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HUMAN MUTATION Mutation in Brief #947(2007) Online


Townes-Brocks Syndrome: Twenty Novel SALL1

Mutations in Sporadic and Familial Cases and
Refinement of the SALL1 Hot Spot Region
Elke M. Botzenhart1, Gabriella Bartalini2, Edward Blair3, Angela F. Brady4, Frances Elmslie5,
Karen L. Chong6, Katie Christy7, Wilfredo Torres-Martinez7, Cesare Danesino8,
Matthew A. Deardorff9, Jean-Pierre Fryns10, Sandrine Marlin11, Sixto Garcia-Minaur4,
Yorck Hellenbroich12, Beverly N. Hay13, Maila Penttinen14, Vandana Shashi15, Paulien Terhal16,
Lionel Van Maldergem17, Margo L. Whiteford18, Elaine Zackai9, and Jürgen Kohlhase1,19*
Institut für Humangenetik und Anthropologie, Universität Freiburg, Freiburg, Germany; 2Department of Pediatrics,
University of Siena, Siena, Italy; 3Department of Clinical Genetics, Churchill Hospital, Headington, Oxford, United
Kingdom; 4Kennedy-Galton Centre, North West Thames Regional Genetics Service, Harrow, United Kingdom; 5South
West Thames Regional Genetics Service, St Georges Hospital Medical School, London, United Kingdom; 6Hospital for
Sick Children, Division of Clinical and Metabolic Genetics, Toronto, Ontario, Canada; 7Indiana University School of
Medicine, Department of Medical and Molecular Genetics, Indianapolis, Indiana; 8Genetica Medica, Universitá di
Pavia, Pavia, Italy; 9Clinical Genetics Center, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania;
Centrum voor menselijke erfelijkheid, Universitaire Ziekenhuizen Leuven, Leuven, Belgium; 11Unite de Genetique
Medicale, Hopital D’enfants Armand Trousseau, Paris, France; 12Institut für Humangenetik, Universitätsklinikum
Schleswig-Holstein, Campus Lübeck, Germany; 13Department of Pediatrics, Umass Memorial Health Care, Worcester,
Massachusetts ; 14Clinical Genetics Unit, Department of Pediatrics, Turku University Central Hospital, Turku, Finland;
Department of Pediatrics, Wake Forest University Health Sciences, Winston-Salem, North Carolina; 16Department of Medical
Genetics, Universitair Medisch Centrum Utrecht, Utrecht, The Netherlands; 17Centre de Genetique Humaine, Institut de
Pathologie et de Genetique, Loverval, Belgium; 18Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospital,
Glasgow, Scotland, United Kingdom; 19Center for Human Genetics, Freiburg, Germany

*Correspondence to: Prof. Dr. J. Kohlhase, Praxis für Humangenetik, Heinrich-von-Stephan-Str. 5, 79100
Freiburg, Germany; Tel.: +49 761 896454-0; Fax: +49 761 896454-9; E-mail: jkohlhase@humangenetik-

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ko1850/7-2.

Communicated by David L. Rimoin

Townes-Brocks syndrome (TBS) is an autosomal dominant malformation syndrome

characterized by renal, anal, ear, and thumb anomalies caused by SALL1 mutations. To date,
36 SALL1 mutations have been described in TBS patients. All but three of those, namely
p.R276X, p.S372X, and c.1404dupG, have been found only in single families thereby preventing
phenotype-genotype correlations. Here we present 20 novel mutations (12 short deletions, five
short duplications, three nonsense mutations) in 20 unrelated families. We delineate the
phenotypes and report previously unknown ocular manifestations, i.e. congenital cataracts with
unilateral microphthalmia. We show that 46 out of the now 56 SALL1 mutations are located
between the coding regions for the glutamine-rich domain mediating SALL protein interactions
and 65 bp 3’ of the coding region for the first double zinc finger domain, narrowing the SALL1
mutational hotspot region to a stretch of 802 bp within exon 2. Of note, only two SALL1
mutations would result in truncated proteins without the glutamine-rich domain, one of which
is reported here. The latter is associated with anal, ear, hand, and renal manifestations,
indicating that the glutamine-rich domain is not required for typical TBS. © 2007 Wiley-Liss,

Received 26 July 2006; accepted revised manuscript 25 September 2006.


DOI: 10.1002/humu.9476
2 Botzenhart et al.


