four genes. We then used single-gene mutants to
identify the causative genes. We show that PPI deficits in Df1??
mice are caused by haploinsufficiency of two genes, Tbx1 and
Gnb1l. Mutation of either gene is sufficient to cause reduced PPI.
Tbx1 is a transcription factor, the mutation of which is sufficient to
cause most of the physical features of 22q11DS, but the gene had
not been previously associated with the behavioral?psychiatric
phenotype. A likely role for Tbx1 haploinsufficiency in psychiatric
disease is further suggested by the identification of a family in
which the phenotypic features of 22q11DS, including psychiatric
disorders, segregate with an inactivating mutation of TBX1. One
family member has Asperger syndrome, an autistic spectrum dis-
order that is associated with reduced PPI. Thus, Tbx1 and Gnb1l are
strong candidates for psychiatric disease in 22q11DS patients and
candidate susceptibility genes for psychiatric disease in the wider
mouse model ? psychiatric disease ? DiGeorge syndrome ?
(1:4,000 live births). Behavioral and psychiatric disorders are a
prominent part of the 22q11DS phenotype. In children, these
disorders include cognitive defects, anxiety, attention deficit disor-
der, and problems of social interaction that are increasingly recog-
nized to meet the criteria of autistic spectrum disorder (1, 2), a
neurodevelopmental disorder. In adults, high rates of psychotic
disorders, especially schizophrenia, have been reported (2–5).
It is likely that the pathophysiological basis of many psychiatric
disorders is heterogeneous involving multiple genes and environ-
mental factors. Therefore, when they occur frequently in associa-
tion with a defined genetic defect, as in the case of 22q11DS (3, 4,
6, 7), it offers a unique opportunity to identify causative or
contributing genes, especially if a good animal model is available.
We developed a mouse model of 22q11DS (8), the Df1?? mouse,
which carries a heterozygous deletion encompassing 22 genes.
aused by a heterozygous multigene deletion, 22q11 deletion
syndrome (22q11DS) is a relatively common genetic disorder
Df1?? mice recapitulate many of the cardiovascular defects asso-
ciated with 22q11DS (8), and they also display abnormal behavior,
including impaired sensorimotor gating, as measured by prepulse
inhibition (PPI) of the startle response (9), a behavioral abnormal-
ity that is associated with several psychiatric and behavioral disor-
ders including schizophrenia and schizotypal personality (reviewed
in ref. 10), 22q11DS (11), and Asperger syndrome (12). In the
present study, we set out to determine whether reduced PPI in
Df1?? mice results from haploinsufficiency of a particular gene or
genes. Results unexpectedly revealed the presence of two adjacent,
dosage-sensitive genes that significantly affect sensorimotor gating.
We also report that psychiatric disorders, in particular Asperger
syndrome, can occur in association with inactivating mutations of
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
day n; ASR, acoustic startle response; VCFS, velocardiofacial syndrome; NLS, nuclear local-
dB.G., A.M., and P.A. contributed equally to this work.
Psychological Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom. E-mail:
Technology, 2121 West Holcombe Boulevard, Houston, TX 77030. E-mail: elindsay@
© 2006 by The National Academy of Sciences of the USA
chromosome 22q11.2 showing selected genes. Blue and violet bars below
these alleles, Df (16)1–Df (16)5 (13, 14), has been abbreviated to Df1–Df5. The
where blue indicates reduced PPI, violet indicates normal PPI, and green
indicates PPI not tested.
Deletion mutants and single-gene mutants. The black line at top is a
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TBX1, rather than with a 22q11 chromosomal deletion, consistent
with our mouse studies.
PPI Analysis of Mouse Mutants. The reflexive response to the
acoustic startle stimulus that is the basis of the PPI assay requires
that mice can hear, and hearing loss could confound the results of
this test. We therefore evaluated auditory function in Df1?? mice
by measuring distortion product otoacoustic emissions (for meth-
odology, see Supporting Text, which is published as supporting
information on the PNAS web site). No difference was found
between mutants and wild-type littermates (see Fig. 5, which is
published as supporting information on the PNAS web site),
indicating that reduced PPI in Df1?? mutants is not secondary to
To identify the gene(s) responsible for impaired sensorimotor
gating in Df1?? mice, we performed the PPI assay on five mutant
mouse lines (13, 14) that carry heterozygous multigene deletions
that are partially overlapping with Df1 (Fig. 1). All mice analyzed
were male and female littermates on a N5–6 C57BL?6c?/c?genetic
background that was generated by backcrossing mutant C57BL?
