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HUMAN MUTATION Mutation in Brief #833 (2005) Online

MUTATION IN BRIEF

SALL1 Mutation Analysis in Townes-Brocks Syndrome:


Twelve Novel Mutations and Expansion of the Phenotype
Elke M. Botzenhart1, Andrew Green2, Helena Ilyina3, Rainer König4, R. Brian Lowry5,
Ivan F. M. Lo6, Mordechai Shohat7,Leah Burke8, Julie McGaughran9, Ronit Chafai10,
Geneviève Pierquin11, Ron C Michaelis12, Margo L. Whiteford13, Kalle O. J. Simola14 ,
Bernd Rösler1, and Jürgen Kohlhase1*
1
Institut für Humangenetik und Anthropologie, Universität Freiburg, Freiburg, Germany; 2 National Centre for
Medical Genetics, Our Lady's Hospital, Crumlin, Dublin, Ireland; 3 Institute for Hereditary Diseases, Minsk,
Belarus; 4 Institut für Humangenetik, Universität Frankfurt/ Main, Frankfurt/ Main, Germany; 5 Department of
Medical Genetics, University of Calgary, Alberta Children's Hospital, Calgary, Alberta, Canada; 6 Clinical
Genetic Service, Department of Health, Hong Kong; 7 Department of Medical Genetics, Rabin Medical Center,
Petah Tikva, Israel; 8 Division of Clinical Genetics, Department of Pediatrics, UVM College of Medicine,
Burlington, Vermont; 9 Queensland Clinical Genetics Service, Royal Children's Hospital and Health District,
Brisbane, Queensland, Australia; 10 Service de Pédiatrie, Centre Hospitalier de Luxembourg, Luxembourg; 11
Centre de Génétique Humaine, Centre Hospitalier Universitaire de Liège, Liège, Belgique; 12 J.C. Self Research
Institute, Greenwood Genetic Center, Greenwood, South Carolina ; 13 Ferguson-Smith Centre for Clinical
Genetics, Yorkhill Hospital, Glasgow, Scotland, United Kingdom; 14 Genetics Clinic, Department of Pediatrics,
Tampere University Hospital, Tampere, Finland

*Correspondence to: PD Dr. J. Kohlhase, Institut für Humangenetik und Anthropologie, Universität Freiburg,
Breisacher Str. 33, 79106 Freiburg, Germany; Tel.: +49 761 2707050; Fax: +49 761 2707041; E-mail:
juergen.kohlhase@uniklinik-freiburg.de

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ko1850/7-1.

Communicated by Iain McIntosh

Townes-Brocks syndrome is an autosomal dominantly inherited disorder, which comprises multiple


birth defects including renal, ear, anal, and limb malformations. TBS has been shown to result from
mutations in SALL1, a human gene related to the developmental regulator SAL of Drosophila
melanogaster. The SALL1 gene product is a zinc finger protein thought to act as a transcription
factor. It contains four highly conserved, evenly distributed C2H2 double zinc finger domains. A
single C2H2 motif is attached to the second domain, and at the amino terminus SALL1 contains a
C2HC motif. Most mutations causing TBS are clustered in the N-terminal third of the SALL1
coding region and result in the production of truncated proteins containing only one or none of the
C2H2 domains and the N-terminal transcriptional repressor domain of SALL1. Twenty-three
SALL1 mutations were reported prior to this work, 22 of which are located in exon 2, 5’ of the
second double zinc finger-encoding region. Here we present 12 novel mutations in SALL1 associated
with Townes-Brocks syndrome in 13 unrelated families. These include three nonsense mutations,
three short insertions and six short deletions. Thus the number of SALL1 mutations increases to 35.
Rare phenotypical features among mutation positive patients include hypothyroidism, vaginal
aplasia with bifid uterus, cryptorchidism, bifid scrotum without hypospadia scrotalis, unilateral
chorioretinal coloboma with loss of vision, dorsal hypoplasia of the corpus callosum, and umbilical
hernia. ©2005 Wiley-Liss, Inc.

Received 22 February 2005; accepted revised manuscript 11 May 2005.

© 2005 WILEY-LISS, INC.


DOI: 10.1002/humu.9362
2 Botzenhart et al.

