An atypical deletion of the Williams-Beuren syndrome interval implicates genes associated with defective visuospatial processing and autism.
ABSTRACT During a genetic study of autism, a female child who met diagnostic criteria for autism spectrum disorder, but also exhibited the cognitive-behavioural profile (CBP) associated with Williams-Beuren syndrome (WBS) was examined. The WBS CBP includes impaired visuospatial ability, an overly friendly personality, excessive non-social anxiety and language delay.
Using array-based comparative genomic hybridisation (aCGH), a deletion corresponding to BAC RP11-89A20 in the distal end of the WBS deletion interval was detected. Hemizygosity was confirmed using fluorescence in situ hybridisation and fine mapping was performed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction.
The proximal breakpoint was mapped to intron 1 of GTF2IRD1 and the distal breakpoint lies 2.4-3.1 Mb towards the telomere. The subject was completely hemizygous for GTF2I, commonly deleted in carriers of the classic approximately 1.5 Mb WBS deletion, and GTF2IRD2, deleted in carriers of the rare approximately 1.84 Mb WBS deletion.
Hemizygosity of the GTF2 family of transcription factors is sufficient to produce many aspects of the WBS CBP, and particularly implicate the GTF2 transcription factors in the visuospatial construction deficit. Symptoms of autism in this case may be due to deletion of additional genes outside the typical WBS interval or remote effects on gene expression at other loci.
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ABSTRACT: The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and non-epithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathologic conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.AJP Lung Cellular and Molecular Physiology 09/2012; · 3.52 Impact Factor
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ABSTRACT: Deletion of the Williams-Beuren syndrome (WBS) critical region (WBSCR), at 7q11.23, causes a developmental disorder commonly characterized by hypersociability and excessive talkativeness and often considered the opposite behavioral phenotype to autism. Duplication of the WBSCR leads to severe delay in expressive language. Gene--dosage effects on language development at 7q11.23 have been hypothesized. Molecular characterization of the WBSCR was performed by fluorescence in situ hybridization and high-resolution single-nucleotide polymorphism array in two individuals with severe autism enrolled in a genetic study of autism who showed typical WBS facial dysmorphism on systematic clinical genetic examination. The serotonin transporter promoter polymorphism (5-HTTLPR, locus SLC6A4) was genotyped. Platelet serotonin levels and urinary 6-sulfatoxymelatonin excretion were measured. Behavioral and cognitive phenotypes were examined. The two patients had common WBSCR deletions between proximal and medial low copy repeat clusters, met diagnostic criteria for autism and displayed severe impairment in communication, including a total absence of expressive speech. Both patients carried the 5-HTTLPR ss genotype and exhibited platelet hyperserotonemia and low melatonin production. Our observations indicate that behaviors and neurochemical phenotypes typically associated with autism can occur in patients with common WBSCR deletions. The results raise intriguing questions about phenotypic heterogeneity in WBS and regarding genetic and/or environmental factors interacting with specific genes at 7q11.23 sensitive to dosage alterations that can influence the development of social communication skills. Thus, the influence of WBSCR genes on social communication expression might be dramatically modified by other genes, such as 5-HTTLPR, known to influence the severity of social communication impairments in autism, or by environmental factors, such as hyperserotonemia, given that hyperserotonemia is found in WBS associated with autism but not in WBS without autism. In this regard, WBS provides a potentially fruitful model with which to develop integrated genetic, cognitive, behavioral and neurochemical approaches to study genotype--phenotype correlations, possible gene--environment interactions and genetic background effects. The results underscore the importance of considering careful clinical and molecular genetic examination of individuals diagnosed with autism.Molecular autism. 08/2013; 4(1):29.
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ABSTRACT: Williams Beuren syndrome (WBS) is a multisystemic disorder caused by a hemizygous deletion of 1.5 Mb on chromosome 7q11.23 spanning 28 genes. A few patients with larger and smaller WBS deletion have been reported. They show clinical features that vary between isolated SVAS to the full spectrum of WBS phenotype, associated with epilepsy or autism spectrum behavior. Here we describe four patients with atypical WBS 7q11.23 deletions. Two carry ∼3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG) genes. Other two carry a shorter deletion of ∼1.2 Mb at centromeric side that excludes the distal WBS genes BAZ1B and FZD9. Along with previously reported cases, genotype-phenotype correlation in the patients described here further suggests that haploinsufficiency of HIP1 and YWHAG might cause the severe neurological and neuropsychological deficits including epilepsy and autistic traits, and that the preservation of BAZ1B and FZD9 genes may be related to mild facial features and moderate neuropsychological deficits. This report highlights the importance to characterize additional patients with 7q11.23 atypical deletions comparing neuropsychological and clinical features between these individuals to shed light on the pathogenic role of genes within and flanking the WBS region.European Journal of Human Genetics advance online publication, 12 June 2013; doi:10.1038/ejhg.2013.101.European journal of human genetics: EJHG 06/2013; · 3.56 Impact Factor
LETTER TO JMG
An atypical deletion of the Williams–Beuren syndrome interval
implicates genes associated with defective visuospatial
processing and autism
Lisa Edelmann, Aaron Prosnitz, Sherly Pardo, Jahnavi Bhatt, Ninette Cohen, Tara Lauriat, Leonid
Ouchanov, Patricia J Gonza ´lez, Elina R Manghi, Pamela Bondy, Marcela Esquivel, Silvia Monge,
Marietha F Delgado, Alessandra Splendore, Uta Francke, Barbara K Burton, L Alison McInnes
............................................................... ............................................................... .....
