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Identification of a frameshift mutation in the gene TWISTin a family affected with Robinow-Sorauf syndrome
  1. Medizinisches Zentrum für Humangenetik, Philipps-Universität Marburg, Bahnhofstrasse 7a, D-35033 Marburg/Lahn, Germany
  2. Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany
    1. Medizinisches Zentrum für Humangenetik, Philipps-Universität Marburg, Bahnhofstrasse 7a, D-35033 Marburg/Lahn, Germany
    2. Institut für Humangenetik, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany

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      Editor—The original report by Robinow and Sorauf1 described a large family with autosomal dominant craniosynostosis and hallucal duplication. The clinical features include craniosynostosis, plagiocephaly, flat face, hypertelorism, thin, long, and pointed nose, shallow orbits, strabismus, and broad great toes with a duplication of the distal phalanx. This autosomal dominant syndrome is listed as a separate entry in the McKusick catalogue2 (MIM 180750), although it is clinically similar to Saethre-Chotzen syndrome (MIM 101400). The most characteristic additional feature in Robinow-Sorauf syndrome is a bifid or partially duplicated hallux. In the past, the relationship between these conditions has been controversial. Carter et al 3 emphasised the differences in the phenotype in two patients, and considered it as a separate entity in accordance with the report of Robinow and Sorauf.1 In a further report, similar clinical findings were described as Saethre-Chotzen syndrome4 or an unusual form of acrocephalosyndactyly.5 Another phenotypically similar phenotype has been described as Pfeiffer syndrome.6 Bifid distal hallucal phalanges have also been observed in auralcephalosyndactyly syndrome, in which brachycephaly, facial asymmetry, delayed suture closure, and small pinnae were associated with cutaneous syndactyly 4/5 of the feet.7 8 Based on the cytogenetic findings of Reardon and Winter9 involving chromosome 7p21, there is now growing consensus that Robinow-Sorauf syndrome is a variant of Saethre-Chotzen syndrome involving the same gene.

      Recently, mutations in the gene TWIST have been identified in patients with Saethre-Chotzen syndrome.10-12 The gene is localised on chromosome 7p21 and encodes a transcription factor containing a basic helix-loop-helix (bHLH) motif.13 Here we report the identification of a frameshift mutation in TWIST in a family with clinical features of Robinow-Sorauf syndrome. This supports the assumption that Robinow-Sorauf syndrome is an allelic variant of the Saethre-Chotzen syndrome. Other TWISTmutations have been identified in the family originally described by Young and Harper5 12 and in another case described by El Ghouzzi et al.10

      The pedigree of the three generation family is shown in fig 1A. The proband (III.1) was referred for diagnostic evaluation and genetic counselling at the age of 15 months because of craniosynostosis and broad thumbs and halluces. He was born at term after an uneventful pregnancy (weight 3450 g, length 55 cm, head circumference 36 cm). A younger brother is healthy. At the age of 2 days, seizures occurred which were treated with phenobarbital. At the age of 4 months the paediatrician noticed a protruding fontanelle without signs of brain pressure. Our first clinical examination at the age of 15 months showed the following abnormalities: plagiocephaly, downward slanting palpebral fissures and marked bilateral ptosis, a protruding anterior fontanelle, and broad thumbs and halluces with valgus deformity (fig 1B). Head circumference was 47 cm (50th centile). Developmental milestones were normal. Radiological examination and CT scan of the skull showed pansynostosis. X ray of the feet showed a partial duplication of the distal phalanx on both sides (fig 1C). The boy underwent neurosurgical treatment at the age of 40 months (biorbital advancement and reconstruction of the forehead and orbits). At our last clinical examination at the age of 6 years, he was in a good condition and head circumference was 52 cm (50th centile). Intellectual development was normal.

      Figure 1

      (A) Abbreviated pedigree of the family studied. (B) Front view of the face of the proband (III.1) and his mother (II.2). Note bilateral ptosis (II.2 and III.1) and downward slanting palpebral fissures (III.1). (C) X ray of the feet; left mother (II.2), right proband (III.1). Note duplication of the distal phalanx of the halluces.