KEY WORDS: SALL1; Townes-Brocks syndrome

INTRODUCTION
Townes-Brocks syndrome (TBS; MIM# 104780) is a rare autosomal dominantly inherited malformation
syndrome, characterized by imperforate anus, preaxial polydactyly and/or triphalangeal thumbs, and dysplastic
ears (Powell and Michaelis, 1999). Further malformations/ anomalies include renal anomalies, hearing loss (either
sensorineural, conductive or both), foot malformations, congenital heart defects and rarely mental retardation
(Surka et al., 2001). TBS is caused by mutations in SALL1, a human gene related to the developmental regulator
gene sal of Drosophila melanogaster (Kohlhase et al., 1998). SALL1 comprises three exons (Kohlhase et al., 1996;
Kohlhase et al., 1998) encoding a zinc finger protein thought to act as a transcriptional repressor (Netzer et al.,
2001). It contains four highly conserved double C2H2 zinc finger domains, which are evenly distributed. A single
C2H2 motif is attached to the second domain. At the amino terminus SALL1 contains a C2HC motif. The protein is
exclusively found in the nucleus where it is localized to pericentromeric heterochromatin (Netzer et al., 2001). In
patients with typical signs of Townes-Brocks syndrome SALL1 mutations have been found in 64.3-83.3%
(Kohlhase et al., 1999; Marlin et al., 1999). To date 20 different SALL1 mutations have been reported in patients
with Townes-Brocks syndrome, the majority of which are frameshift mutations. Two additional mutations were
found in two families the affected members of which do not suffer from clinical Townes-Brocks syndrome but
show a Branchio-Oto-Renal syndrome-like phenotype (Albrecht et al., 2004; Engels et al., 2000). Another
mutation was found in a girl who showed features overlapping with TBS and Goldenhar syndrome (Gabrielli et al.,
1993; Kohlhase et al., 1999) adding up to a total of 23 mutations previously reported. In this report we present 12
novel mutations in SALL1 associated with Townes-Brocks syndrome.

MATERIALS AND METHODS

Patients
Venous blood was collected from patients with Townes-Brocks syndrome and unaffected relatives after
obtaining their informed consent. A skin biopsy and mouth brush was performed to get DNA from different germ
layers in order to examine one parent for mosaicism.

Genetic analysis
Genomic DNA was prepared from peripheral lymphocytes and other samples by routine procedures. Mutation
analysis of exons 1, 2, and 3 of SALL1 was performed as described previously (Kohlhase et al., 1999). Identified
sequence aberrations were confirmed by a second PCR and sequencing of amplicons. Where possible further
family members were analyzed for segregation of the aberration. Mutations were numbered according to GenBank
NM_002968.1 (SALL1 mRNA sequence).

Case reports
Family A shows one affected boy who presented with a left ear tag at birth and was subsequently found to have
a degree of anal stenosis and showed small multicystic kidneys at ultrasound examination with impaired renal
function. He has normal hands, a 2/3 syndactyly and overriding toes of both feet. The father had a right bifid
thumb and bilateral abnormal ear pinnae.
In family B, the affected child presented with mild IUGR (birth weight 2510g at 37 weeks), bilateral
triphalangeal thumbs, low-set, dysplastic ears, high imperforate anus with perianal fistula and sensorineural
hearing loss (~ 40dB). Further noticeable problems were neonatal jaundice and recurrent hypoglycemia, which
resolved later on. Ultrasound examination of the brain revealed mild asymmetry of head and ventricles. Physical
examination additionally revealed a high arched palate and undescended right testis. The mother also showed
bilateral triphalangeal thumbs and sensorineural hearing loss (~ 45dB). No other features of Townes-Brocks
syndrome were seen.
Family C shows an affected female infant born at 41 weeks of gestation with birth parameters within the normal
range (weight 3400g, length 49cm and head circumference 34.5 cm). Noticeable problems were imperforate anus
with rectovaginal fistula, bilateral preaxial polydactyly with triphalangeal thumb on the right hand and dysplastic
Novel SALL1 Mutations 3