J Med Genet 2007;44:136–143. doi: 10.1136/jmg.2006.044537
Background: During a genetic study of autism, a female child
who met diagnostic criteria for autism spectrum disorder, but
also exhibited the cognitive–behavioural profile (CBP) asso-
ciated with Williams–Beuren syndrome (WBS) was examined.
The WBS CBP includes impaired visuospatial ability, an overly
friendly personality, excessive non-social anxiety and language
Methods: Using array-based comparative genomic hybridisa-
tion (aCGH), a deletion corresponding to BAC RP11-89A20 in
the distal end of the WBS deletion interval was detected.
Hemizygosity was confirmed using fluorescence in situ
hybridisation and fine mapping was performed by measuring
the copy number of genomic DNA using quantitative poly-
merase chain reaction.
Results: The proximal breakpoint was mapped to intron 1 of
GTF2IRD1 and the distal breakpoint lies 2.4–3.1 Mb towards
the telomere. The subject was completely hemizygous for
GTF2I, commonly deleted in carriers of the classic ,1.5 Mb
WBS deletion, and GTF2IRD2, deleted in carriers of the rare
,1.84 Mb WBS deletion.
Conclusion: Hemizygosity of the GTF2 family of transcription
factors is sufficient to produce many aspects of the WBS CBP,
and particularly implicate the GTF2 transcription factors in the
visuospatial construction deficit. Symptoms of autism in this
case may be due to deletion of additional genes outside the
typical WBS interval or remote effects on gene expression at
1:7500, as estimated in a recent study.1Three region-specific
low-copy repeats (LCRs), referred to as centromeric, medial and
telomeric blocks A, B and C, flank the WBS deletion site (fig 1).
Most WBS deletions are 1.5 Mb in length (95%) and result
from unequal crossing over between the centromeric and
medial portions of the block B LCR containing GTF2I and NCF1,
but sparing GTF2IRD2.2GTF2IRD2 is deleted in the much rarer
1.84 Mb deletion cases mediated by portions of the block A
The cognitive–behavioural profile (CBP) of WBS includes
severely impaired visuospatial ability, mental retardation, an
overly friendly personality accompanied by excessive non-social
anxiety, attention deficit, hyperacusis and/or love of music.3
Language delay is usually present, but verbal IQ is generally,
although not always, higher than performance IQ in WBS
cases.3–8By contrast, although language delay is common to
illiams–Beuren syndrome (WBS) results from a
,1.5 Mb deletion in chromosome 7q11.23 that con-
tains about 20 genes. The disorder occurs at a rate of
both disorders, subjects with autism tend to have higher
performance IQs than verbal IQs and display excessive social
versus non-social anxiety. However, difficulty reading emo-
tions from the face and voice, an inability to get along with
peers and poor social judgement are common features of both
WBS cases with atypical CBPs and nested proximal deletions
have helped generate genotype–phenotype correlations. For
instance, there have been multiple reports of WBS cases with
normal visuospatial ability that carry atypical deletions sparing
the GTF2transcription factors GTF2IRD1
Furthermore, a recent report described a female child carrying
a WBS deletion that extended through intron 1 of GTF2IRD1,
but spared GTF2I, whose visuospatial abilities were less severely
impaired than those of full-deletion carriers.16This finding
suggests that hemizygosity of both GTF2I and GTF2IRD1 is
necessary to produce the severe visuospatial deficits seen in
carriers of the classic WBS deletion, a hypothesis supported by
Here, we describe a female child with many aspects of the
WBS CBP, who also meets diagnostic criteria for autism
spectrum disorder, as assessed by the Autism Diagnostic
Interview–Revised17and the Autism Diagnostic Observation
Schedule,18but lacks the classic medical and physical features
of WBS. She carries a novel, de novo deletion between 2.4 and
3.1 Mb in length extending towards the telomere from intron 1
of GTF2IRD1, which results in hemizygosity of the genes at the
distal end of the WBS deletion, including GTF2IRD1, GTF2I and
Abbreviations: aCGH, array-based comparative genomic hybridisation;
BAC, bacterial artificial chromosome; CBP, cognitive–behavioural profile;
FISH, fluorescence in situ hybridisation; HIP1, Huntingtin-interacting protein
1; LCR, low-copy repeat; PCR, polymerase chain reaction; UPL, Universal
Probe Library; WBS, Williams–Beuren Syndrome; YWHAG, tyrosine 3-
monooxygenase/tryptophan 5-monooxygenase activation protein c
N Hemizygosity of GTF2I and GTF2IRD1 results in the
WBS cognitive-behavioural profile including a severe
visuospatial construction deficit and an overly friendly
N GTF2I and GTF2IRD1 oppositely regulate Goosecoid, a
gene involved in craniofacial development.
