In this study, we have identified a further 8 families with mutations in the RAB23 gene, nearly doubling the number of different mutant alleles known. These additional patients have enabled us to establish a clearer picture of the phenotypic spectrum in Carpenter syndrome. We provide the first direct evidence that truncating mutations in RAB23 are unstable, suggesting a role for NMD in pathogenesis, and describe the first homozygous mutations in RAB23 that encode single amino acid changes. These mutations are associated with a similar phenotypic spectrum to nonsense mutations, thereby identifying key residues that are essential for protein function.
The cases presented here, together with those previously published by Jenkins et al. 
and Alessandri et al. 
provide an expanded series in which to examine the phenotypic spectrum of Carpenter syndrome. The only universal features are craniosynostosis and soft-tissue syndactly, usually accompanied by insertional/preaxial polydactyly of the feet and often postaxial polydactyly of the hands; cryptorchidism/hypoplastic external genitalia were observed in almost all male cases (Supp. Table S1). Where CT scanning of the skull was performed, fusion of the sagittal suture was observed in all patients, and bicoronal synostosis was almost always present. Whereas all patients in the study by Jenkins et al. 
had high birth weight, the birth weights in the current group of patients were in the normal range, although they did go on to exhibit postnatal obesity. Polysplenia/accessory spleens, a characteristic laterality defect, was previously reported by Alessandri et al. 
in a single patient. One patient in the present study (subject 4080) exhibited this phenotype, confirming this as a specific association with mutation of RAB23
, and suggesting a role for RAB23 in the regulation of left-right patterning.
Greig syndrome, which is caused by heterozygous loss-of-function mutations in GLI3
(MIM# 165240), is also characterized by polysyndactyly, as well as other features seen in Carpenter syndrome, including agenesis of the corpus callosum and cryptorchidism in boys [Johnston et al., 2005
]. Recently, synostosis of the metopic or sagittal sutures has been reported as a rare association with Greig syndrome [Johnston et al., 2010
; Kini et al., 2010
; McDonald-McGinn et al., 2010
], further confusing accurate diagnosis. Indeed, three patients referred to us for RAB23
mutation testing proved instead to be positive for 6.0-8.3 Mb deletions including GLI3
[Hurst et al., 2011
]. Currently, only midline synostosis has been reported in Greig syndrome, so the presence of bicoronal synostosis in almost all patients with Carpenter syndrome, together with other frequent features such as obesity and umbilical hernia, help to distinguish between the two disorders. However, patients with either diagnosis should be considered for both GLI3
The identification of a homozygous missense mutation (p.M12K) and a homozygous amino acid deletion (p.Y79del) in RAB23, which were both associated with fairly typical features of Carpenter syndrome, highlights two residues important for normal biochemical function. Like other Rab proteins, the core structure of RAB23 comprises 6 buried β-strands [Eathiraj et al., 2005
]. Both the p.M12K mutation (Supp. Figure S1A
) and the previously reported p.C85R mutation [Jenkins et al., 2007
], represent non-conservative substitutions from sulfur-containing amino acids located within this core, to charged residues with more bulky side chains. These mutations are therefore likely to disrupt protein folding. The p.Y79del is located within one of the switch domains of RAB23. Both Rab3a and Rab5c contain a triad of hydrophobic residues, including a tyrosine residue in the switch domain, equivalent to Y78 in RAB23, that interact with effector proteins [Merithew et al., 2001
]. The Y78 residue is adjacent to the deleted Y79, which normally points in the opposite direction and is conserved in both Rab3a and Rab5c (Supp. Figure S1B
). Therefore, deletion of the Y79 residue is likely to disrupt interaction of RAB23 with effector proteins. Because patients homozygous for both p.M12K and p.Y79del exhibit the full Carpenter syndrome phenotype, it is likely that these mutations result in complete loss-of-function.
Notwithstanding these considerations, most patients with Carpenter syndrome reported to date are homozygous for truncating (nonsense or frameshift) mutations in RAB23. Interestingly, these truncating mutations seem to be distributed unevenly, being absent in the final third of the protein (). If the C-terminal prenylation motif of RAB23 were essential for function, as in other Rabs, then truncating mutations located anywhere in the gene would be expected to generate null-alleles. Although further mutation screening will be required to confirm that the spectrum of truncating mutations is non-random, this may be explained by the fact that the prenylation motif of RAB23 is atypical (see Introduction). This suggests that alternative mechanisms, other than disruption of prenylation, may also be critical for pathogenesis; NMD represents one such mechanism.
We evaluated NMD by analyzing the relative levels of mutant and wild-type transcripts in individuals heterozygous for RAB23
mutations, and found that transcripts encoding truncated forms of RAB23
are unstable. This was demonstrated both for a splice site mutation (PTC located in the third of six coding exons) and for the most 3′ (and most common) mutation in RAB23
(p.L145X; PTC in fourth coding exon), suggesting that all truncating mutations in Carpenter syndrome are likely to undergo NMD. Because PTCs in the last exon and 3′ part of the penultimate exon of RAB23
encoding residues downstream of p.L145, are expected to escape NMD [Khajavi et al., 2006
], the absence of mutations in this region may also reflect a critical role for NMD in pathogenesis. Although more N-terminally located truncating mutations might be expected to give rise to more severe phenotypes by disrupting more of the functional domains, no genotype-phenotype correlations are apparent with respect to the location of truncating mutations in RAB23
. This evidence also points to an important role for NMD in pathogenesis.