ears with overfolded helices. Other distinct features were a rightsided preauricular skin tag, a VSD, a sacral
dimple, sparse eyebrows and a weakness of the left facial nerve. Further features uncommon for TBS were a small
umbilical hernia and a broad nasal bridge. The father of the child was unavailable for examination.
In family D birth parameters of the affected boy were within the normal range (weight 3400g, length 52cm and
head circumference 33.5cm). The child presented with duplication of both thumbs, imperforate anus with perianal
fistula, balanic hypospadias, dysplastic ears with thick helices, and seizures (type not specified). Radiologically an
abnormal aspect of the S1 vertebra (having the shape of a lumbar vertebra) and 13 pairs of ribs were seen.
Additionally the child showed a severe gastroesophageal reflux, a mild hearing loss of approximately 50dB (type
not specified), which required hearing aids, and a mild mental delay with ADHD (age at diagnosis unknown).
The second child of healthy parents in family E was born with imperforate anus, preaxial polydactyly of both
hands and bilateral preauricular ear pits and tags with overfolded helices on both ears. As to his feet the left hallux
was fused to the left second toe. The right second toe was absent and he had a syndactyly of his right third and
fourth toes. The right hallux seemed to be a fusion product of the first and second toe (those features were
previously only seen with the p.R276X mutation). Additionally he showed a bifid scrotum with undescended
testes. As a particular feature he shows an umbilical hernia.
The next patient (family F) was a male, who was diagnosed at the age of nine with sporadic Townes-Brocks
syndrome. He had shown imperforate anus at birth as well as long thumbs, overlapping toes and first degree
hypospadias. A right-sided preauricular tag was seen. He also shows mild bilateral sensorineural hearing loss and
polycystic kidneys with moderate chronic renal failure. He further exhibits some ocular abnormalities, which
include a chorioretinal coloboma of the right eye with no vision on the right eye and bilateral Brushfield spots, and
has bilateral epicanthic folds. Attention deficit disorder was assumed in the first years of life but normal
development was reported later on. The mother has a very short left thumb and deficient cartilage in her left ear.
Hand radiographs and metacarpophalangeal pattern profiles (MCPP) were performed on the patient and his
mother. The latter had shortened proximal phalanges of both thumbs (L>R) while her son had shortened distal
phalanges of both thumbs.
The female sporadic patient of family G presented at birth with an anteriorly placed anus and anal stenosis,
malformed ear pinnae, a bifid left thumb (no abnormality of right thumb reported), right foot 2/3 syndactyly,
hypoplastic kidneys and impaired renal function. A mild high frequency hearing loss in at least one ear could not
be excluded due to inability of the child to cooperate (age at examination 18 months).
Another female sporadic patient (family H) showed imperforate anus and rectovestibular fistula at birth (birth
weight 3400g, length 50cm). She furthermore had dysplastic ears with left preauricular sinus and fibrochondral
tag, and a bifid thumb on the left, for which she underwent surgery. No abnormalities of the right hand were seen.
Ultrasound examination revealed hypoplastic kidneys with cortical cysts. The patient shows moderate chronic
renal insufficiency with difficulties with oral intake which required gastric button feeding. She furthermore showed
growth retardation and received carbohydrates and fat supplements. Other physical features not described so far to
be associated with TBS included complete bifid uterus and vaginal aplasia.
The index patient of family I presented after an uneventful pregnancy with imperforate anus and perianal
fistula, bilateral triphalangeal thumbs with leftsided preaxial polydactyly, dysplastic ears, right sided dysplastic
kidney and sensorineural hearing loss. Birth parameters were within the normal range (weight 3920g, length 54cm
and head circumference 36cm, APGAR-Score 8-10-10). As an unusual feature a dorsal corpus callosum
hypoplasia was noted. The father of the index patient had imperforate anus and bilateral preaxial polydactyly at
birth. Additional physical features in the father included dysplastic ears with sensorineural hearing loss, which was
corrected with hearing aids, overlapping toes and dysplastic kidneys. Ophthalmological examination revealed a
bilateral Duane anomaly.
Another male patient in family J was born to nonconsanguineous unaffected parents after an uneventful
pregnancy at 41 weeks of gestational age. Delivery was spontaneous and uneventful. Birth weigth was 3550g.
After birth the baby was found to have imperforate anus and surgery was performed. An elevated TSH level was
detected in the cord blood by the neonatal screening program, and congenital hypothyroidism was later confirmed.
The thyroid scan result showed mildly increased perfusion. Thyroxine replacement therapy was started.
Additionally abnormal appearance of external ears was noted and a hearing test (BAER) was performed which
showed normal results. Development was normal. Further phenotypical features included epicanthic folds, broad
nasal bridge, flat nasal tip, short nasal septum, long philtrum, thin upper lip, bilateral small dysplastic ears and
small preauricular tags. The fingers appeared to be short and stubby with broad thumbs. X-Ray examination of the
4 Botzenhart et al.