KEY WORDS: SALL1; Townes-Brocks syndrome

Townes-Brocks syndrome (TBS; MIM# 104780) is a rare autosomal dominant 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 36 different SALL1 mutations have been reported in patients
with Townes-Brocks syndrome and overlapping phenotypes, all resulting in premature termination codons
(Botzenhart et al., 2005; Walter et al., 2006). In a previous study (Kohlhase et al., 1999), it was suggested that
most mutations occur in a “hot spot region” 5' to the coding region for the most aminoterminal double zinc finger
domain. Here we report the detection of 20 novel SALL1 mutations and the associated phenotypic features and
present evidence for a refined “hot spot region” in the SALL1 gene.


Venous blood was collected from patients with Townes-Brocks syndrome and unaffected relatives after
obtaining their informed consent.

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
The female patient in family 1 was born following an uneventful pregnancy and delivery. At birth, imperforate
anus with anteposition of the anus, duplication of the left thumb and bifid tragus with bilateral preauricular tags
were seen. Subsequent to surgical correction, the patient suffered from constipation but was otherwise well and
had a normal developmental course. Follow-up investigations revealed a decreased creatinine clearance indicating
impaired renal function.
In family 2, the affected girl was born to a 35 year-old healthy woman. Amniocentesis was performed because
of maternal age and chromosomes were normal (46,XX). The child was born at 39 weeks gestation by standard
vaginal delivery with Apgars 8 and 9, birth weight 2987g and length 51cm. At birth, she was noted to have an
imperforate low anterior positioned anus, which was repaired by transposition anoplasty. Also noted were a left
preauricular tag, left preauricular pit, and small squared left helix. She had slight facial asymmetry noted at 11
months of age. There was a deep sacral dimple but ultrasound examination was normal. She also had a left
triphalangeal bifid thumb with single proximal phalanx and a right trifid thumb with duplicated proximal phalanx.
The carpal bones, radius and ulna were normal bilaterally. Surgery was performed at 1 year of age to remove the
radial duplicate thumb and excision of the proximal interphalangeal joint was performed. Postnatal abdominal
ultrasound revealed left renal agenesis but normal appearance of the right kidney, adrenals, uterus, ovaries and
bladder. She had a transient creatinine elevation perinatally. There was no vesicoureteral reflux. Interestingly,
unilateral renal agenesis was also reported as an isolated finding in one of her cousins. The chest was asymmetric
Novel SALL1 Mutations 3

