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Townes–Brocks syndrome (TBS, OMIM #104780) is a rare autosomal dominant malformation syndrome characterised by anal, renal, limb, and ear anomalies.1 TBS is caused by mutations in SALL1, a human putative zinc finger transcription factor gene related to the developmental regulator sal of Drosophila melanogaster.2 The SALL1 gene product is a zinc finger protein thought to act as a transcription factor. It contains four highly conserved C2H2 double zinc finger domains that are evenly distributed. A single C2H2 motif is attached to the second domain, and at the amino terminus SALL1 contains a C2HC motif.3 The protein is exclusively found in the nucleus and localises to pericentromeric heterochromatin, acting as a transcriptional repressor.4
Twenty three of 24 SALL1 mutations known to date are located in exon 2.[2, 5–11 (and unpublished results)] These are short insertions and short deletions as well as one large intraexonic deletion, and nonsense mutations, one of which (c.826C→T, Arg276X) was found in seven independent families with sporadic TBS.5–6,12 One mutation within intron 2 creates an aberrant splice site.7 All mutations lead to premature stop codons and have been thought to cause the phenotype via haploinsufficiency. However, neither heterozygous nor homozygous Sall1 knockout mice show the TBS phenotype, but homozygotes die from kidney malformations like those commonly seen in TBS,13 raising the possibility that SALL1 mutations in humans might have a dominant or dominant negative effect.
In a previous report, Marlin et al6 described the nucleotide 826C of the SALL1 cDNA as a mutational hotspot. They noted that the c.826C→T (Arg276X) mutation never occurred in a case of inherited TBS and speculated that this might be explained by infertility in carriers. In our cohort, we detected SALL1 mutations in 24 cases with sporadic TBS. Among these, 11 had the Arg276X mutation; three published5 and eight new cases. Combining these findings together with detection of this mutation in three out of four families with sporadic TBS by Marlin et al6 and the single case reported by Keegan et al,12 the Arg276X mutation was found in 15 out of 29 (51.7%) families with sporadic TBS. However, it was not detected in 20 familial cases with TBS and detectable SALL1 mutation2,5–8,10,11(and unpublished results).
One of the eight new patients with the Arg276X mutation was a 19 year old woman who presented in the 7+2 gestational week of her first pregnancy. She was diagnosed with TBS due to the presence of the following anomalies: preaxial polydactyly and triphalangeal thumbs on the right hand, broad, short thumb on the left, dysplastic ears, anteriorly placed anus, severe sensorineural hearing loss, overlapping toes on the right foot. A combined atrial septum defect/ventricular septum defect had been diagnosed previously. Her parents were unaffected. Molecular analysis of the SALL1 gene was performed as described,5 and the mutation Arg276X was detected in the patient. She was informed of the 50% risk that her child would also be affected with Townes–Brocks syndrome, bearing in mind the fact that this mutation had never been seen in a familial case. The patient opted for prenatal diagnostics. Amniocentesis was performed at 15+1 gestational weeks. Karyotype was normal female (46,XX) on the 300 band level. DNA was prepared from cultured amniocytes by routine procedures, the 5′ part of exon 2 of the SALL1 gene was PCR amplified from this DNA as described,5 and direct sequencing of DNA fragments was performed with primers S2 and TR1.1.5 On both strands, the mutation Arg276X was confirmed. DNA was prepared from the father of the fetus, and genotyping of informative polymorphic markers on DNA of mother, father, and fetus excluded that the result reflected contamination of amniocytes by maternal cells.
The mutation (c.826C→T; Arg276X) is the most common SALL1 mutation in Townes–Brocks syndrome (TBS), being previously detected in seven unrelated patients. We report eight additional cases and provide a genotype–phenotype correlation for this mutation.
Arg276X has been demonstrated as the cause of TBS in 15 of 29 cases of sporadic TBS with detectable SALL1 mutations but has not been observed up to now in familial cases. The absence of the mutation in familial cases of TBS with SALL1 mutations had raised the question of whether the Arg276X mutation results in infertility.
Here we present the first two generation family with the Arg276X mutation. Both the mother and her newborn daughter have typical TBS but the daughter is more severely affected.
Comparison of all known cases with the Arg276X mutation suggests a higher incidence of congenital heart defects (including two cases with tetralogy of Fallot not previously observed with SALL1 mutations) compared with other SALL1 mutations. Infertility may still explain the low frequency of the Arg276X mutation in familial cases but it is clearly not present in all mutation carriers. Reduced genetic fitness of mutation carriers could be another explanation.