N A gene related to Goosecoid, called Goosecoid-like
resides in the 22q11 DiGeorge/Velocardiofacial syn-
drome region also associated with autism and a
visuospatial construction deficit.
GTF2IRD2 (figs 1, 2), as well as roughly 14 other genes which lie
downstream of a gap in the genomic sequence that has yet to be
resolved. Two of the 14 genes have previously been implicated
in neuropsychiatric disorders, Huntingtin-interacting protein 1
(HIP1)19and tyrosine 3-monooxygenase/tryptophan 5-mono-
oxygenase activation protein c (YWHAG).20Therefore, the
symptoms of autism observed in our patient may be caused
or exacerbated by hemizygosity of another gene or genes in the
deletion interval or result from remote effects on gene
expression at other loci.
MATERIALS AND METHODS
This project was approved under the guidelines of the Ministry
of Health of Costa Rica, the ethical committee of the National
Children’s Hospital in San Jose ´, California, USA, and the
institutional review board at Mount Sinai School of Medicine,
New York, USA, in accordance with the Declaration of Helsinki.
These approvals remain active. The parents of patient 41A
provided written informed consent.
We are conducting an ongoing genetic study of autism in the
isolated founder population of the Central Valley of Costa
Rica.21Occasionally, we are referred atypical cases including
patient 41A, a female child that smiled often and was overly
friendly with strangers. She had a normal karyotype (G
banding at 550), was negative for fragile X and lacked a
constellation of physical features consistent with a known
syndrome. However, we hypothesised that despite the normal
karyotype data, mental retardation or atypical behavioural
features could be indicative of an underlying submicroscopic
chromosomal abnormality, so we decided to screen this patient
using array-based comparative genomic hybridisation (aCGH).
Array-based comparative genomic hybridisation
Labelling of DNA samples, including a dye swap, and
hybridisation to the Spectral Chip 2600 (Spectral Genomics,
Houston, Texas, USA) whole-genome bacterial artificial chro-
mosome (BAC) array, was carried out at Spectral Genomics,
according to the manufacturer’s protocol. Hybridised micro-
array slides were analysed with GenePix 4000B scanner (Axon
Ins, Union City, California, USA), and data obtained were
analysed using the Spectralware V.2.24 software (Spectral
Genomics). Normalisation of the Spectral aCGH data was
performed using the global linear regression and pin linear
options provided in the Spectralware V.2.24 software. Clones
were considered to be positive for a duplication or deletion if
the log2fluorescence intensities of the spots were .2 standard
deviations (SDs) beyond the mean ratio of intensities for the
Fluorescence in situ hybridisation confirmation of aCGH
BAC DNAs for validation of aCGH results and LB agar stabs of
individual BAC clones were obtained directly from Spectral
Genomics and BAC PAC Resources Center (San Francisco,
41A. The thick horizontal arrows represent the three large blocks of low-copy repeats (LCRs), labelled as blocks A, B and C, with centromeric (c), medial (m)
and telomeric (t) sequence indicated. Single-copy regions flank the LCRs, between Cm and Bm, and between Am and Bt (which contains the gene
WBSCR16). The common WBS deletion interval (approximately 1.5 Mb) is depicted as a solid line. The extended boundaries of the rare 1.84 Mb deletion
are shown as dotted lines. Roughly 28 genes lie in the WBS deletion interval; only the distal genes that have been the focus of genotype–phenotype
correlations are labelled for clarity. Atypical WBS deletions are displayed below the case 41A deletion interval.
Schematic representation of the classic 7q11.23 William–Beuren syndrome (WBS) deletion region and the atypical deletion detected in patient
lies in intron 1 of GTF2IRD1, completely deleting GTF2I, NCF1 and GTF2IRD2, the distal genes in the WBS deletion interval. Fluorescence in situ
hybridisation (FISH) clones with hatch marks indicate that the subject is hemizygous for that sequence. Clones labelled with half-hatch marks gave
diminished signals owing to cross hybridisation. All Universal Probe Library (UPL) probes listed in table 2 are displayed as upright arrows. A line crossing the
arrow signifies that the subject is hemizygous for the sequence corresponding to the UPL probe. The double-headed upright arrow marks the location of
microsatellites D7S1870 and D7S2490 used to determine the deletion that occurred de novo on a paternal chromosome. The location of segmental
duplications, indicating highly repetitive sequence, has been approximated from the Database of Genomic Variants.