at birth but radiological investigations were normal. Twelve pairs of ribs and normal vertebrae were identified. At
2.5 years, bilateral sensorineural hearing loss was detected, with the right progressing more than the left. She had a
normal ophthalmology exam at 4 months. She had a minor delay in motor skills but cognitively she was felt to be
appropriate. Postnatal chromosomes were 46,XX, with normal FISH studies for 22q11 and the subtelomeric
The female patient in family 3 was born at 38 weeks of gestation after an uneventful pregnancy. After delivery
she was noted to have imperforate anus. On physical examination, low set ears with overfolded superior helices,
bilateral preauricular tags and bilateral triphalangeal thumbs with hypoplastic thenar eminences were evident.
Heart and kidneys were normal. At age 8 months. growth and developmental milestones were normal. She had
failed a hearing screen on the left side and thus was suspected to have a hearing loss in that ear. Further
audiological evaluation was pending.
The affected boy in family 4 was born at 41 weeks of gestation. At birth, the following congenital anomalies
were noted: imperforate anus and rectal atresia, severe hypospadias, bilateral preaxial polydactyly, right sided
triphalangeal thumb and a left sided bifid thumb. He had dysplastic ears with overfolded helices. Birth parameters
were within the normal range (birth weight 3380g, head circumference 34,1cm). Echocardiography did not reveal
any abnormalities. Renal ultrasound at day 4 showed bilateral small kidneys. Repeat renal ultrasound at the age of
11 months revealed kidney sizes on the 10th percentile. Renal function was normal. No foot malformations were
seen. X-ray examination of the child revealed thirteen ribs on the right side. Developmental milestones at the age
of one year were normal.
In family 5, the affected boy is the second child of healthy non-consanguineous parents. He was noted at birth
to have bilateral duplicated triphalangeal thumbs, dysplastic ears and bilateral 3/4 cutaneous syndactyly of his toes.
He also had anal stenosis requiring anoplasty surgery in the neonatal period. At the age of 4 years, he had further
surgery to correct a right-sided strabismus. His renal function was normal and ultrasound examination of his
urinary tract revealed no abnormality. He had normal hearing but required speech therapy for generalised speech
delay. There was no family history of thumb, ear, renal or anal anomalies but his father also required correction of
a strabismus in childhood.
In family 6 the affected boy (6.1) presented with a left duplicated thumb, three preauricular skin tags and
sensorineural hearing loss. Additionally he had a dermoid cyst of the left eye, a left vesicoureteral reflux with left
ureter reimplantation surgery and was operated on for hypospadias and chordee. He has bilateral pes planus and
bilateral overlapping of his second and third toes. He furthermore has gynecomastia and attention deficit disorder.
The mother of the patient (6.2), who was also shown to carry the SALL1 mutation detected in her son has a right
sided ear tag and a hearing loss which was not further specified.
The male index patient in family 7 (7.1; Fig. 1) was first diagnosed with TBS at the age of four years with a
history of anal atresia treated at birth, profound bilateral sensorineural hearing loss, dysplastic ears, overlapping
toes (II and IV on III) and slightly broad thumbs. At the same time, the diagnosis was also made in his siblings. His
elder sister (7.2) has bilateral sensorineural hearing loss, dysplastic ears, short thumbs and short third toes. Two
other sisters (7.3 and 7.4), identical twin girls, both have anteriorally placed anus, anal stenosis treated at birth,
dysplastic ears, senorineural hearing loss, congenital lamellar cataracts and microphthalmia with the right eye
being smaller than the left eye and overlapping toes (II and IV on III). One of the twins additionally showed facial
asymmetry with the right side of the face being smaller than the left side. She also had short thumbs, a short third
left toe and a small nail on the second right toe. The thumbs of the other twin girl were short and squared off. The
father of the children (7.5; I.1 Fig. 1), who was also found to have the SALL1 mutation, had a long-standing history
of hearing loss. He had short, broad thumbs and short second toes. At the age of 50 years he was found to have
faint lamellar cataracts in both eyes. He also had some dot cortical lens opacities. None of these were visually
The affected boy in family 8 presented at birth with dysplastic ears and a preauricular tag. Audiometry was
performed and showed neither sensorineural nor conductive hearing loss. Furthermore, he had imperforate anus,
preaxial polydactyly of both hands and syndactyly of the second and third toes. No urogenital or cardiac
abnormalities were seen. Chromosomes were normal (46,XY). There was only mild delay of developmental
milestones at the age of one year. The child was able to walk at the age of 15 months. At 2 years and 8 months he
seemed to have a normal mental and normal speech development.
The female patient in family 9 was the second child of healthy parents. At the 18th week of gestation,
oligohydramnios was noted and at the 35th week of gestation abnormal kidneys were diagnosed. She was born at
4 Botzenhart et al.