6c?/c?;129S5?SvEvBrd mice with C57BL?6c?/c?mice for 5–6 gen-
be significantly impaired in Df1?? mice (P ? 0.044; Fig. 2). In
addition, Df3?? and Df4?? mice also had reduced PPI (P ? 0.004
and 0.001, respectively), whereas the other two deletion mutants
tested, Df2?? and Df5??, had normal PPI (P ? 0.871 and 0.92,
respectively; Fig. 2). Collectively, these results identified a PPI
and the proximal Df5 deletion breakpoint (Fig. 1). The generation
of these two chromosomal deletions has been described (13, 14).
Briefly, we used retroviral insertion of an Hprt3? chromosome
engineering cassette (15), using as a substrate for the insertion
embryonic stem cell lines in which an Hprt5? cassette (15) had been
previously inserted into the genes Es2el (for Df2) and Hira (for Df4
and Df5). The proximal Df5 breakpoint has been localized to the
Df2 breakpoint had previously only been mapped by in situ hybrid-
ization by using a large genomic clone. To localize more precisely
the Df2 breakpoint, we used long-range PCR to amplify the
breakpoint region (15). Cloning and sequencing of the PCR prod-
ucts identified the breakpoint to be in intron 2 of the Txnrd2 gene.
The published mouse sequence (http:??www.ensembl.org) indi-
cated that the refined PPI critical region spanned 300 kb of DNA
Impairment was most apparent at the lower prepulse sound levels, as previously noted (9). The increased PPI seen in Cdcrel1?/?mice did not reach statistical
significance. The magnitude of the ASR was significantly greater (*) in Df3??, Tbx1?/?, and Cdcrel1?/?mice than in their respective wild-type littermates, but
overall there was no relationship between ASR and PPI.
PPI of the acoustic startle response (ASR). Reduced PPI was seen in deletion mutants Df1??, Df3??, and Df4?? and in Tbx1?/?and Gnb1l?/?mice.
www.pnas.org?cgi?doi?10.1073?pnas.0600206103Paylor et al.
and encompassed four genes: Gnb1l, Tbx1, Gp1b?, and Cdcrel1
(Fig. 1). Interestingly, this critical region excluded two genes that
as being responsible for PPI impairments in the affected deletion
mutants. A third candidate behavioral gene that maps within the
Df2 deletion is Comt. Several population-based studies have re-
ported genetic association between COMT and schizophrenia,
although results have been inconsistent. Recently, a COMT low-
activity allele, COMT158met (18), was reported to correlate with
increased severity of psychosis and reduced prefrontal cortex gray
matter volume in a small longitudinal study of 22q11DS patients
(19). Interestingly, in mice, a genetic interaction between Comt and
Prodh has been recently demonstrated (20). Our finding of normal
PPI in Df2?? mice, which are heterozygous for Comt, is consistent
with a previous study that reported normal PPI in Comt?/?and
Comt?/?mice (21) and excludes a role for the gene in the Df1??
gene(s), we performed the PPI assay in Tbx1?/?, Cdcrel1?/?, and
Gnb1l?/?mice. We excluded GpIb? from our analysis because
of function causes Bernard–Soulier disease, a bleeding disorder
that has no known association with psychiatric disease (22). The
generation of Tbx1 and Cdcrel1 mutants has been reported (13).
Gnb1l mutant mice were obtained from Lexicon Genetics Inc.
(The Woodlands, TX). The Gnb1l gene was inactivated by
insertion of a gene-trapping cassette into intron 2,476 base pairs
upstream of exon 3, which is the first coding exon. The insertion
results in aberrant splicing to ?-gal and loss of downstream
mRNA (data not shown). Gnb1l?/?mice, which were provided
on a mixed C57BL?6c?/c?;129S5?SvEvBrd background, were
healthy and fertile. Loss of Gnb1l function is lethal in early
embryogenesis, and no Gnb1l?/?embryos were recovered after
embryonic day (E)6.5. To test the single-gene mutations on the
same background as the deletion mutants, Tbx1?/?, Cdcrel1?/?,
and Gnb1l?/?mice were first backcrossed to C57BL?6c?/c?mice
for 5–6 generations. Unexpectedly, the PPI assay showed that
both Tbx1?/?and Gnb1l?/?mice had reduced PPI (P ? 0.013
and 0.046, respectively; Fig. 2), similar to that identified in the
(P ? 0.341). Thus, normal gene dosage of both Tbx1 and Gnb1l
is required for normal sensorimotor gating in mice. We conclude
that the deletion of these two genes causes impaired sensorimo-
were significant increases in the acoustic startle response (ASR)
in one of the deletion mutants (Df3??) and in two single-gene
mutants, Tbx1?/?and Cdcrel1?/?(P ? 0.001, 0.002, and 0.02,
respectively). Differences in startle responses have been re-
ported between male and female mice, and estrous can affect
PPI. However, in the two-way ANOVA for ASR and the
three-way ANOVA for PPI, we did not detect a main effect of
gender, and more importantly, no gender-specific effect in any
of the genotypes. Thus, it is unlikely that the increased ASR in
the abovementioned mutants is caused by gender effects. Over-
all, there was no direct relationship between levels of PPI and
acoustic startle amongst the various mutant mouse lines. Such
dissociation between PPI and acoustic startle has been docu-
mented by others (reviewed in ref. 23).