hands revealed no bony dysplasia. The second and third toes were hypoplastic. There was no family history of anal
problems or hearing deficit.
A further family K with two affected individuals was seen which later was shown to have the same mutation as
the family extensively reported by Kurnit et al. 1978 (family L) (Kurnit et al., 1978). The female index patient in
family K was the second child in the family. She was born at term after a normal pregnancy with birth weight
2800g and length 50cm. At birth small dysplastic ears with two right skin tags, bilateral thumb duplication and
imperforate anus with rectovaginal fistula were seen, on which she was operated successfully. At 10 years bilateral
cochlear neuritis with grade 2 hearing loss was established. Renal ultrasound examination at 15 years revealed
bilateral renal hypoplasia. The girl has normal facial features, small lateral upper incisors with otherwise normal
dental status and normal intellectual and physical development. Her brother was the first child in the family. He
was born at term after an uneventful pregnancy with low birth weight (2600g, lenght 49cm). He furthermore
showed double right thumb, 1-2 and 3-4 right foot syndactyly, severe anal stenosis, bilateral cryptorchidism and
hypospadias. Anal stenosis was treated successfully. At the age of ten years sensorineural hearing loss of first
degree was established. At 17 years bilateral renal hypoplasia was noted. The boy had normal physical and
intellectual development, narrow shoulders, kyphosis, and low set protruding posteriorly rotated dysplastic ears
with overfolded helices. The mother of both children is intellectually normal. There are no noticeable facial
features exept for the typical dysplastic ears already seen in her son. She exhibits a mild hearing loss of unknown
type, as she did not want to be examined further. Additionally a bifid right thumb, a left renal hypoplasia and a
right mobile kidney were seen. No anal problems were noticed.
For the Family L, the data presented in Table 1 was collected when the family was restudied in 1997, and
includes the original index patient as well as nine other confirmed affected individuals on whom blood samples
were obtained and physical examinations performed (Burke et al. 1997). The patriarch of the family (L.1) had
bilateral hearing loss, anal stenosis, broad thumbs, and renal failure requiring a unilateral renal transplant. He had
three affected daughters. The first daughter (L.2) of the second generation had an anteriorly placed anus, bilateral
lop ears, preauricular tags, bilateral hearing loss and had vesicoureteral reflux that led to bilateral renal transplants.
She had two affected sons (the third generation), the first of which (L.5) had broad thumbs, lop ears, preauricular
tags, and hearing loss. Her other son (L.6) had an imperforate anus, overlapping toes with syndactyly, and
vesicoureteral reflux with a unilateral dysplastic kidney. The second of the three daughters in the second
generation (L.3) had an anterior placed anus, bifid thumbs, a hooked third toe, preauricular tags and bilateral
hearing loss. She had no children. The third daughter (L.4) had a history of bowel blockage, a broad thumb, a
history of multiple renal infections and preauricular tags. This daughter had an affected daughter (L.7) and an
affected son (L.8). Her affected daughter (L.7) had anal stenosis, broad and deviated thumbs, lop ears, preauricular
tags and hearing loss. She had one stillbirth with multiple congenital anomalies and two affected sons (L.9 and
L.10 - the fourth generation), each of whom had anal stenosis, bifid deviated thumbs, lop ears and hearing loss and
one had a dysplastic kidney (L.10). The affected son (L.8 – the second generation) of the third daughter (L.4) had
anal stenosis, broad thumbs, lop ears, preauricular tags, unilateral microtia and hearing loss.
The propositus of family M is a boy, who had mild anal atresia and largish preauricular tags at birth.
Subsequent examinations revealed conductive deafness. Clinical examination additionally showed a deep
preauricular fistula on the left cheek as well as rather broad thumbs. His mental development has been normal at
the time of examination. The propositus mother had bilateral preauricular tags and a left lateral mouth cleft at birth.
She also has camptodactyly and an anteriorly placed anus. The maternal grandfather was reported to have
congenital anal atresia, preauricular tags and bifid thumbs. Similar malformations were seen in his sibs, too.