39 weeks of gestation with birth weight of 2940g and length of 45cm. APGAR scores were 7/8/9. The serum
creatinine was 250μmol/l and Urea was 12,7mmol/l. At birth multiple dysmorphic features were seen including
bilateral dysplastic deep set ears with bilateral preauricular tags, a leftsided preauricular pit, a narrow left auditory
canal, a bifid right thumb and an adducted left thumb. Additionally the patient exhibited left sided retinal coloboma
and bilateral dimples at shoulders, elbows and knees. She had an anteriorly placed anus and hypoplastic kidneys.
She developed a severe feeding difficulty necessitating G-tube placement and was never able to eat orally.
Gastroesophageal reflux and hiatus hernia were noticed and operated on. Hearing loss with delayed onset of speech
was recognized. She developed renal failure and received a renal transplant. After transplantation, pseudotumor
cerebri occurred and was treated with shunt implantation. Despite operative treatment the patient developed
repeated episodes of nausea, hypertonia and cerebral edema. Cerebellar tonsils were suspected to be herniated in
the foramen magnum but this could not be confirmed definitely by MRI. The patient had repeated ear and
nasopharyngeal infections, delayed motor but normal cognitive development.
The index patient of family 10 is a girl who presented at birth with cupped ears, right sided preaxial
polydactyly, bilateral triphalangeal thumbs, a low grade imperforate anus and rectovaginal fistula. Additionally she
showed bilateral mild syndactyly of the 3rd and 4th toes and right sided syndactyly of the 1st and 2nd toes which was
only recognizable on the plantar side of the foot. The toes overall appeared short and the second toes appeared to
be hypoplastic bilaterally. A renal ultrasound at birth revealed normal size and structure of both kidneys. Another
ultrasound examination at the age of 9 months however showed small echogenic kidneys. The child had a small
ASD secundum type, which had resolved at 1 year of age. She had gastroesophageal reflux managed by
medication and chronic constipation managed by diet. She had poor weight gain. At age 9 months she showed
normal mental development. She began walking at 14 months of age. At 20 months of age she had bilateral hearing
aids. A cranial CT at 21 months demonstrated a choroid plexus cyst in the left lateral ventricle already seen by
ultrasound imaging done as a neonate. An evaluation of her development at 20 months placed her at an 18-month
level for motor skills. An evaluation by a speech and language team at 21 months indicated she was at the 17
month level for communications kills. A repeat evaluation by Pediatric Endocrine at 21 months notes her height to
be at the 6th percentile, her weight to be at the 1st percentile, and that she has had normal interval growth. An IGF-
1 (insulin-like growth factor 1) was low normal previously but was well within the normal range at 21 months. Her
PTH (parathyroid hormone) and thyroxine levels were also normal. A repeat renal ultrasound again showed small
echogenic kidneys but they have had interval growth. Her creatinine and BUN remain mildly elevated. She has had
normal blood pressure measurements.
The index patient of family 11 presented at the age of four months with a history of anal stenosis and a mucous
crest along the anal-scrotal line at birth. He exhibited unilateral cryptorchidism and unilateral renal agenesis. Other
features suggestive of TBS were bilateral triphalangeal thumbs, bilateral overriding of the fourth on the third toes
and mildly dysplastic ears. Head circumference was on the 10th percentile and length at the 50th percentile.
Chromosome analysis showed a normal male karyotype.
The female patient in family 12 presented with outer ear malformations, bilateral triphalangeal thumbs,
imperforate anus and renal failure with mild mental retardation.
The index patient of family 13 is a boy, who presented at birth with anal ectopy and stenosis, bilateral
triphalangeal thumbs and small dysplastic ears. Renal ultrasound showed a normal right kidney and a left kidney in
pelvic position. At the age of 3 months BERA-test revealed a mild left-sided hearing loss (15 db) and moderate
hearing loss (40 db) on the right side, with normal MRI of the brain and inner ear. At the age of 2.5 years
psychomotor development is normal. His mother has small ears with mild hearing loss and bilateral thumb
hypoplasia. She has two normal younger sisters and normal parents.
In family 14, a female patient was born to non-consanguineous parents and at birth several congenital
abnormalities were recognized including imperforate anus with recto-vaginal fistula, a triphalangeal thumb, and
preaxial polydactyly on the right side. She was felt to be dysmorphic with mild hypotelorism and small retroverted
ears. At five years mild mental retardation and mild perceptive hearing loss, especially in the high tones were
recognized. On both feet, the toes are long, and the second toe is overlapping the third, Her elder sister died shortly
after birth. She had a short neck, malformed ears with absence of the external auditory canal, absence of both
thumbs and malformed radii. Esophageal atresia, tetralogy of Fallot and unilateral dysplastic kidney were also
seen. Chromosome analysis was normal.
The affected girl in family 15 was the product of the second pregnancy of healthy normal parents. A first
pregnancy ended in a first trimester spontaneous miscarriage. Birth weight was 3560 g, length was 50 cm and head
Novel SALL1 Mutations 5

