American Journal of Medical Genetics 136A:390–394 (2005) Clinical Report Pallister–Hall Syndrome: Unreported Skeletal Features of a GLI3 Mutation T. Roscioli, 1,2 D. Kennedy, 2 J. Cui, 3 B. Fonseca, 4 G.F. Watson, 5 J. Pereira, 6 Y-G. Xie, 3 and D. Mowat 1,7 * 1 South Eastern Sydney Genetics Service, Sydney Children’s Hospital, Sydney, Australia 2 Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital, University of Sydney, Sydney, Australia 3 Disciplines of Laboratory Medicine, Genetics, Pediatrics, Memorial University of Newfoundland, St. John’s, NL, Canada 4 St. George Hospital Paediatric Unit, Sydney, Australia 5 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia 6 Department of Radiology, Sydney Children’s Hospital, Sydney, Australia 7 School of Women’s and Children’s Health, University of New South Wales, Sydney, Australia We describe two patients with Pallister–Hall syndrome (PHS), both with evidence of a general- ized skeletal dysplasia as typified by upper and lower acromesomelic limb shortening and the previously unreported fibular hypoplasia, radio- ulnar bowing, and proximal epiphyseal hypopla- sia. Genomic DNA was only available for sequen- cing analysis in patient 2 and the mutation, c.3386_3387delTT was detected in exon 14 of the GL13 gene. It is also possible that the findings in patient 1 represent the phenotypic expression of a novel GLI3 mutation. This report further expands the PHS phenotype and raises the possibility of specific GLI3 mutations resulting in more severe skeletal features. It also suggests that PHS should be included in the differential diagnosis of antena- tally ascertained acromesomelic limb shortening and bowing with fibular hypoplasia particularly in the presence of polysyndactyly. ß 2005 Wiley-Liss, Inc. KEY WORDS: Pallister–Hall; polydactyly; ham- artoma; acromesomelia; skeletal dysplasia; GLI3; antenatal ultra- sound INTRODUCTION The Pallister–Hall syndrome (PHS) was initially described as the association of hypothalamic hamartoma, post-axial polydactyly, and imperforate anus with the presence of other anomalies [Clarren et al., 1980; Hall et al., 1980]. Review of these and other published patients has enabled diagnostic criteria for PHS to be developed which include the presence of a hypothalamic hamartoma, central polydactyly usually involv- ing polysyndactyly of the third or fourth digits, and the presence of these features in a first-degree relative [Biesecker et al., 1996]. The PHS, Greig Cephalopolysyndactyly (GCPS), Preaxial polydactyly type IV, and polysyndactyly type A (PAP-A) are allelic conditions with mutations having been found in the GLI3 zinc finger protein [Vortkamp et al., 1991; Wild et al., 1997; Kang et al., 1997a; Rhadkrishna et al., 1997b, 1999]. We present two patients with PHS with a generalized skeletal dysplasia. Limb shortening has previously been noted in some reports of PHS [Hall et al., 1980; Graham et al., 1983; Iafolla et al., 1989; Pennman Splitt et al., 1994; Verloes et al., 1995] and also in digenic inheritance in GCPS with GLI3 and COL2A1 mutations [Sobetzko et al., 2000]. The findings presented here however of fibular hypoplasia, radio-ulnar bowing, and proximal epiphyseal hypoplasia are novel. A causative mutation was detected in Patient 2, GLI3 c.3386_3387delTT. MATERIALS AND METHODS Blood samples were only available from Patient 2. Genomic DNA was isolated from the peripheral blood using standard methods. The entire coding sequences including partial flanking intron sequences of GLI3 gene were PCR amplified and subjected to automated sequencing. The PCR amplifica- tion was performed in a reaction volume of 30 ml. Approxi- mately 100–200 ng of genomic DNA was used in each PCR amplification. The DNA amplification was performed in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 200 mM dNTPs (ATP, GTP, CTP, and TTP), 0.05 mM of each primer set (Table I), and 1 unit of AmpliTaq DNA polymerase. 35 PCR cycles were performed with varying annealing temperatures (see the Web Only Supplement Table at http://www.interscience.wiley.com/ jpages/1552-4825/suppmat/index.html). The PCR amplified fragments were sequenced in both sense and anti-sense directions using a CEQ 8000 (Beckman Coulter) automatic sequencer. Resulting DNA sequences were aligned and compared with the published sequence of GLI3 gene (NM_000168 mRNA; NT_007819 genomic DNA; AJ250408 protein) by using computer software, Sequencher (Beckman Coulter). RESULTS Clinical Findings The clinical findings reported here compared with those in the literature are summarized in Table I. The first patient was a This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/ index.html. T. Roscioli’s present address is Department of Clinical and Molecular Genetics, Royal Prince Alfred Hospital, University of Sydney, Missenden Rd, Sydney, Australia. D. Kennedy’s present address is Statewide Medications in Pregnancy and Lactation Advisory Service, Royal Hospital for Women, Sydney, Australia. *Correspondence to: Dr. D. Mowat, Sydney Children’s Hospital Genetics Department, Sydney Children’s Hospital, High St. Randwick, 2031, Australia. E-mail: d.mowat@unsw.edu.au Received 25 February 2005; Accepted 14 April 2005 DOI 10.1002/ajmg.a.30818 ß 2005 Wiley-Liss, Inc.