Atypical deletion of the distal William–Beuren syndrome (WBS) deletion interval of approximately 2.4–3.1 Mb. The proximal deletion breakpoint
Atypical WBS deletion137
California, USA), respectively, and are listed in supplementary
supplemental). BAC DNA was prepared according to the
product manual of the Nucleobond Plasmid Maxikit (BD
Biosciences Clontech, San Jose, California, USA). Each BAC
was labelled using the Nick Translation Reaction Kit (Vysis,
Downer’s Grove, Illinois, USA) and Spectrum Green-11-dUTP
(Vysis) or Spectrum Red-11-dUTP (Vysis). Single or dual-
coloured fluorescence in situ hybridisation (FISH) was per-
formed on metaphase spreads from the peripheral blood of the
participant or established lymphoblastoid lines dropped on to
precleaned glass slides using a standard protocol. Images were
captured with an ImagePoint cooled CCD video camera
(Photometrics, Tuscon, Arizona, USA) through a Labophot-2A
fluorescence microscope (Nikon, Melville, New York, USA)
using Cytovision FISH software (Applied Imaging, San Jose,
Genomic quantitative polymerase chain reaction for
We adapted the Universal Probe Library (UPL) system from
Roche (Roche, Nutley, New Jersey, USA) to perform genomic
quantitative polymerase chain reaction (PCR). The Roche
system offers a library of eight-nucleotide fluorescence-labelled
probes containing locked nucleic acids to ensure tight binding
with the target sequence. Probes are chosen according to the
primer sequences specified using ProbeFinder V.2.04 software
(Roche, http://www.universalprobelibrary.com). This system is
a precise and much cheaper alternative to traditional TaqMan
assays, as the complete UPL covers .99% of the transcriptome
using only 90 probes paired with transcript-specific primers.
Primers to be used with the UPL probes were designed using
gene sequences from the UCSC Genome Browser (NCBI Build
35, http://genome.ucsc.edu/) and the ProbeFinder application.
Supplementary table B includes all primers and probes used for
www.jmg.bmjjournals.com/supplemental). Reactions were per-
formed with a volume of 10 ml containing 25 ng DNA, 400 nM
of each primer, 100 nM UPL probe (Roche) and 16 Platinum
Quantitative PCR SuperMix-uracil-N-glycosylase, with Rox
(Invitrogen, Carlsbad, California, USA). All reactions were
performed in quadruplicate with an ABI Prism 7900HT
sequence detection system (Applied Biosystems, California,
USA). In addition to the case and parental DNA (case 41A, 41B
father and 41C mother) and the genes to be assayed for copy
number, we included a trio (child and two parents) from the
CVCR as control genomic DNA samples (C1, C2 and C3) and
four reference genes (COBL, GUSB, PPIA and SNCA) on each
plate. PCR conditions were as follows: 2 min uracil-N-
glycosylase activation at 50˚C, then 2 min denaturation at
95˚C, followed by 40 cycles of 95˚C for 15 s and 60˚C for 1 min.
Quantitative PCR to evaluate cDNA expression
cDNA was generated with SuperScript III First Strand kit
according to the manufacturer’s instructions. The products
were diluted either 1:250 (for reference probes) or 1:25 (for all
other probes), and 4.5 ml of the diluted reaction was used in the
Four genes within the deletion interval and three reference
genes were assayed by TaqMan gene expression probes
Expression probes are listed in supplementary table C (available
online at http://jmg.bmj.com/supplemental).
Quantitative PCR data analysis
The data were first evaluated using the Sequence Detection
Software V.2.1 (Applied Biosystems). The baselines and
thresholds for each gene were adjusted to measure Ct values
at the most linear range possible. Quadruplicates having a SD
.0.2 were evaluated and outlying Ct values .2 SDs from the
mean of the quadruplicate were excluded from further analysis.
The remaining Ct values were then fed into the qBase program22
and analysed using the default options and settings. The
reference genes were excluded from the analysis one at a time
on the basis of the coefficient of variation, until two remained.23
For genomic quantitative PCR experiments, relative quantifica-
tion was performed and then calibrated by setting control DNA
sample C2 to a value of 1. Single-copy deletions were detected
when relative copy number values were ,0.75. In the
quantitative PCR expression assays, relative quantification
was performed, calibrated by setting 41C to a value of 1, then
all values were normalised to the mean of 41B and 41C as 1.
(For raw data from both the genomic quantitative PCR
Note the abnormal external rotation of the ear on the lateral photograph.
Parental consent was obtained for publication of this figure.
Anteroposterior (A) and lateral (B) photographs of patient 41A.
138Edelmann, Prosnitz, Pardo, et al
mapping assays and quantitative PCR expression assays, refer
to supplementary tables D and E, respectively, available online
The microsatellite primer pairs D7S1870 in GTF2I and D7S2490
at the distal end of HIP1 were synthesised by Integrated DNA
Technologies (Coralville, Iowa, USA). PCR conditions are
available on request. The microsatellites were genotyped using
the ABI 31-30 XL (Applied Biosystems) and genotype calls were
performed using Genemapper V.3.7 (Applied Biosystems).
Patient 41A (age 6 years and 6 months at the time of
The mother of patient 41A (41C, 18K years old at the time of
the child’s birth) experienced bleeding in both the first and
third trimesters and gained ,7 kg during the pregnancy. The
child was delivered at term without complications, although
she was born with an orbital hemangioma that required surgery
at 2 years and 8 months of age, resulting in strabismus. Her
birth weight, height and head circumference were 2930 g (20th
centile), 47 cm (10th centile) and 32 cm (,5th centile),
respectively. (These norms are from the National Center for
Health Statistics, as none are available for Costa Rica.) She has
one younger male sibling who is in good health and had no
perinatal complications. She did not have a social smile until
she was 1 year old and had delayed acquisition of phrase
speech (3K years of age). She developed sphincter control at
42 months; other motor milestones were generally normal, or
only mildly delayed. Her parents placed her in first grade in a
regular school, but she had to be removed because of aggression
against her peers and other disruptive behaviour. She is very
interested in music, and certain sounds, but does not have
hyperacusis according to parental reports, and clinical observa-
tion, although hyperacusis was not formally tested.