circumference was 35 cm. She was noted at birth to have a duplicated right thumb, a broad left thumb with normal
terminal phalanx on X-ray, a right preauricular appendix on the right side, everted, simple ears, an anteriorly
placed anus, a deep presacral dimple and complete cutaneous syndactyly of toes III-IV of the right foot. Renal and
cardiac ultrasound examinations were normal and hearing assessment was normal at the age of 2 years.
Psychomotor development was mildly retarded: Developmental level of 18 months at the age of 26 months and of
30 months at the age of 38 months (Bayley scales of development). At the age of 3 years she was integrated in a
normal preschool.
In family 16, the father and his two daughters were shown to carry a SALL1 mutation. The first daughter (16.1)
was born with imperforate and anteriorly placed anus and hypoplastic kidneys. Later on vesicoureteral reflux was
demonstrated. She had dysplastic ears with overfolded helices and broad thumbs. Her younger sister (16.2)
presented with anteriorly placed anus, hypoplastic kidneys and vesicoureteral reflux. She had large ears and a
broad triphalangeal thumb. Information on the father’s phenotype was not available.
The affected girl in family 17 showed anteposition of the anus and a unilateral preauricular tag with normal
The index patient of family 18 (18.1) was born at term as the second child of the family after an uneventful
pregnancy. His birth parameters were in the normal range (birth weight 3960g,). On neonatal examination he was
noted to have an imperforate anus, a heart murmur and dysplastic ears. The anal malformation consisted of an anal
membrane that was corrected surgically without any further problems. Cardiologic evaluation revealed a PDA and
a small VSD which closed spontaneously at three years of age. A hearing assessment at the age of 12 months was
reportedly normal but further assessment at 5.5 years revealed mild bilateral sensorineural hearing loss. On
physical examination both thumbs and great toes were rather broad, with clinodactyly and overlapping of the
second and third toes and pes planus. Chromosome analysis showed no abnormalities. The father of the patient
(18.2) was born with a duplicated right thumb. His left thumb and great toes were rather broad. He underwent anal
dilatation procedures as a young infant. He has mild to moderate hearing loss corrected with hearing aids.
The index patient of family 19 (19.1) was born at 27 weeks of gestation and subsequently developed
intracerebral haemorrhage. He was noted at birth to have anal atresia, dysplastic ears with overfolded helices,
hypospadias, vesicoureteral reflux and bilateral renal dysplasia. Additionally, he exhibited a right sided club foot
and severe psychomotor retardation. His younger brother (19.2) had a right sided ear tag, anal stenosis and
clubfeet. The mother of both children (19.3) had a duplicated distal phalanx of the right thumb and an anteriorly
positioned anus with anal stenosis.
The index patient of family 20 (20.1) presented at birth with right and left sided preaxial polydactyly and a left
sided triphalangeal thumb. He had imperforate anus and malformed ears. There were no other abnormalities seen
in this patient. The patient’s brother (20.2) had a duplicated right thumb and a triphalangeal left thumb. He had a
sacral dimple, imperforate anus, kidney length asymmetry, bilateral overfolded ear helices and asymmetric nipple
placement. The mother of both children (20.3) was reported to have extra bones in both thumbs and has hearing
difficulties. A maternal uncle of the index patient (20.4) was reported to have an overfolded ear helix. A cousin of
the index patient (20.5) and son of 20.4 was reported to have club feet, unspecified anal problems and unspecified
ear abnormalities. Another cousin of the index patient (20.6) was reported to have unspecified ear problems. The
maternal grandfather of the index patient (20.7) was reported to have hearing difficulties and the maternal great
grandfather (20.8) was also reported to have unspecified hearing problems.


In the index patients and other affected members of the families mentioned above, we identified 20 novel
SALL1 mutations in exon 2 of SALL1 among the 20 unrelated families (Fig. 2). The mutations c.313delA,
c.814C>T, c.901C>T, c.995delC, c.1045dupA, c.1084_1085delGC, c.1119_1197del79, c.1134delT,
c.1174_1175delCT, c.1228G>T, c.1415_1425del11, c.1503_1504delGA, and c.2256delC were found in sporadic
cases, whereas the mutations c.1047dupC, c.1061dupA, c.1273delC, c.1516_1517dupAT, c.3019_3032dup14,
c.3249_3255del7 and c.3414_3415delAT were detected in familial cases.
6 Botzenhart et al.

