Nonsense mutations in AAGAB cause punctate palmoplantar keratoderma type Buschke-Fischer-Brauer

Abstract

Punctate palmoplantar keratodermas (PPKPs) are rare autosomal-dominant inherited skin diseases that are characterized by multiple hyperkeratotic plaques distributed on the palms and soles. To date, two different loci in chromosomal regions 15q22-15q24 and 8q24.13-8q24.21 have been reported. Pathogenic mutations, however, have yet to be identified. In order to elucidate the genetic cause of PPKP type Buschke-Fischer-Brauer (PPKP1), we performed exome sequencing in five affected individuals from three families, and we identified in chromosomal region 15q22.33-q23 two heterozygous nonsense mutations-c.370C>T (p.Arg124(∗)) and c.481C>T (p.Arg161(∗))-in AAGAB in all affected individuals. Using immunoblot analysis, we showed that both mutations result in premature termination of translation and truncated protein products. Analyses of mRNA of affected individuals revealed that the disease allele is either not detectable or only detectable at low levels. To assess the consequences of the mutations in skin, we performed immunofluorescence analyses. Notably, the amount of granular staining in the keratinocytes of affected individuals was lower in the cytoplasm but higher around the nucleus than it was in the keratinocytes of control individuals. AAGAB encodes the alpha-and gamma-adaptin-binding protein p34 and might play a role in membrane traffic as a chaperone. The identification of mutations, along with the results from additional studies, defines the genetic basis of PPKP1 and provides evidence that AAGAB plays an important role in skin integrity.

Figure 1

Linkage Intervals and Mutation Analysis of AAGAB (A) In the upper panel, part of chromosome 15 shows the identified linkage intervals and the corresponding flanking microsatellites. The genomic positions of these markers are indicated in Mb. AAGAB resides within the region that was found by Martinez-Mir et al. (2003) and Bchetnia et al. (2009). In the lower panel is the genomic structure of AAGAB; ten exons are indicated as black bars. The noncoding parts of exons 1 and 10 are indicated with smaller bars. (B) In the left panel is the c.481C>T transition in exon 5 from individual 60698 (pedigree B) and individual 61926 (pedigree C). In the right panel is the c.370C>T transition in exon 4 from individual 54187 (pedigree A).

Figure 2

Clinical Picture Disseminated hyperkeratotic papules on palms (A) and soles (B) of a 57-year-old index person of pedigree B.

Figure 3

Pedigrees of Families A, B, and C Pedigrees of families A (A), B (B), and C (C). Unaffected and affected family members are indicated by clear symbols and blackened symbols, respectively. Asterisks denote individuals genotyped for the variants in AAGAB. To maintain confidentiality, we have not shown genotypes of unaffected individuals. Diagonal bars through the symbols denote deceased individuals. Question marks in the symbols denote lacking information about the phenotype.

Figure 4

Immunoblot and Immuofluorescence Analysis of COS7 Cells Transiently Expressing Wild-Type and Mutant AAGAB and Allele-Specific PCR (A) COS7 cells were transiently transfected with V5-tagged AAGAB wild-type or mutant (p.Arg124^∗ or p.Arg161^∗) constructs with the use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and were harvested 30 hr after transfection. After lysis of cells in ripa buffer and sonication, lysates were subjected to electrophoresis and immunoblotting. We used monoclonal V5 antibody (R960-25, Invitrogen, 1:4.000) to detect AAGAB proteins. For normalization, we used a monoclonal GAPDH antibody (G8795, Sigma-Aldrich, 1:16.000). The wild-type AAGAB has a size of around 35 kDa, whereas the two truncated proteins have the anticipated sizes of 14 kDa and 18 kDa. However, we observe slightly larger bands, which might be due to the added tag and to glycosylation or phosphorylation of the protein. (B) Allele-specific PCR was performed with cDNA that was generated from RNA extracted from the blood of affected individuals. We used the same forward primer (5′-ATGATGCTGTGAGATTTTATCCC-3′) for all reactions. The reverse primers used for studying AAGAB expression in individual 54187 were 5′-CACCATTCTTGAGCTTTTTGGCG-3′ for amplifying the wild-type allele and 5′-CACCATTCTTGAGCTTTTTGGCA-3′ for amplifying the mutant c.370C>T allele. The reverse primers used for studying AAGAB expression in individual 60698 were 5′- CATTCAGGGCTTGGACAATGCG-3′ for amplifying the wild-type allele and 5′-CATTCAGGGCTTGGACAATGCA-3′ for amplifying the c.481C>T mutant allele. We readily observed the wild-type allele in both cases, whereas the mutant allele was only present at reduced levels. (C) Sequencing results of cDNA samples of affected individuals with gene-specific AAGAB primers. We could only detect a very small peak for the c.481C>T allele (arrow on the right), and we could not detect the c.370C>T allele at all (arrow on the left), indicating no presence of the respective mutant transcripts. (D) Immunofluorescence analysis of transiently transfected HaCaT cells with the above-mentioned V5 antibody. No differences in staining were observed between cells expressing the wild-type (left) or the mutant (center = mutant p.Arg124^∗; and right = mutant p.Arg161^∗) AAGAB.