The patient met the Autism Diagnostic Interview–Revised
criteria for autism in all three domains. Her most severe
symptoms included deficits in the comprehension of simple
language, articulation, reciprocal conversation, attention to
voice, empathy and socialising with peers. She met the Autism
Diagnostic Observation Schedule criteria for autism in the
social, play and restricted behaviour domains, but was 2 points
below the cut-off score in the communication domain. She
smiles easily and is superficially friendly with strangers,
although she exhibits minimal reciprocal social interaction.
She also displays excessive non-social anxiety and moderately
severe attention deficits.
Her cognitive profile on the Wechsler Preschool and Primary
Scale of Intelligence—third edition24showed a verbal IQ of 53, a
performance IQ of 57 and a Full-Scale composite IQ of 49,
which places her in the extremely low range when compared
with her peers. She had an IQ of 67 using the Leiter-R,25a non-
verbal IQ test. Her verbal and performance abilities were also
both in the extremely low range. We noted no significant
differences in performance on the verbal subtests, suggesting
that her verbal cognitive abilities are similarly developed.
Notably, she showed an extreme weakness in visuospatial
construction (block design score of 1; see supplementary tables
Fa and Fb for full details of the IQ results, available online
at http://jmg.bmj.com/supplemental). Developmental testing
showed an adaptive behaviour composite score of 50, a
communication skills summary score of 44, a daily living skills
domain summary score of 64 and a social skills summary score
of 54 (Vineland Adaptive Behavioral Scales).
At the time of the evaluation, the patient weighed 21 kg
(50th centile), measured 120 cm in height (50th centile), and
had a head circumference of 51 cm (50th centile). The results of
our medical evaluation did not show any cardinal medical
features of WBS, such as heart defects (negative echocardio-
gram and no heart murmur), joint laxity, loose skin or
although more extensive diagnostic testing might uncover such
abnormalities. She also had no endocrine or serum calcium
abnormalities and did not have feeding or sleeping difficulties
as an infant. Her blood pressure was normal. Her head and
facial features were remarkable for asymmetrical, abnormally
shaped external ears, most prominent on the left side and
iatrogenic strabismus due to the orbital hemangioma (fig 3).
The only facial features consistent with WBS that she displayed
include widely spaced teeth, a wide mouth and prominent lips.
Specifically, she did not display dolichocephaly, bifrontal
upturned nose, a bulbous nasal tip or a long philtrum.
Array-based comparative genomic hybridisation
Analysing the aCGH data, clone RP11-89A20 showed a signal
consistent with a deletion of this region (table 1). The two
flanking clones, RP11-137M14 (proximal) and RP11-451M14
(distal), located approximately 3.3 Mb apart, were not deleted.
Only the clones in the WBS deletion interval were considered
for further follow-up. Refinement of the deletion interval was
conducted with a combination of FISH and genomic qualitative
PCR with UPL probes.
Deletion mapping by FISH and genomic quantitative PCR
Given the genomic location of the deleted BAC within the
classic WBS deletion interval, we first tested the ,180 kb
probe LSI ELN (Vysis) used to screen for the WBS deletion. The
LSI ELN probe gave two clear signals on both metaphase
Patient 41A: results of an array-based comparative genomic hybridisation from Spectral Genomics
Clone nameCyto band
Cy5 test log
Cy3 test log
Known genes in the
CTC-232B231pter 1.5420.803G CENTB5, SCNN1D, B3GALT6, DVL1, PUSL1,
TNFRSF18, UBE2J2, TNFRSF4
Observed in 14/20 Costa Rican cases
Cluster of BTN genes in the extended MHC
Solute carrier family 39 (metal ion transporter)
BAC, bacterial artificial chromosome; G, gain (duplication); L, loss (deletion).
The data were analysed using the global linear and pin lowess options of the Spectral V.2.24 software. The known CNV column indicates whether this particular clone
was duplicated or deleted in normal people. We indicate a new CNV because this clone was duplicated in 14/20 Costa Rican cases, with autism hybridised in the
original study (results to be published elsewhere). Importantly, we tested many of the putative CNVs indicated by the array data and found them to be false-positive
results, therefore we only followed up the most convincing findings in our patients.