Table 1: Summary of Clinical Features in Mutation Positive Patients

Patient Mutation Anus Hand Ears Hearing Kidneys IRF Feet UG Heart
1 c.313delA + + + - - + - - -
2 c.814C>T + + + + + + - + -
3 c.901C>T + + + + - - - - -
4 c.995delC + + + - + - - + -
5 c.1045dupA + + + - - - + - -
6.1 c.1047dupC - + + + - - + + -
6.2 c.1047dupC - - + + - - - - -
7.1 c.1061dupA + + + + - - + - -
7.2 c.1061dupA - + + + - - + - -
7.3 c.1061dupA + + + + - - + - -
7.4 c.1061dupA + + + + - - + - -
7.5 c.1061dupA - + - + - - + - -
8 c.1084_1085delGC + + + - - - + - -
9 c.1119_1197del79 + + + + + + - - -
10 c.1134delT + + + + + + + - +
11 c.1174_1175delCT + + + - + - + + -
12 c.1228G>T + + + - - + - - -
13.1 c.1273delC + + + + + - - - -
13.2 c.1273delC - + + + - - - - -
14 c.1415_1425del11 + + + + - - + - -
15 c.1503_1504delGA + + + - - - + - -
16.1 c.1516_1517dupAT + + + - + - - + -
16.2 c.1516_1517dupAT + + + - + - - + -
17 c.2256delC + - + - - - - - -
18.1 c.3019_3032dup14 + + + + - - + - +
18.2 c.3019_3032dup14 + + - + - - + - -
19.1 c.3249_3255del7 + - + - + - + + -
19.2 c.3249_3255del7 + - + - - - + - -
19.3 c.3249_3255del7 + + - - - - - - -
20.1 c.3414_3415delAT + + + - - - - - -
20.2 c.3414_3415delAT + + + - + - - - -
20.3 c.3414_3415delAT - + - + - - - - -

Summary of phenotypic features in the mutation positive patients reported here. See case reports for further details. Patients and
families are indicated by numbers referring to the designation used in the case reports. Index patients and further affected
family members are indicated by number and consecutive numbering after the decimal. Abbreviations: IRF: Impaired renal
function; UG: Urogenital abnormalities; “+” or “-” indicate whether the physical features mentioned above are present or not.
Mutations were numbered according to GenBank NM_002968.1 (SALL1 mRNA sequence). Mutations were numbered
according to GenBank NM_002968.1 (SALL1 mRNA sequence). Numbering is from the A of the ATG initiation codon.

Figure 1. Pedigree of family 7. Note that the affected identical twins II.2 and II.3 both
showed congenital lamellar cataracts and microphthalmia, whereas I.1 was found to have
faint lamellar cataracts at the age of 50 years. No eye involvement was noted in the other
family members.
Novel SALL1 Mutations 7

