Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget disease of bone

Abstract

Paget disease of bone (PDB) is a common disorder characterized by focal and disorganized increases of bone turnover. Genetic factors are important in the pathogenesis of PDB. We and others recently mapped the third locus associated with the disorder, PDB3, at 5q35-qter. In the present study, by use of 24 French Canadian families and 112 unrelated subjects with PDB, the PDB3 locus was confined to approximately 300 kb. Within this interval, two disease-related haplotype signatures were observed in 11 families and 18 unrelated patients. This region encoded the ubiquitin-binding protein sequestosome 1 (SQSTM1/p62), which is a candidate gene for PDB because of its association with the NF-kappaB pathway. Screening SQSTM1/p62 for mutations led to the identification of a recurrent nonconservative change (P392L) flanking the ubiquitin-associated domain (UBA) (position 394-440) of the protein that was not present in 291 control individuals. Our data demonstrate that two independent mutational events at the same position in SQSTM1/p62 caused PDB in a high proportion of French Canadian patients.

Figure 1

Haplotype analysis in French Canadian families with PDB (common, DD, PT, CT, and GE families) and allele combinations of informative sporadic cases (UN096, UN054, UN070, and UN010). Markers are indicated on the left, with the 1215C→T mutation and two informative SNPs at position 916 and 976 in the coding region of SQSTM1/p62. The marker order is based on haplotype analysis of 24 families (379 individuals), allowing the lower level of recombination. Shared haplotypes are represented by vertical black bars (PDB3H1) or are boxed (PDB3H2). Because haplotypes of sporadic cases could not be determined, both alleles for heterozygotic markers are depicted.

Figure 2

Physical map of the PDB3 locus at 5q35-qter and genomic organization of SQSTM1/p62. We retrieved a contig of candidate BACs, overlapping the PDB interval from the Human Genome Project Working Draft (Golden Path). We localized Généthon microsatellite markers (Dib et al. 1996), present within this interval, using an ePCR approach (Schuler 1998). Genotyping additional markers using the same approach confirmed order of the BAC clones. A, Chromosome 5. B, BAC contig of the PDB3 linkage region and localization of microsatellite markers. Eight new markers were developed for this study; NL5 and NL25 defined the 300-kb PDB critical interval. The asterisk (*) indicates the NL7–NL5 subcontig being in a different order relative to the Golden Path (August 2001 release). C, Genes present within the PDB3 linkage region. D, Genomic structure of SQSTM1/p62. Exons are represented as boxes and intronic sequences as thin bars. E, cDNA and mutation localization. The segments encoding the Phox and Bem1p domain (PB1), zinc finger ZZ, and UBA domains (Schultz et al. ; SMART Web site) are shown by black boxes.

Figure 3

SQSTM1/p62 mutation P392L. Sequence electrophoregram of a wild-type unaffected subject (top panel) and heterozygous or homozygous patients (middle and lower panels). Nucleotide and predicted amino acid changes are indicated on the left. The arrows point to the mutation. The nucleotide sequences of primers annealing to exon 8–flanking intronic DNA used for amplification and sequencing of exon 8 were as follows: forward, 5′-CACTGTGGCCTGTGAGGAC-3′, and reverse, 5′-CAGTGAGCCTTGGGTCTCG-3′. A full description of the primers used for amplification and sequencing is available at the CHUL Research Center Web site.