Atypical WBS deletion 139
chromosomes and interphase nuclei, indicating that patient
41A does not carry a deletion of the ELN gene (fig 4A). We then
selected a tiling path of FISH clones across the immediate
interval surrounding RP11-89A20 to confirm the Spectral
Genomics array data. The deletion was confirmed using the
clones in supplementary table A (available online at http://
jmg.bmj.com/supplemental), and the results are depicted
schematically in fig 2. Figure 4B shows FISH results for
RP11-89A20 (clone D in fig 2) that gave one normal signal and
a second weak signal of varying intensity, although this clone
was clearly deleted according to the Spectral Genomics
software. A diminished signal could indicate that this segment
of DNA was only partially deleted or that the BAC was partially
hybridising to non-allelic homologous sequence elsewhere. The
second alternative is most likely, given that others have
observed a similar hybridisation pattern using FISH clones in
the block B LCR26; the results obtained with the adjacent
proximal and distal overlapping clones, the UPL probe data and
the original Spectral Genomics array data all indicate a
deletion. Hybridisation of RP11-89A20 (clone D) to paternal
and maternal metaphase chromosomes and interphase nuclei
showed two clear signals, indicating that this deletion is de
novo in patient 41A (fig 5C,D). In addition, parental nuclei
often displayed a second weak signal adjacent to a strong signal
in many of the nuclei, as did the normal chromosome 7 from
41A. However, this second signal was often too close to resolve
from the stronger signal hybridising to its identical sequence in
block B and was only clearly visible as a discrete albeit weak
signal on the deleted chromosome 7 of 41A.
BACs RP11-813J7 (clone C) and RP11-219M8 (clone E) also
gave a normal signal and a second weaker signal when
hybridised to patient 41A (fig 5A, B, respectively). BAC RP11-
813J7 shares substantial overlap with RP11-89A20 and there-
fore displays a similar hybridisation pattern. Dual-colour FISH
with PAC RP5-1186P10 (clone B) gave one signal indicating
that this segment of DNA is deleted. BAC RP11-451K15 (clone
G) gave a strong signal and a slightly diminished signal (fig 5C),
consistent with partial cross hybridisation to other repetitive
sequences as has been reported by Antonell et al.26Dual-colour
FISH results for PACs RP4-665P5 (clone A) and RP5-962D3
(clone F) also showed two signals for each PAC, but again, the
signals appear diminished on one chromosome 7 (fig 5D).
Clone A is not completely deleted, as indicated by the UPL
probe data (fig 6). Figure 5 (E, F, G) shows the results of
interphase FISH with BAC CTB-16K1427(clone H) for patient
41A, her mother, and her father, respectively. CTB-16K14 is an
unsequenced BAC from the Caltech BAC library. Nuclei of
patient 41A exhibit a single hybridisation signal, indicating a
deletion of the sequence containing HIP1, whereas both parents
have normal hybridisation patterns, with two signals of equal
These data are also consistent with the fine-mapping UPL
probe data (fig 6, raw data in supplementary data table D,
available online at http://jmg.bmj.com/supplemental) that place
the centromeric breakpoint of the deletion within the first
intron of GTF2IRD1 in clone A, and the telomeric breakpoint
somewhere between gene UPK3B and the putative gene
KIAA1505. Note that a gap in the genomic sequence exists
The ELN probe (orange signal) is labelled with Spectrum Orange dUTP and LSI D7S486, D7S522 (green signal) is labelled with spectrum green dUTP. (B)
FISH results for patient 41A performed with the SGI BAC RP11-89A20 (green) labelled with spectrum green dUTP, and the control probe for the 7q
subtelomere (orange, TelVysion 7q (Vysis)) labelled with spectrum orange dUTP. A normal green signal and a second weaker green signal (indicated with
arrowheads) of varying intensity are observed on both metaphase chromosomes and interphase nuclei. Control probe signals appear equal in intensity on
both chromosomes 7. (C, D). FISH results for the father and mother, respectively, of patient 41A, performed with the SGI BAC RP11-89A20 (green) labelled
with spectrum green dUTP, and the control probe for the 7q subtelomere (orange, TelVysion 7q (Vysis)) labelled with spectrum orange dUTP. Two normal
green signals are observed, and control probe signals are also of equal intensity on both chromosomes 7.
(A) Fluorescence in situ hybridisation (FISH) results for patient 41A performed with the William–Beuren syndrome (WBS) probe LSI ELN (Vysis).
140Edelmann, Prosnitz, Pardo, et al
between WBSCR16 and HIP1; therefore, placement of additional
UPL probes in this region was not possible. Supplementary
table B contains a list of the genes in the proximal portion of
the deleted interval, upstream of the gap in the genomic
sequence and downstream of the gap, respectively (http://
Analyses of gene expression
Quantitative reverse transcriptase-PCR analyses of GTF2IRD1,
GTF2I, HIP1 and YWHAG were performed on cDNA generated
from RNA extracted from transformed lymphocytes derived
from patient 41A, her father (41B) and her mother (41C).