Together with the 36 previously known mutations this report tallies to 56 known SALL1 mutations (Fig. 2;
Table 2). 43 (77%) are frameshift mutations, i.e. small deletions <100bp, one bigger deletion (1150bp) within exon
2, three short insertions and seven short duplications. 12 mutations (21%) are nonsense changes. One point
mutation was detected within intron 2 creating an aberrant splice acceptor site (Blanck et al., 2000).
In a previous report it was noted that most mutations cluster in exon 2 5’ of the region encoding the first double
zinc finger (i.e. nucleotides 1351-1497), and the region was therefore described as a “hot spot region” (Kohlhase et
al., 1999). A closer analysis of the now available mutational spectrum reveals that 42 mutations (75%) are
positioned within this region. Interestingly, five additional mutations were found 3’ to this region between
nucleotides 1498 and 1565, but the next mutation is situated further 253 bp 3’. Based on the presumed importance
of the glutamine-rich interaction domain encoded by nucleotides 687-750, which was found to mediate interaction
of truncated csal1 (chick SALL1 homologue) with other csal proteins (Sweetman et al., 2003), it appears that
mutations 5’ of this region are also rarely detected. Taking this into account, we suggest that the mutational “hot
spot region” should be refined to the region between nucleotide 764, just 3’ of nucleotides 687-750 that encode the
glutamine-rich interaction domain, and nucleotide 1565, 69 bp 3’ of the region encoding the most aminoterminal
double zinc finger domain. This region of 802 bp contains 45 of 56 (80%) SALL1 mutations.
The glutamine-rich domain would be present in nearly all of the truncated proteins possibly resulting from
mutations in SALL1. Experimental evidence from a mouse carrying a TBS-typical truncating SALL1 mutation
suggested that truncated SALL proteins could achieve a dominant negative effect by heterodimerization with wild
type SALL protein through the glutamine-rich domain, leading to disruption of target gene regulation by altering
cellular localization of wild type SALL1 or other SALL proteins (McLeskey Kiefer et al., 2003). In contrast, the
mutations c.313delA and c.419delC, localized 5’ of the glutamine-rich domain, would both lead to truncated
proteins of 181 amino acids in length without the glutamine-rich domain. Instead, both would contain a poly-
alanine tract. The mutation c.419delC has been proposed to encode a shorter protein that is also able to interact
with other SALL proteins albeit to a much lower affinity despite of the missing glutamine-rich domain (McLeskey
Kiefer et al., 2003). The same should apply for the mutation c.313delA. Further experiments will be necessary to
elucidate the different molecular effects leading to TBS in humans.
With respect to the phenotype it is noteworthy that congenital lamellar cataracts and unilateral microphthalmia
as seen in the monozygous twins and their father of family 7 have not been reported in TBS. Ocular anomalies in
Townes-Brocks syndrome are not common but have been detected in a few patients. The eye findings previously
described in Townes-Brocks patients include iris and chorioretinal coloboma observed in a single case (Kohlhase
et al., 1999; Rossmiller and Pasic, 1994), the daughter of whom had Duane anomaly, and in another patient
presenting with ipsilateral loss of vision and bilateral Brushfield spots (Botzenhart et al., 2005). Furthermore, optic
nerve atrophy was reported in one patient (Blanck et al., 2000). The coincidence of chorioretinal coloboma and
Duane anomaly in the same family is especially interesting with regard to the phenotypic overlap between TBS
and Okihiro/ acro-renal-ocular syndrome caused by SALL4 mutations (Kohlhase et al., 2002). Structural eye
anomalies of various kinds can be caused by SALL4 mutations (Kohlhase et al., 2003), and it is important to note
that truncated SALL1 proteins alter the intracellular localization of full length SALL4 (Sakaki-Yumoto et al.,
2006), possibly explaining that SALL1 mutations may lead to eye anomalies by reducing SALL4 protein at its site
of action. However, Sall1 is strongly expressed in the developing mouse eye (Buck et al., 2001). Therefore, we
cannot rule out that eye anomalies can also be caused by SALL1 haploinsufficiency or that a reduction of
functional SALL1 protein itself contributes to the phenotype. Since structural eye anomalies are rare in TBS and
more common (but still rare) in Okihiro syndrome, it seems that either different mutations affect SALL4 in
different ways and/or that other factors modify this phenotypic feature.
We therefore would interprete the eye findings in the above mentioned family as a result of the identified
SALL1 mutation, and our data suggests that since ocular anomalies like cataract, colobomata or microphthalmia
occur in a percentage of TBS patients, a careful ophthalmologic examination of children and adults with TBS is
recommended. However, we cannot exclude that the lamellar cataracts in this family are due to a mutation in a
gene causing autosomal dominant cataracts segregating together with the SALL1 mutation.
8 Botzenhart et al.

Figure 2. Schematic representation of the SALL1 protein (1324 amino acids) and localization of the mutations identified to
date (previously reported mutations are indicated with upward symbols, mutations described here with downward symbols).
Zinc fingers are indicated as ovals. (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.1403_1404insG was
found in two unrelated families (2). All other mutations have been found only once. The red horizontal bar marks the refined
“hot spot region”, the blue bar assigns the glutamine rich domain. Positions of the introns are indicated.

Rib anomalies in Townes-Brocks syndrome like the aforementioned hypoplasia of the left costal cartilage and
additional ribs were described only once before to our knowledge (Botzenhart et al., 2005). In that case, a bilateral
additional pair of ribs as well as vertebral malformations were seen. Thus rib anomalies seem to be a rare finding
in TBS but may complicate the differentiation from VACTERL (MIM# 192350) in some cases especially if
additional vertebral malformations occur. Considering all the phenotypical information on TBS patients with
proven SALL1 mutations known to date it has to be stated that in addition to classical TBS findings consisting of
renal, anal, ear and thumb anomalies there are additional rare phenotypic features which are associated with TBS
and should be addressed by the attending physician. These include ocular abnormalities, hypothyroidism,
urogenital malformations, rib and vertebral malformations and abdominal hernia. As the majority of TBS patients
known to date were seen and diagnosed in early childhood, careful physical examination and long term follow up
will be necessary to gain more detailed information about the long term come outcome of these patients.