      The family history showed that the proband’s mother (II.2, fig 1B) had been surgically treated for premature closure of the coronal and sagittal sutures at the age of 3 years. She had similar facial features to her son (downward slanting palpebral fissures, ptosis) and broad big toes in valgus position. The combination of craniosynostosis, facial features, and duplication of the big toes was suggestive of Robinow-Sorauf syndrome. The maternal grandmother was reported to have bilateral ptosis without other signs of the disorder. She refused to participate in molecular studies.

      EDTA blood was obtained from the index patient (III.1) and his mother (II.2) and DNA extracted according to standard procedures. Primers used for the PCR amplification of the coding region of theTWIST gene were those described by Howardet al 11 (TW1f: 5′-GAG GCG CCC CGC TCT TCT CC-3′ and TW1r: 5′-AGC TCC TCG TAA GAC TGC GGA C-3′; amplicon 378 bp) and El Ghouzzi et al 11 (TW2f: 5′-GCA AGC GCG GCA AGA AGT CT-3′ and TW2r: 5′-GGG GTG CAG CGG CGC GGT C-3′; amplicon 461 bp) following their protocols. The resulting PCR products were run on a 1.2% agarose gel, excised from the gel, and the DNA was isolated with a Gel Extraction Kit (Qiagen). Isolated PCR products were sequenced in both directions using an ABI PRISM Dye terminator cycle sequencing ready reaction kit (Perkin Elmer) on a model 373 automated DNA sequencer (Applied Biosystems).13

      Sequence analysis of the patient’s DNA showed a single base insertion (460-461 insA) localised in the second triplet of the helix II domain of the transcription factor gene TWIST. This frameshift mutation leads to a stop at position 864 and elongates the putative protein product by 88 amino acids. This mutation was confirmed by a restriction enzyme digestion of the 461 bp PCR product (TW2f/r), because the insertion generates a new BsaJI restriction site. The normal sized fragments resulting from digestion are 231, 201, and 30 bp (fig 2, lane C). The 231 bp fragment is split into a 190 bp and 41 bp fragment by the mutation. Both mother and son showed the wild type fragments in addition to the aberrant 190 bp DNA fragment (fig 2, lanes 1 and 2). DNA fragments 41 and 30 bp were not resolved by the electrophoretic conditions applied.

      Figure 2

      Confirmation of the frameshift mutation (460-461insA) by gel electrophoresis. DNA fragments generated by BsaJI digestion of the PCR product representing the 3′ portion of the TWIST gene (product size 461 bp10) (lanes 1, 2, C). M: size marker. Mother (II.2, lane 1) and son (III.1, lane 2) showing the mutant DNA fragment of 190 bp in addition to the wild type fragments (231 and 201 bp). C: unaffected subject showing the absence of mutation. DNA fragments 42 and 30 bp were not resolved by electrophoresis.

      In Saethre-Chotzen syndrome, nonsense, missense, duplication, and deletion mutations have been identified in the coding region ofTWIST in familial and isolated cases.15-17 These aberrations occurred in different functional domains of the gene. Most of the known mutations are detected within the DNA binding helix I or loop domain.15 16

      The mutation identified in the family reported here is caused by an insertion of a single adenosine at position 460-461 (460-461insA) which leads to a frameshift. Recently, three additional missense mutations affecting the helix II domain (460A→G, 475C→T, 481C→T) have been described.10 12 15 None of these patients showed hallucal reduplication of the distal phalanx. Furthermore, in the family with Robinow-Sorauf syndrome originally described by Young and Harper,5 a mutation in the coding region ofTWIST has been described.12 In another patient with a mutation in TWIST, a hallucal reduplication was also observed.10

      In conclusion, the mutational spectrum in Saethre-Chotzen/Robinow-Sorauf syndrome does not allow phenotype-genotype correlation. It is not clear whether phenotypic expression is influenced by pleiotropy as suggested by mutantM-twist heterozygous mice.18


      The authors would like to thank Dr Lothar Karolyi for helpful discussion. This work was supported by the Deutsche Forschungsgemeinschaft and the Stiftung P E Kempkes, Marburg, Germany.


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