Expression of GTF2I was decreased to about 60%, and
expression of YWHAG was decreased to about 50%, in patient
41A compared with both parents (supplementary data fig A,
supplementary table C, raw data in supplementary data table
Expression of HIP1 was unchanged in either patient 41A or in
the WBS control. However, expression of GTF2IRD1, using
probes targeting exons 2 and 11, deleted in patient 41A, was
increased in both patient 41A and the WBS control (NIGMS cell
line GM13460, Coriell) and was at least 2.5 times the
expression observed in 41C. The father, 41B, exhibited an
intermediate increase in expression for both GTF2IRD1 probes
that fell between the levels of the deletion carriers and 41C. The
abnormal expression values for GTF2IRD1 in the father may be
Beuren syndrome (WBS) region (fig 2). (A) BAC RP11-813J7 labelled with spectrum green dUTP. A normal green signal and a second weaker signal are
observed on both metaphase chromosomes and interphase nuclei. (B) BAC RP11-219M8 labelled with spectrum green dUTP. A normal green signal and a
second faint signal are consistently observed on both metaphase chromosomes and interphase nuclei. (C) Dual-colour FISH results with PAC RP5-1186P10
(spectrum red dUTP) and BAC RP11-451K15 (spectrum green dUTP). A single red signal and two green signals of equal intensity are present. (D) Dual-
colour FISH results for PACs RP4-665P5 (spectrum green dUTP) and RP5-962D3 (spectrum red dUTP). (E, F, G) Interphase FISH results with BAC CTB-
16K1427(spectrum green dUTP) for patient 41A, her mother and her father, respectively. Nuclei of patient 41A exhibit a single green hybridisation signal,
whereas both parents have normal hybridisation patterns, with two signals of equal intensity.
Fluorescence in situ hybridisation (FISH) results for patient 41A performed with BAC and PAC clones that span the distal end of the William–
UPL Map 01
Relative copy number
UPL Map 02
UPL Map 03
numbers and standard deviations (SDs) from the qBase program. The control average series is the mean of the values and SDs of the three control DNA
samples (C1, C3 and the calibrator C2) as well as the parents of patient 41A (41B and 41C). Single-copy number deletions were detected in the patient
when relative quantification values dropped to ,0.75. The deletion spans from probe GTF2IRD1-2 to UPK3B.
Graph of the Universal Probe Library (UPL) probe data used to fine map the deletion carried by patient 41A, displaying relative genomic copy
Atypical WBS deletion 141
indicative of some kind of genomic rearrangement; however,
we were unable to detect the parental inversion that has been
described previously in the father.28
Data from microsatellites upstream, D7S1870 and downstream
of the gap, D7S2490 (fig 2), indicate that the deletion in our
patient occurred de novo on a paternal chromosome (data not
Our results show that hemizygosity of the GTF2 transcription
factors is sufficient to produce multiple aspects of the WBS
CBP, including impaired visuospatial construction abilities, an
overly friendly personality accompanied by excessive non-social
anxiety and language delay. Although mental retardation and
autistic traits in patient 41A might well be a consequence of a
reduced dosage of the GTF2 transcription factors, these
symptoms might also be caused or exacerbated by other genes
deleted in patient 41A, the prime candidates being HIP1 and
YWHAG. Expression of GTF2I and YWHAG in transformed
lymphocytes from patient 41A was reduced to 60% and 50% of
the parental values, respectively. Expression of GTF2IRD2 was
not evaluated, as this gene has an extra copy in humans that is
transcribed, although it is not clear if it is functional. The
expression data for GTF2I in patient 41A falls within the range
described in a previous analysis of gene expression of
transformed lymphocytes from 10 full WBS deletion carriers
and their parents using semiquantitative reverse transcriptase-
PCR (range 42–70%).29We found increased expression of
GTF2IRD1 in both our cases and in the WBS control cell line.
These data are consistent with a recent report documenting
decreased expression of GTF2IRD1 in cultured skin fibroblasts
from cases with typical WBS deletions, but normal or even
increased expression of GTF2IRD1 in transformed lympho-
cytes.30Only one group has reported decreased expression of
GTF2IRD1 in a typical WBS deletion carrier16; the same group
also found decreased expression of GTF2IRD1 in the atypical
deletion carrier HR using semiquantitative reverse transcrip-
tase-PCR. The reason for this discrepancy is unclear; however,
expression of GTF2IRD1 in transformed lymphocytes is not
representative of gene expression in fibroblasts, and different
regulatory elements are probably important for region-specific
expression. Finally, expression of HIP1 was reduced in both
transformed lymphocytes and skin fibroblasts from typical
WBS deletion carriers who have two copies of HIP130; however,
we did not observe decreased expression of HIP1 in our patient
or in the WBS typical deletion carrier used as a control. This
discrepancy might have arisen because our Taqman probe was
designed to the 59 untranslated region of HIP1, whereas the
probe used by Merla et al30targeted exon 31 of HIP1. Thus, it is
possible that we have measured different splice forms and that
expression of only one of the splice forms is altered.