Brain Expression of Tbx1 and Gnb1l. Tbx1 encodes a member of the
T-box family of transcription factors. We and others have shown
that Tbx1 haploinsufficiency is responsible for cardiovascular,
craniofacial, thymic, and parathyroid defects in mouse models of
gene through the identification of mutations in patients with a
classical 22q11DS phenotype but without the common chromo-
somal deletion (26). Whether TBX1 haploinsufficiency is respon-
sible for neurodevelopmental or psychiatric disorders in 22q11DS
patients is not known because neuropsychiatric assessments on
patients with the three TBX1 point mutations identified to date
have not been reported.
Tbx1 has been shown by RT-PCR to be expressed in postnatal
mouse brain (27), but regional brain expression has not been
reported. We analyzed Tbx1 expression at various developmental
stages by RT-PCR and real-time quantitative RT-PCR and found
it to be very low in preterm embryonic brain, whereas levels
increased steadily from birth to 3 months (Fig. 3 A and B). To
analyze regional brain expression we used a lacZ-knockin allele
(28), which showed expression to be limited to the vasculature in
term embryos (Fig. 3C) and in adult mice (not shown). A role for
the microvasculature in the pathophysiology of schizophrenia has
been proposed on theoretical grounds, because microvascular
damage could satisfy developmental and degenerative models of
schizophrenia (reviewed in ref. 29). Such a proposal is on the basis
of numerous clinical studies that have reported cerebral blood flow
abnormalities and increased prevalence of minor physical abnor-
increases steadily between E17.5 and 12 weeks as measured by real-time quan-
endothelial cells lining of blood vessels (arrow in Right) but not in the vascular
smooth muscle (arrowheads in Right). Gnb1l expression remains steady at the
an adult Gnb1l?/?mouse showing Gnb1l expression. bg, basal ganglia; t, thala-
Brain expression of candidate behavioral genes. Tbx1 brain expression
Paylor et al.
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malities in schizophrenia patients and the increased prevalence of
schizophrenia in individuals who suffered perinatal problems,
Gnb1l encodes an evolutionarily conserved peptide of unknown
function, which contains six putative WD40 repeats but no other
recognizable functional domains (30, 31). The gene is required for
embryonic development and Gnb1l loss of function causes embry-
onic lethality by E6.5 (P.A. and P.J.S., unpublished data). We
analyzed Gnb1l expression by real-time quantitative RT-PCR and
found it to be uniformly expressed between E17.5 and 12 weeks
(Fig. 3D). The gene is widely expressed in adult mouse brain with
striking regional distribution in forebrain, midbrain, and hindbrain
structures, including the thalamus, hypothalamus, amygdala, hip-
pocampus, pons (Fig. 3E), medulla, and cerebellum (not shown).
Because Gnb1l and Tbx1 lie only 17 kb apart, we considered the
possibility that the engineered mutation of either gene may affect
expression of its neighbor. We analyzed Gnb1l expression in the
brain of Tbx1?/?embryos at term by in situ hybridization and by
real-time quantitative RT-PCR and found no difference between
mutant and wild-type littermates (data not shown), indicating that
the expression of Gnb1l is not regulated by Tbx1 nor is it compro-
mised by the Tbx1 targeting. The complementary experiment
the onset of Tbx1 expression. However, we have shown that
development of the fourth pharyngeal arch artery is a sensitive
indicator of Tbx1 dosage reduction, and 100% of Tbx1?/?embryos
has fourth pharyngeal arch artery hypoplasia at E10.5 (32). This
phenotype is also observed in embryos heterozygous for a hypo-
Tbx1, we would expect to see similar defects in Gnb1l?/?mutants.
