Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1

Abstract

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous, autosomal recessive disorder of peroxisomal metabolism that is clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of patients with RCDP, because of their mislocalization to the cytoplasm. We report the mutational spectrum in the PEX7 gene of 78 patients (including five pairs of sibs) clinically and biochemically diagnosed with RCDP type I. We found 22 different mutations, including 18 novel ones. Furthermore, we show by functional analysis that disease severity correlates with PEX7 allele activity: expression of eight different alleles from patients with severe RCDP failed to restore the targeting defect in RCDP fibroblasts, whereas two alleles found only in patients with mild disease complemented the targeting defect upon overexpression. Surprisingly, one of the mild alleles comprises a duplication of nucleotides 45-52, which is predicted to lead to a frameshift at codon 17 and an absence of functional peroxin 7. The ability of this allele to complement the targeting defect in RCDP cells suggests that frame restoration occurs, resulting in full-length functional peroxin 7, which leads to amelioration of the predicted severe phenotype. This was confirmed in vitro by expression of the eight-nucleotide duplication-containing sequence fused in different reading frames to the coding sequence of firefly luciferase in COS cells.

Figure 1

Alignment of peroxin 7 orthologues from four different phyla, represented by human, Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae. Alignment was determined using the Clustal W program (Thompson et al. 1994). Amino acids that are identical and conserved in at least three sequences are indicated in blackened and shaded boxes, respectively. The horizontal black lines underneath the alignment indicate the positions of the six WD repeats. Mutations identified in the patients and affecting the human peroxin 7 sequence are indicated above the alignment as amino acid substitutions (one-letter code), nonsense mutations (*), and frameshift mutations (f). The FDW64–66VALR mutation is indicated (-&-), and the overlined residues represent the 370-369del27nt mutation (deletion of amino acid residues 124–132). For details of these mutations, see table 2.

Figure 2

Functional complementation of PTS2-mediated peroxisomal protein import by PEX7 alleles. Ten different PEX7 alleles identified in the patients were coexpressed with PTS2-tagged GFP to test their ability to restore PTS2-mediated peroxisomal protein import in skin fibroblasts from a patient homozygous for the L292X mutation. A, Expression of control PEX7 resulted in punctate peroxisomal fluorescence in >90% of GFP-expressing cells, with 20%–40% of the cells showing cytosolic fluorescence in addition to punctate fluorescence. B, None of the eight alleles derived from patients with severe RCDP (see Subjects and Methods) were able to complement the PTS2-mediated protein import defect, and PTS2-tagged GFP fluorescence was invariably cytosolic (the L70W allele, which is representative for all other seven alleles, is shown). C and D, Expression of the 8-nt duplication PEX7 allele resulted in punctate peroxisomal fluorescence in 90% of the cells. In 40%–60% of the GFP-expressing cells, however, cytosolic fluorescence and punctate fluorescence were evident, indicating that complementation by this allele is less efficient than complementation by the control allele. E and F, Expression of the H285R PEX7 allele resulted in punctate peroxisomal fluorescence in 50%–70% of GFP-expressing cells, but this was always against a background of cytosolic fluorescence; no cells were found in which fluorescence was exclusively punctate.

Figure 3

In vitro transcription and translation of PEX7 alleles. The same 10 constructs used for the complementation studies were used to express the PEX7 alleles in a T7-coupled reticulocyte lysate expression system. Each allele produced a polypeptide of approximately the expected size, with the exception of the 8-nt duplication allele, which gave no detectable product.

Figure 4

Predicted folding topology of peroxin 7 based on WD-repeat secondary structure features and locations of PEX7 mutations. A, Folding of an individual WD repeat. Each WD repeat is composed of four β-strands named “a,” “b,” “c,” and “d” (arrows) separated by loops and turns (lines), which together make up one blade of a propeller. The top region of a folded WD repeat is defined by the tight turn between β-strands b and c. B, Topology model showing the structural arrangement of the six WD repeats predicted for peroxin 7 and mapping of the various amino acid residues affected by the missense mutations identified in the patients with RCDP. The WD repeats (indicated as WD-1–WD-6) are alternately shaded and blackened and are separated by connecting loops.