At 33+2 gestational weeks the patient was submitted to the hospital because of polyhydramnion and cervix insufficiency. Owing to a pathological cardiotocogram, a primary caesarian section was performed at 34+1 gestational weeks and the child transferred to the intensive care unit. APGAR was 6/8/8, weight 1980 g, head circumference 29.9 cm. The following abnormalities were noted: bilateral preaxial polydactyly with triphalangeal thumbs, bilateral dysplastic ears with preauricular tags, imperforate anus with rectovaginal fistula, bilateral split feet with 1–2 syndactyly and aplasia of the third ray (fig 1A). Cardiac evaluation revealed pulmonary atresia and a ventricular septal defect with right aortic arch (fig 1B). Multiple major aortopulmonary collateral arteries were seen, and aplasia of the central pulmonary arteries was noted. The initial hearing test, renal ultrasound, and urea/creatinine measurements were normal.
This is the first report of a two generation family with TBS due to the SALL1 mutation c.826C→T (R276X). Previously, this mutation had been reported in seven independent families with sporadic TBS, and it has been assumed that the mutation is not inherited because it might not allow fertility, although no data supporting this hypothesis were presented.6 Our results show that infertility is not generally present at least in females with this mutation. Fertility studies in other adult carriers of the R276X mutation would be helpful to further clarify this issue. Even so, this mutation is the most abundant SALL1 mutation in TBS and was found in 15 of 29 families with sporadic TBS. Among these are 24 families (11 with R276X) analysed by our group. Ten of these have already been reported,5,7,9 eight are presented in this report, and six others are unpublished (Kohlhase et al, in preparation). Four sporadic cases (three with R276X) have been described by Marlin et al,6 and a further one with R276X by Keegan et al.12 The phenotype of all patients with the R276X SALL1 mutation is shown in table 1. Except for the child (224/2; table 1), all patients reported here are sporadic cases with unaffected parents, and with the exception of three patients for whom parental samples were unavailable, the mutation R276X was found in the patient’s but not in the parents’ DNA. All sixteen patients (100%) with this mutation show anal abnormalities; among these, 11/16 (68.8%) have imperforate anus. In addition, 15/16 (93.8%) show characteristic thumb malformations, all (100%) have dysplastic ears, 12/16 (75%) have hearing loss of some kind, 2/16 (12.5%) have mental retardation, 8/16 (50%) have renal malformations, 5/16 (31.3%) have impaired renal function, 12/16 (75%) have foot malformations (with four patients showing split feet), 8/16 (50%) have congenital heart defects, and 4/9 (44.4%) males show hypospadias. Notably, two patients reported here show a tetralogy of Fallot.
Comparison of these findings with the incidence of malformations in SALL1 mutation positive cases as reported previously10 indicates that the R276X mutation is associated with a higher rate of anal malformations (17/22 (77%))10 and a lower rate of hearing loss (19/22 (86%))10. Surka et al summarised heart defects in 7/22 patients with SALL1 mutations, including two patients (5/15 and TB66, see table 1) with the R276X mutation. This means that 5/20 (25%) of TBS patients with other SALL1 mutations have congenital heart disease compared with 50% (8/16) with the R276X mutation. However, because of the small sample size this difference does not reach statistical significance (χ2 test). Our comparison of 16 patients shows that in the vast majority of cases the R276X mutation causes the typical TBS phenotype. However, two malformations, tetralogy of Fallot and split feet resulting from missing second or third toes/rays or both, have not been observed with other SALL1 mutations,.
The rare occurrence of this mutation in familial TBS is still unexplained. Our results show that female infertility at least is not generally caused by this mutation. However, infertility cannot be excluded for all carriers, although there is currently no indication that any other SALL1 mutation results in fertility problems. Heart defects seem more common with the R276X mutation, and this is the only SALL1 mutation currently known to be associated with tetralogy of Fallot. Therefore, reduced genetic fitness might be another explanation for the rare familial occurrence of the R276X mutation. The family presented here shows a more severe phenotype in the second generation. However, this is just a single observation, and increased phenotypic severity was also observed in families with other SALL1 mutations.10 Therefore, we cannot show that this is a true anticipation, and we cannot present a hypothesis to explain how such an anticipation might occur. Increasing severity of phenotype in succeeding generations has been observed for several genetic diseases, but an explanation for cases without dynamic mutations is still sought.14
In conclusion, we have presented the first familial case of TBS caused by the most common SALL1 mutation R276X, thereby demonstrating that infertility is not necessarily associated with this mutation. We compared the phenotype of 16 patients with TBS due to the R276X mutation and noticed a higher rate of congenital heart defects compared with patients with other SALL1 mutations. The mutation is the first SALL1 mutation to be associated with tetralogy of Fallot and split feet. This comparison also allows the first genotype–phenotype correlation for the most common SALL1 mutation in TBS.
This study was funded by the Wilhelm-Sander-Stiftung (grant 98.075.2 to J Kohlhase). We thank W Engel for his support and U Sancken for statistical advice.
The first two authors contributed equally to this work.
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