However, this was not the case and intracardiac ink injection
revealed normal fourth pharyngeal arch artery development (data
not shown). Although this experiment cannot exclude a tissue-
specific effect of Gnb1l mutation on Tbx1 expression in brain, these
results make it unlikely that the mutation causes a generalized
reduction of Tbx1 expression.
Mutation Analysis in Patients. Thefewpatientsidentifiedsofarwith
TBX1 mutations have not undergone neuropsychiatric assessment.
We have identified a cohort of patients with a 22q11DS-like
phenotype but without the common 22q11.2 microdeletion. From
this cohort, we selected for mutational analysis of TBX1 a family
clinical information). Briefly, the index patient, V39?02 (Fig. 4A),
has the characteristic facial appearance of VCFS and hypernasal
speech. She has no known cardiac defect. Both her surviving
children have the characteristic facial appearance of VCFS and
congenital heart disease: V39?04, a 17-year-old male, has pulmo-
nary stenosis; and V39?03, a 13-year-old male, was diagnosed at
birth with tetralogy of Fallot (Fig. 4A). A recent psychiatric
assessment of both boys (see Supporting Text for methodology)
UTRs. Shown at the bottom is a DNA sequence of a patient with the 1320–1342del23bp mutation (upper panel) and the wild-type TBX1 sequence in an unrelated
individual (lower panel); the position of the mutation is boxed. (C) Subcellular localization of wild-type and mutant TBX1 constructs expressed in U2-OS cells. hTbx1,
wild-type TBX1; 1320–1342del, del23bp mutation described here; G145R, predicted null mutation; 1250delC, point mutation described by Yagi et al. (26). Constructs
were detected with anti-TBX1 antibody. Cell nuclei were stained with DAPI. (D) Transcriptional activation of the CAT reporter gene by wild-type and mutant TBX1.
Significant differences in transcriptional activation between wild-type TBX1 and a TBX1 construct are indicated by***(P ? 0.001). Data were normalized for
transfection efficiency and depicted as average values ? SEM.
www.pnas.org?cgi?doi?10.1073?pnas.0600206103 Paylor et al.
resulted in the diagnosis of Asperger syndrome in V39?03.
Asperger syndrome is an autistic spectrum disorder that is charac-
terized by stereotyped and obsessional behavior and pervasive
abnormalities in socioemotional and communicative behavior (34–
36). Impaired PPI has been reported in some individuals with
Asperger syndrome (12). The presence of Asperger syndrome in a
single family member is consistent with the high variability of other
features of the 22q11DS, even within families. In addition, psychi-
atric disorders often appear in adulthood.
Screening of the TBX1 coding sequence identified a 23-bp
frameshift deletion (1320–1342del23bp) in patient V39?02 and in
both her sons. The deceased daughter of V39?02 also carried the
diagnosis of VCFS, but no DNA was available for analysis. The
mutation occurred at the 3? end of the TBX1 transcript (counting
A of the initiation codon as 1; Fig. 4B). This mutation was not
detected in 716 controls. The frameshift created by 1320–
the mutation does not affect the T-box, it disrupts the central
domain (amino acids 439–448) of a highly conserved nuclear
it changes the conserved residues PYP to WPR (see Fig. 6, which
is published as supporting information on the PNAS web site).
To identify the functional significance of this mutation, we
engineered a human TBX1 cDNA carrying the 1320–1342del23bp
mutation and tested the ability of the mutant protein to localize to
the nucleus and to transactivate a T-box-binding element construct
in a tissue culture system (see Supporting Text for methodology). In
parallel, we tested two other engineered constructs: 1250delC,
which encodes a previously identified mutant form of TBX1 (26),
and G145R, which is the equivalent of a loss-of-function TBX5
T-box mutation G80R that prevents DNA binding (38). Immuno-
cytochemical investigation of transfected cells showed tight nuclear
localization of the wild-type TBX1 and G145R proteins (Fig. 4C),
whereas the 1250delC mutant protein was mainly found in the
cytoplasm, consistent with the findings of Stoller and Epstein (37)
(note that 1250delC in our numbering is equivalent to 1223delC
in ref. 26). Unexpectedly, the 1320–1342del23bp mutant protein
also localized to the nucleus, despite lacking the NLS (Fig. 4C). We
therefore examined this peptide sequence for other potential NLS
sequences (39) and found that the frameshifted protein contains
the sequence RGRRRRCR at amino acids 465–472 (see Fig. 7,
which is published as supporting information on the PNAS
web site). This sequence corresponds to a known NLS sequence
R[GVLIP]RRRRxR that is found in a variety of animal protamine
sequences, as well as Epstein–Barr nuclear antigen (http:??cubic.