RESULTS
Mutation analysis was carried out in 13 unrelated families with tentative diagnosis of Townes-Brocks
syndrome. We identified 12 novel mutations (c.764delT (p.L255fs), c.778C>T (p.Q260X), c.940A>T (p.K314X),
c.1028_1029delTA (p.I343fs), c.1124delC (p.S375fs), c.1145_1146insTA (p.L383fs), c.1263delC (p.L422fs),
c.1321dupA (p.T441fs), c.1327delG (p.D443fs), c.1374delT (p.F458fs), c.1404dupG (p.R469fs) and c.1543G>T
(p.E515X)) in exon 2 of SALL1 among the 13 unrelated families (original data have been reviewed but are not
shown). The mutations c.940A>T (p.K314X), c.1028_1029delTA (p.I343fs), c.1124delC (p.S375fs),
c.1145_1146insTA (p.L383fs), c.1263delC (p.L422fs), c.1374delT (p.F458fs) and c1543G>T (p.E515X) were
found in sporadic cases, whereas the mutations c.764delT (p.L255fs), c.778C>T (p.Q260X), c.1321dup
(p.T441fs)A, c.1327delG (p.D443fs) and c.1404dupG (p.R469fs) were detected in familial cases. Both families
Novel SALL1 Mutations 5

carrying the c.1404dupG mutation were reported to originate from Eastern Europe (Ukraine and Belarus),
suggesting a possible relationship. However, haplotype analysis excluded a common ancestor (data not shown). In
family F, the mutation c.1145_1146insTA was neither found in the DNA of the mother’s skin fibroblast nor in the
DNA of her oral mucosa cells although the presence of a short thumb indicated a likely mosaic state for the
mutation.
Interestingly, some of the affected patients showed physical features that were not clearly associated with
Townes-Brocks syndrome as yet. These include hypothyroidism in a male patient, vaginal aplasia with bifid uterus
and a small umbilical hernia in a female patient and cryptorchidism in three patients one of which additionally
showed an umbilical hernia. For details see case reports and Table 1.

DISCUSSION
In combination with the 23 previously known mutations this report increases the number of known SALL1
mutations to 35. 27 (77%) are frameshift mutations, i.e. small deletions <100bp, one bigger deletion (1150bp)
within exon 2, and five short insertions. Seven mutations (20%) are nonsense changes, and one point mutation was
detected within intron 2 creating an aberrant splice acceptor site (Blanck et al., 2000).
Previous findings suggested that most point mutations cluster 5’ of the region encoding the first double zinc
finger (i.e. nucleotides 1351-1497) (Kohlhase et al., 1999; Marlin et al., 1999). Our results reported here confirm
this observation. SALL1 mutations resulting in TBS still seem to occur only in exon 2 and intron 2, and most
(30/35, 85.7%) mutations are found 5’ to or within the region encoding the first double zinc finger. This suggests
either that this region is especially prone to mutations or that mutations further 3’ in the gene result in weaker
phenotypes. However, the known 3’ mutations do not differ significantly from the 5’ mutations with respect to the
phenotypic severity. The positions of the mutations with respect to the SALL1 protein are shown in Fig. 1.
Significant intra- and interfamiliar variability in the clinical presentation of patients with Townes-Brocks
syndrome has been noted in many reports. With the exception of the most common mutation c.826C>T (p.R276X),
which was found in 15 sporadic (one unpublished) and one familial case (Kohlhase et al., 2003), mutation analysis
of SALL1 in TBS has failed as yet to demonstrate a clear genotype-phenotype correlation. The R276X mutation
seems to show less clinical variability than other mutations, in that it is associated predominantly with the classical
TBS phenotype and a possibly more severe outcome due to a higher rate of congenital heart defects. Otherwise,
there is no obvious correlation among the clinical presentation and the nature or location of specific point
mutations except for the mutations c.1277_1278delGA causing a phenotype overlapping with Goldenhar syndrome
(Gabrielli et al., 1993; Kohlhase et al., 1999) or c.967C>T and c.1819delG resulting in a phenotype overlapping
with Branchio-Oto-Renal syndrome (Albrecht et al., 2004; Engels et al., 2000). The mutation c.1404dupG reported
here was found in two independent, unrelated families. As in the family reported by Kurnit et al. (1978), the
affected family members suffer from renal disease, suggesting that this is a true feature caused by the truncated
protein resulting from the mutation. Renal disease should therefore also be associated with the same mutation in
other families. Since all but one of the mutations reported here were found in only one family it remains unclear if
the different phenotypes are actually caused by different mutational effects or are potentially due to epigenetic
factors.
It has been speculated for quite some time that SALL1 mutations cause Townes-Brocks syndrome via
haploinsufficiency (Kohlhase, 2000), as expected from the fact that all but one mutation in TBS patients are
predicting premature termination codons and expected to result in unstable transcripts. This hypothesis was first
questioned by the phenotype of a Sall1 knock out mouse model (Nishinakamura et al., 2001), which has no
phenotype in the heterozygous and shows only isolated kidney defects in the homozygous mutants, but no TBS-
like pattern of malformations. Another animal model mimicking a typical TBS-causing nonsense mutation showed
a TBS-like phenotype already in the heterozygotes and indicated that SALL1 mutations could lead to truncated
proteins (as detected in the mutant) with a dominant negative effect and thereby cause the disease (McLeskey
Kiefer et al., 2003). Therefore, it is quite possible that each mutation has its own specific consequence on the
phenotype, as illustrated for the c.826C>T (p.R276X) mutation (Kohlhase et al., 2003). The novel SALL1
mutations reported here are all truncating and – like the mutations already known - would be expected to lead to
nonsense-mediated decay. Thus, our data presented here do not help to further clarify how SALL1 mutations lead
to the phenotype.
6 Botzenhart et al.