We thank the patients and their families for their cooperation, and Bernd Rösler and Tanja Velten for expert
technical assistance.
Novel SALL1 Mutations 9

Table 2. SALL1 Mutations

Base change Amino acid change Position Phenotypea Reference
c.313delA p.T105fs Exon 2 TBS This article
c.419delC p.S141fs Exon 2 TBS Kohlhase et al. 1999
c.764delT p.L255fs Exon 2 TBS Botzenhart et al. 2005
c.778C>T p.Q260X Exon 2 TBS Botzenhart et al. 2005
c.792_793delGC p.L264fs Exon 2 TBS Surka et al. 2001
c.814C>T p.Q272X Exon 2 TBS This article
c.817delG p.G273fs Exon 2 TBS Salerno et al., 2000
c.826C>T p.R267X Exon 2 TBS Kohlhase et al. 1999
c.840delC p.L282fs Exon 2 TBS Blanck et al., 2000
c.901C>T p.Q301X Exon 2 TBS This article
c.940A>T p.K314X Exon 2 TBS Botzenhart et al. 2005
c.967C>T p.Q323X Exon 2 Bor syndrome like Albrecht et al., 2004
c.995delC p.P332fs Exon 2 TBS This article
c.1028_1029delTA p.I343fs Exon 2 TBS Botzenhart et al. 2005
c.1045dupA p.T349fs Exon 2 TBS This article
c.1047dupC p.T350fs Exon 2 TBS This article
c.1061dupA p.V356fs Exon 2 TBS This article
c.1084_1085delGC p.A362fs Exon 2 TBS This article
c.1115C>A p.S372X Exon 2 TBS Kohlhase et al. 1999
c.1115C>G p.S372X Exon 2 TBS Kohlhase et al. 1998
c.1119_1197del79 p.S375fs Exon 2 TBS This article
c.1124delC p.S375fs Exon 2 TBS Botzenhart et al. 2005
c.1134delT p.F387fs Exon 2 TBS This article
c.1145_1146insTA p.L383fs Exon 2 TBS Botzenhart et al. 2005
c.1146delT p.L383fs Exon 2 TBS Kohlhase et al. 1999
c.1174_1175delCT p.L392fs Exon 2 TBS This article
c.1200_1206del7 p.V401fs Exon 2 TBS Kohlhase et al. 1999
c.1228G>T p.G410X Exon 2 TBS This article
c.1255delT p.L419fs Exon 2 TBS Devriendt et al., 2002
c.1263delC p.L422fs Exon 2 TBS Botzenhart et al. 2005
c.1268delC p.Q424fs Exon 2 TBS Kohlhase et al. 1998
c.1273delC p.Q425fs Exon 2 TBS This article
c.1277_1278delGA p.R426fs Exon 2 TBS/Goldenhar Kohlhase et al. 1999
c.1291_1300del10 p.P431fs Exon 2 TBS Kohlhase et al. 1999
c.1321dupA p.T441fs Exon 2 TBS Botzenhart et al. 2005
c.1327delG p.D443fs Exon 2 TBS Botzenhart et al. 2005
c.1347_1348delCA p.H449fs Exon 2 TBS Marlin et al. 1999
c.1374delT p.F458fs Exon 2 TBS Botzenhart et al. 2005
c.1404dupG p.R469fs Exon 2 TBS Botzenhart et al. 2005
c.1411_1412insA p.H471fs Exon 2 TBS Marlin et al. 1999
c.1415_1425del11 p.G473fs Exon 2 TBS This article
c.1479_1480insAG p.V494fs Exon 2 TBS Marlin et al. 1999
c.1503_1504delGA p.K500fs Exon 2 TBS This article
c.1509C>A p.Y503X Exon 2 TBS Blanck et al. 2000
c.1516_1517dupAT p.Q507fs Exon 2 TBS This article
c.1543G>T p.E515X Exon 2 TBS Botzenhart et al. 2005
c.1565delC p.T522fs Exon 2 TBS Marlin et al., 1999
c.1819delG p.L608fs Exon 2 BOR-like Engels et al., 2000
c.1966_1975del10 p.T656fs Exon 2 TBS Marlin et al. 1999
c.1487_2048del562 p.Q497fs Exon 2 TBS Marlin et al. 1999
c.2256delC p.Y753fs Exon 2 TBS This article
c.2779C>T p.Q927X Exon 2 TBS Walter et al. 2006
c.3019_3032dup14 p.C1012fs Exon 2 TBS This article
c.3249_3255del7 p.D1085fs Exon 2 TBS This article
c.3414_3415delAT p.c1139fs Exon 2 TBS This article
IVS2-19T p.V1179fs Intron 2 TBS Blanck et al. 2000
Phenotype: TBS, Townes Brocks syndrome; BOR, Branchio-Oto-Renal syndrome.
10 Botzenhart et al.

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Novel SALL1 Mutations 11

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