We have shown previously that TBX1 is a transcriptional acti-
vator that can induce expression of a CAT-reporter protein under
the control of a Brachyury consensus binding site sequence,
1T-CAT (40). Using the same experimental system, we found that
wild-type TBX1 activated the CAT reporter, confirming our pre-
vious finding (Fig. 4D), whereas the mutant constructs G145R,
1250delC, and 1320–1342del23bp did not. Overall, these data
suggest that the mutation identified in V39?02 and her children is
The strong association between common psychiatric disorders and
more genes in the region confers susceptibility to these disorders.
Three candidate genes from the region, COMT, PRODH, and
ZDHH8C, have been shown to cause behavioral abnormalities
when the genes are mutated in mice. PPI defects have been
reported in Prodh and Zdhhc8 null mutants (very mild in the latter
case), but not in heterozygotes, whereas Comt heterozygous and
homozygous mice have normal PPI. Our study shows that in a
uniform genetic background, combined heterozygosity of all three
normal PPI in Df2?? mice results from a combined effect of two
or more genes that positively and negatively modulate PPI. How-
ever, our data are consistent with published data that show normal
PPI in Zdhh8c and Comt heterozygotes [PPI levels have not been
reported for Prodh heterozygotes, but they have normal L-proline
levels (20)]. Thus, in the context of the PPI phenotype, there is no
evidence of a genetic interaction between any of these genes at
heterozygous gene dosage levels. In the future, it will be interesting
to see whether a Tbx1 transgene can rescue PPI defects in Df1??
mutants. Currently, this cannot be tested because the only Tbx1
transgenic lines available carry transgenes that contain multiple
genes, including Tbx1.
Our strategy to genetically dissect the Df1 deletion unexpectedly
revealed the presence of two adjacent, dosage-sensitive genes that
affect sensorimotor gating, and PPI defects are genetically heter-
ogeneous. Importantly from the disease perspective however, Tbx1
and Gnb1l represent previously unrecognized examples of genes
that affect PPI in the heterozygous mutant state. Both genes are
hemizygous in 22q11DS patients, making them strong candidates
for the associated psychiatric and behavioral phenotypes. The two
genes are apparently unrelated and have distinct expression pat-
terns in brain, suggesting that they are unlikely to function in the
same genetic pathway. Particularly intriguing is our finding of an
inactivating mutation in TBX1 in an individual with Asperger
with TBX1 mutations, it is difficult to evaluate the relative contri-
bution of TBX1 haploinsufficiency to behavioral disorders and
psychiatric disease in 22q11DS. However, our mouse studies show
PPI impairment in the heterozygous state. Therefore, we propose
that these two genes are major contributors to the 22q11DS
population, the 88-kb genomic segment region that harbors TBX1
and GNB1L may represent a susceptibility locus for schizophrenia
and other psychiatric disorders characterized by PPI impairment.
Future studies into the functions of these two genes in brain should
clarify the genetic pathways affected by their mutation and, poten-
tially, may lead to the identification of drug targets aimed at
prevention and or treatment of the psychiatric symptoms in
expression in brain increases postnatally, suggesting that early drug
intervention may prevent the onset of Tbx1-related psychiatric
Mouse Strains, Breeding, and Genotyping. Behavioral testing was
performed on a total of 502 mice (n ? 25–35 mutant and wild-type
similar numbers of males and females were tested. The mice were
all on a N5–6 C57BL?6c?/c?genetic background generated by
backcrossing C57BL?6c?/c?;129S5?SvEvBrd mixed-background
mutant mice with C57BL?6c?/c?mice for 5–6 generations.
Gnb1l?/?mice were provided by Lexicon Genetics Inc., and were
generated by blastocyst injection of stem cells from OST35527
(OmniBank). Mice were genotyped by PCR by using DNA ex-
tracted from tail biopsies (see Supporting Text for primer
PPI Assay. PPI was measured by using the SR-Lab system (San
Diego Instruments, San Diego) as described (41). Animals were
tested at age 8–16 weeks. Before testing, each mouse was acclima-
tized to the Plexiglas cylinder for 5 min, during which time the
background noise level (70 dB) was continually present. Individual
mice were then exposed to six blocks of seven trial types that were
presented in a pseudorandom order with an average intertrial
Paylor et al.