Figure 1. Schematic representation of the SALL1 protein (1324 amino acids) and localization of the mutations identified to
date. Zinc fingers are indicated as oval symbols. SALL1 encodes four C2H2 double zinc finger domains distributed over the
protein. A single C2H2 domain is attached to the second double zinc finger. At the aminoterminus, a single C2HC domain is
found. The horizontal bar indicated with “del” shows the position of combined 1150bp deletion within exon 2. (17) indicates
that the c.826C>T mutation has been found in 15 sporadic and two familial cases. At position c.1115, two different nonsense
mutations have been detected (2), and the mutation c.1404dupG was found in two unrelated families (2). All other mutations
have been found only once. Positions of the introns are indicated. Mutations were numbered according to GenBank
NM_002968.1 (SALL1 mRNA sequence).

Mutation analysis of SALL1 has confirmed that TBS shows complete penetrance, and the families reported here
confirm this assumption. However, while it is known that germline mosaicism has been involved in a number of
reported cases of TBS, it is still unclear how prevalent germline mosaicism is in cases of TBS. Our studies so far
revealed two out of 39 families with proven mutation in which the unaffected mother either carried the mutation in
a detectable mosaic state (Kohlhase et al., 1999) or gave birth to two affected children while the mutation could not
be detected in her DNA from peripheral lymphocytes (Blanck et al., 2000). In the latter, it was demonstrated later
that both children inherited the mutation from the mother (Kohlhase et al., unpublished results). In family F we
were also unable to detect the mutation in the mother on either DNA from peripheral lymphocytes or buccal cells
samples. As she shows a unilateral short thumb, and both thumbs were abnormal on radiological examination, it
may be possible that she carries a germ cell mosaic for the mutation. In another family, the father of an affected
child was found to have the second and fourth toes overriding the third. The mutation causing TBS in his child was
only found in paternal mucosa cells but not in lymphocytes (Devriendt et al., 2002). Further studies are required to
elucidate the true proportion of germline mosaicism as well as a true genotype-phenotype correlation, the latter of
which requires that further mutations will be found in more than one family. Some phenotypic features are
reported here for the first time to our knowledge, that is vaginal aplasia with bifid uterus, cryptorchidism, umbilical
hernia, and bifid scrotum without hypospadia scrotalis. Cryptorchidism seems to be a true feature of SALL1
mutations, being present in two patients within this small group of patients. Dorsal hypoplasia of the corpus
callosum has not been reported before, but might be more commonly observed if TBS patients will be more
carefully investigated for brain anomalies. Hypothyroidism has been reported only once before (Kohlhase et al.,
1999), and the finding in another family makes it a true but rare association, warranting thyroid function testing in
TBS children. A chorioretinal coloboma has also been published in a single case to our knowledge, as well as
Duane anomaly (Kohlhase et al., 1999; Rossmiller and Pasic, 1994). This is especially interesting in view of the
Novel SALL1 Mutations 7

occasional phenotypic overlap between TBS and Okihiro syndrome caused by SALL4 mutations (Kohlhase et al.,
2002), showing that such anomalies are less common but present in TBS patients.