GTF2 transcription factors, visuospatial ability and
symptoms of autism
In addition to evidence from atypical WBS deletion carriers,
other data also implicate GTF2I in visuospatial ability. Holinger
et al31examined neurons in the brain of five patients with WBS
and five age-matched controls and observed a lack of staining
for GTF2I in neurons from the posterior parietal lobe,
Brodmann area 7. These neuropathological data are consistent
with a functional MRI study showing isolated hypoactivation of
the parietal portion of the dorsal visual stream in 13 patients
with WBS (and normal IQ) on a task designed to test the
integrity of both the dorsal and ventral visual streams.32
Additionally, a recent study showed down regulation of GTF2I
in association with age-dependent deficits of hippocampal-
dependent spatial memory in mice.33Furthermore, defects in
the anterior hippocampus have been noted in a structural and
functional imaging study of patients with WBS.34Therefore,
reduced expression of GTF2I in the hippocampus may
contribute to impaired spatial cognition and navigation
observed in patients with WBS.35
Other evidence suggests that the GTF2 family of transcription
factors might contribute to abnormalities in speech and
language observed in patients with autism and WBS. For
example, Laws and Bishop5studied language impairment and
social deficits in traditional WBS deletion carriers and found
many similarities between autism spectrum disorders and
WBS, especially in the realm of pragmatic language. In fact,
patients with WBS resemble patients with Asperger’s disorder
in that they often fail to discriminate relevant from irrelevant
information in conversation, and their conversational content is
often incongruent with the conversational intention of others.
They may also convey a paucity of information despite apparent
volubility and an extensive vocabulary. Another group found
that children and adults with WBS did poorly on a basic test of
emotional recognition from facial and vocal cues, especially
negative emotions, despite the fact that people with WBS are
drawn to faces and seek eye contact.8Finally, a duplication of
the WBS deletion interval has been associated with abnorm-
alities in language,29and delayed acquisition of language is
characteristic of both WBS and autism; therefore the GTF2
transcription factors could also have a role in normal speech
and language development.
Other candidate genes for autism outside the WBS
Of the roughly 14 genes deleted downstream of the WBS
deletion interval, HIP1 and YWHAG are the most compelling
candidates for susceptibility to autistic traits. HIP1 is a
multifunctional nucleocytoplasmic protein that was first
Huntingtin.19It is a proapoptotic mediator involved in the
differentiation and growth of hippocampal neurons36and also
mediates the endocytosis of GluR1-containing AMPA receptors
in primary hippocampal neurons.37Hip1-null mice show no
gross changes in brain morphology, or hippocampal or striatal
cell densities and appear normal during embryogenesis, yet
develop a neurological phenotype by 3 months of age,
characterised by wasting, tremor and gait ataxia secondary to
a rigid thoracolumbar kyphosis. (Patient 41A does not have a
history of spinal abnormalities.) HIP1 exhibits dosage sensitiv-
ity in mice.
The other gene in the deletion interval that might confer
aspects of the autism phenotype is YHWAG, a member of a
conserved multigene family of phosphopeptide-binding pro-
teins involved in signal transduction, protein localisation, cell-
cycle checkpoint control and apoptosis.
In summary, our most important finding is that, in addition to
GTF2IRD1, hemizygosity of GTF2I is necessary for full manifes-
tation of the visuospatial construction deficit that is a core
component of the WBS CBP. GTF2I may also contribute to
impaired hippocampal-dependent spatial navigation, and other
roles for this gene related to language or the overly friendly
personality are also possible. Finally, several genes both
flanking and within the WBS deletion interval could be
candidate genes for autism susceptibility owing to their role
screens targeting the genes discussed in this paper are
142Edelmann, Prosnitz, Pardo, et al
We thank Kathryn Castelle for her help in editing and formatting this
manuscript and Safiana Katz for her careful processing of immortalised
cell lines. We also thank Dr Olga Arguedas from the Bioethical Board
and Dr Abdon Castro, Presidente de la Fundacio ´n Pro Hospital Nacional
de Ninos ‘‘Dr Carlos Sa ´enz Herrera.’’
Above all, we thank the family of patient 41A, as well as all the other
families who have participated in our study, and the Autism Parents’
Association of San Jose ´, Costa Rica.
The supplementary tables are available at
A Prosnitz, T Lauriat, L Ouchanov, L A McInnes, Department of Psychiatry,
Mount Sinai School of Medicine, New York, New York, USA
L Edelmann, S Pardo, J Bhatt, N Cohen, L A McInnes, Department of
Genetics and Genomic Sciences, Mount Sinai School of Medicine, New
York, New York, USA
P J Gonza ´lez, M Esquivel, S Monge, M F Delgado, Hospital Nacional de
Ninos ‘‘Dr Sa ´enz Herrera’’, CCSS, Child Developmental and Behavioral
Unit, San Jose ´, Costa Rica
E R Manghi, P Bondy, University of Illinois at Chicago, Chicago, Illinois,
A Splendore, U Francke, Department of Genetics and Pediatrics, Stanford
University School of Medicine, Stanford, California, USA
B K Burton, Division of Genetics, Northwestern University Feinberg School
of Medicine, Chicago, Illinois, USA
Funding: LAM is supported by NINDS grant number R01 043540, a Young
Investigator Award from the Seaver Center for Excellence in Autism
Research and a grant from the General Clinical Research Center at the
Mount Sinai School of Medicine, New York, USA.
Competing interests: None.
Correspondence to: Assistant Professor L A McInnes, One Gustave L Levy
Place, Box 1229, New York, NY 10029, USA; email@example.com
Received 7 June 2006
Revised 8 August 2006
Accepted 1 September 2006
Published Online First 13 September 2006
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