May 16, 2006 ?
vol. 103 ?
no. 20 ?
interval of 15 seconds. The seven trial types comprised the follow-
ing: trial 1 (startle-only trial), 40 ms, 120 dB sound burst; trials 2–6
(prepulse trials), 120 dB startle stimulus preceded 100 ms by 20 ms
prepulse sounds of 74, 78, 82, 86, or 90 dB; and trial 7, 70 dB
background noise. The startle response was recorded for 65 ms,
measuring every 1 ms from the onset of the startle stimulus. The
maximum startle amplitude recorded during the 65-ms sampling
window was used as the dependent variable. We calculated % PPI
trials?startle response-alone trials) ? 100]. Data for each deletion
and mutation were analyzed independently. Acoustic response
amplitude data were analyzed by using two-way (genotype ?
gender) ANOVAs. PPI data were analyzed by using a three-way
(genotype ? gender ? prepulse sound level) ANOVA with re-
Gene Expression Analyses. To visualize ?-gal activity, 4% paraform-
aldehyde-fixed brains were stained in X-gal substrate according to
standard procedures. Two- to 3-mm-thick brain sections were
photographed as whole-mount specimens and then embedded in
paraffin. Sections (10 ?m) were counterstained with Nuclear Fast
Red. RNA in situ hybridization was performed on 10-?m 4%
paraformaldehyde-fixed sections (42). Labeled sense and antisense
in the presence of35S-UTP (MP Biomedicals, Irvine, CA).
Control Subjects. UnrelatedBritishCaucasiancontrolsubjectswere
recruited from the Blood Transfusion Service in Wales and En-
gland (482 males and 234 females; mean age 41.5 years, SD ? 11.5
years). The sample was not specifically screened for psychiatric
Mutation Detection and Sequencing. A transcript map of TBX1 was
created by using the Golden Path Genome Browser (May 2004
freeze) (http:??genome.ucsc.edu?). The coding sequence of the
TBX1 isoform C [National Center for Biotechnology Information
(NCBI) accession no. NM?080647], TBX1 isoform B (NCBI acces-
sion no. NM?005992), and TBX1 isoform C (NCBI accession no.
to each exon were screened for sequence variants in patient
V39?02. Primers were constructed by using the PRIMER3 program
Mutation detection analysis was performed by denaturing high-
performance liquid chromatography (43). All samples with hetero-
duplex traces were subsequently sequenced with BigDye Termina-
(PE Applied Biosystems).
Genotyping of TBX1 deletions. The identified deletion was typed by
using 5?-fluorescently labeled PCR primers (FAM dye), and PCR
products were resolved on an ABI3100 sequencer. The data were
analyzed with GENESCAN 3.7 and GENOTYPER 2.5 software.
CAT assays. U2-OS cells were grown in 12-well dishes to 90%
confluency and transfected in quadruplicate with Lipofectamine
2000 (Invitrogen). The 1T-CAT reporter construct (44) was co-
transfected with TBX1 DNA and the ?-gal expression vector,
pCH110 (Amersham Pharmacia), which was used for normaliza-
tion. The concentration of CAT protein in cell lysates was deter-
mined by using the CAT-ELISA kit (Roche). Differences in
transcriptional activation between wild-type and mutant TBX1
constructs were evaluated by using a Kruskal–Wallis test (SPSS,
Chicago) because the assumption of normality for the variable
transcriptional activation was not given.
Immunocytochemistry. U2-OS cells were grown on poly(D-lysine)-
with TBX1 constructs in pCDNA3. Cells were fixed in 4% para-
formaldehyde after 24 h and permeabilized with 0.05% Nonidet
P-40 in PBS. Cells were incubated with rabbit anti-Tbx1 antibody
(Molecular Probes) at 1:200. Cells were mounted in Vectashield
(Vector Laboratories) with DAPI and photographed by using a
Zeiss AxioVision microscope.
We thank M. Reese, P. Terrell, G. Ji, and C. Gerken for technical support
and S. Reed for clinical assistance. This work is supported by the National
Institutes of Health, National Institutes of Mental Health (E.L. and R.P.),
the March of Dimes (E.L.), and the Ministero dell’Instruzione,
dell’Universita ` e della Ricerca (E.L.). This work was also supported by the
robehavioral Core, Baylor College of Medicine. E.L. is an Associate
Telethon Scientist. P.A. and P.J.S. were supported by the British Heart
were supported by a grant from the Wellcome Trust. Gnbl1 mutant mice
were generated by and purchased from Lexicon Genetics Inc.
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