Table 1. Summary of clinical features


Patient Mutation (DNA) Protein Anus Hands Ears Hearing Kidneys IRF Feet UG Heart
Family A
A.1 c.764delT p.L255fs + - + - + + + - -
A.2 c.764delT p.L255fs - + + - - - - - -
Family B
B.1 c.778C>T p.Q260X + + + + - - - + -
B.2 c.778C>T p.Q260X - + - + - - - - -
Family C
C.1 c.940A>T p.K314X + + + - - - + + VSD
Family D
D.1 c.1028_1029delTA p.I343fs + + + + - - - + -
Family E
E.1 c.1124delC p.S375fs + + + - - - + + -
Family F
F.1 c.1145_1146insTA p.L383fs + + + + + + - + -
Family G
G.1 c.1263delC p.L422fs + + + + + - + - -
Family H
H.1 c.1321dupA p.T441fs + + + - + - - + -
Family I
I.1 c.1327delG p.D443fs + + - + + - - - -
I.2 c.1327delG p.D443fs + + + + - - + - -
Family J
J.1 c.1374delT p.F458fs + + + - - - + - -
Family K
K.1 c.1404dupG p.R469fs + + + + + - - + -
K.2 c.1404dupG p.R469fs + + + + + - + + -
K.3 c.1404dupG p.R469fs - + + + + - - - -
Family L
L.1 c.1404dupG p.R469fs + + - + + + - - -
L.2 c.1404dupG p.R469fs + - + - + + - - -
L.3 c.1404dupG p.R469fs + + + + + + + - -
L.4 c.1404dupG p.R469fs + + + - - + - - -
L.5 c.1404dupG p.R469fs - + + + - - - - -
L.6 c.1404dupG p.R469fs + - - - + + + - -
L.7 c.1404dupG p.R469fs + + + + - - - - -
L.8 c.1404dupG p.R469fs + + + + - - + - -
L.9 c.1404dupG p.R469fs + + + + - - + - -
L.10 c.1404dupG p.R469fs + + + + + - - - -
Family M
M.1 c.1543G>T p.E515X + + + + - - - - -
M.2 c.1543G>T p.E515X + + + - - - - - -
M.3 c.1543G>T p.E515X + + + - - - - - -
Summary of phenotypic features in the patients reported here. See case reports for further details. Patients and families are
termed in capital letters refering to the designation used in the case reports. Index patients and further affected family members
are indicated by capital letters and consecutive numbering. Abbreviations: IRF: Impaired renal function; UG: Urogenital
abnormalities; VSD: Ventricular septal defect; “+” or “-“ refer to whether the physical features mentioned above are present or
not. Anomalies occuring in the family L described by Kurnit et al. 1978 are given as “+” if at least one family member shows
the respective feature. Mutations were numbered according to GenBank NM_002968.1 (SALL1 mRNA sequence).
8 Botzenhart et al.

However, we would like to stress that radius hypoplasia or aplasia has never been seen in any of our patients with a
SALL1 mutation, suggesting that the presence of radial involvement argues more strongly in favor of the diagnosis
of Okihiro syndrome rather than TBS. One of the patients reported here has vertebral malformations (the S1
vertebra having the shape of a lumbar vertebra, and 13 pairs of ribs), strengthening the difficult differentiation
from VACTERL in some cases. As with Okihiro syndrome, an X-ray of the forearms is warranted in such
situations. The same child had severe gastroesophageal reflux, and this is now the third family, in which this
problem has been noticed (Albrecht et al., 2004; Engels et al., 2000), making it a true feature of TBS. However, the
anatomical or physiological reason for the reflux disease is not known, but one might speculate that there is either
a problem of innervation or of an anatomical anomaly. Like with constipation, which is often a problem for TBS
patients, more research is needed to understand this feature.

ACKNOWLEDGMENTS
We thank the patients and their families for their cooperation and Manuela Liebers for technical assistance.

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