Carpenter syndrome: extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay

Abstract

Carpenter syndrome, a rare autosomal recessive disorder characterized by a combination of craniosynostosis, polysyndactyly, obesity, and other congenital malformations, is caused by mutations in RAB23, encoding a member of the Rab-family of small GTPases. In 15 out of 16 families previously reported, the disease was caused by homozygosity for truncating mutations, and currently only a single missense mutation has been identified in a compound heterozygote. Here, we describe a further 8 independent families comprising 10 affected individuals with Carpenter syndrome, who were positive for mutations in RAB23. We report the first homozygous missense mutation and in-frame deletion, highlighting key residues for RAB23 function, as well as the first splice-site mutation. Multi-suture craniosynostosis and polysyndactyly have been present in all patients described to date, and abnormal external genitalia have been universal in boys. High birth weight was not evident in the current group of patients, but further evidence for laterality defects is reported. No genotype-phenotype correlations are apparent. We provide experimental evidence that transcripts encoding truncating mutations are subject to nonsense-mediated decay, and that this plays an important role in the pathogenesis of many RAB23 mutations. These observations refine the phenotypic spectrum of Carpenter syndrome and offer new insights into molecular pathogenesis.

Figure 1

Mutation spectrum in RAB23. At top, the exon/intron organization of RAB23, with the coding part of the cDNA in black and the untranslated regions (UTRs) in white (only exons 2-7 are shown; alternatively spliced 5′ noncoding exons are omitted). Upright numbering refers to the first nucleotide of each exon, starting from the initiation codon, and italic numbering indicates the length of introns. Below, the functional domains in the protein are depicted according to the key. The location of all human mutations described to date that cause Carpenter syndrome are indicated by arrowheads. Black and white arrowheads refer to mutations reported by Jenkins et al. [2007] and Alessandri et al. [2010], respectively. Grey arrowheads refer to novel alleles reported in the current study, and mutations affecting single amino acids are underlined. The asterisk refers to the fact that the p.R28X mutation was co-inherited with the p.L145X mutation, giving a p.[R28X;L145X] allele. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (http://www.hgvs.org/mutnomen). The initiation codon is codon 1.

Figure 2

Clinical appearance of RAB23 mutation-positive Carpenter syndrome at different ages. (A-C) Subject 4388, pregnancy terminated at 19.5 weeks' gestation. (D-I) Childhood pictures of subjects 4206/7 aged 3 days (D,E), 4203 aged 11 mo (F) and 3 yr (G) and 4119 aged 7 yr (H,I). (J,K,L) Subject 4121 aged 29 years. Note dysmorphic craniofacial appearance (A,D,J) and variable combinations of polydactyly, syndactyly and brachydactyly (B,C,H,I,K,L). However polysyndactyly was absent in subject 4203 homozygous for the p.M12K missense mutation (F,G).

Figure 3

Analysis of transcripts encoded by a splice-site or nonsense mutation. (A) Sequencing of genomic DNA from subject 4154 showing a heterozygous T>G substitution (arrowhead) 3 bp upstream of exon 3. (B) RT-PCR with primers in exons 2 and 4 using cDNA extracted from fresh blood of the father of subject 4154. As well as a wild-type fragment at 336 bp, a second fragment is also observed at 250 bp, which is absent in control cDNA. Note that the smaller fragment is much weaker than the wild-type product. (C) Sequencing of the 250 bp fragment, demonstrating skipping of exon 3 in the cDNA product. (D) Pyrosequencing assay used to measure the relative levels of c.434T>A (p.L145X) mutant and wild-type transcripts. The dispensation order and peaks for comparison are indicated, below which is a table listing the sequence of nucleotides incorporated into mutant and wild-type transcripts, and the position at which each peak is produced (for example T2 indicates that a T is incorporated at the second dispensation). Representative pyrograms are shown using cDNA originating from peripheral blood of a wild-type individual (left) and a parent heterozygous for the mutation (right). Pairs of arrows link peaks (black: wild-type; grey, mutant) used for comparative quantification.