The authors have no conflict of interest
Two Novel Mutations at Exon 8 of the Sequestosome 1 (SQSTM1) Gene in an Italian Series of Patients Affected by Paget's Disease of Bone (PDB)†
Article first published online: 2 FEB 2004
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 6, pages 1013–1017, June 2004
How to Cite
Falchetti, A., Di Stefano, M., Marini, F., Del Monte, F., Mavilia, C., Strigoli, D., De Feo, M. L., Isaia, G., Masi, L., Amedei, A., Cioppi, F., Ghinoi, V., Bongi, S. M., Di Fede, G., Sferrazza, C., Rini, G. B., Melchiorre, D., Matucci-Cerinic, M. and Brandi, M. L. (2004), Two Novel Mutations at Exon 8 of the Sequestosome 1 (SQSTM1) Gene in an Italian Series of Patients Affected by Paget's Disease of Bone (PDB). J Bone Miner Res, 19: 1013–1017. doi: 10.1359/JBMR.040203
- Issue published online: 2 DEC 2009
- Article first published online: 2 FEB 2004
- Manuscript Accepted: 2 FEB 2004
- Manuscript Revised: 20 NOV 2003
- Manuscript Received: 5 SEP 2003
- Paget's disease;
- metabolic bone disease;
- genetic research;
- sequestosome gene;
- genetic test;
- mutational analysis
PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Paget's in patients of French Canadian and British descent. Mutational analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder.
Introduction: Paget's disease of bone (PDB) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age. The disorder is genetically heterogeneous, and at present, there is scientific evidence that at least eight different human chromosomal loci are correlated with its pathogenesis. Mutations of the sequestosome1 (SQSTM1) gene were identified as responsible for most of the sporadic and familial forms of Paget in patients of French Canadian and British descent. Such mutations were located at exon 7 and 8 levels, encoding for the ubiquitin protein-binding domain (UBA) and representing a mutational hot spot area.
Materials and Methods: To verify the involvement of this gene in Italian subjects affected by PDB, we performed mutational analysis in 62 sporadic PDB cases.
Results: We described three different mutations at exon 8 level: P392L, already described in the French Canadian population and families predominantly of British descendent, and two novel mutations consisting of the amino acid substitutions M404V and G425R. No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without.
Conclusions: Even though our findings suggest a minor involvement of the SQSTM1 gene in the pathogenesis of sporadic Italian Paget's cases, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlations. Our findings confirm the evidence of a clustered mutation area at this level in this disorder.
PAGET'S DISEASE OF bone (PDB; MIM 167250, 602080) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age.(1) PDB mostly runs asymptomatically, although increased bone turnover can be present, and in ∼30% of patients, bone abnormalities, such as bone pain and deformities, pathological fractures, and deafness may occur. PDB is a genetically heterogeneous disorder, and evidence of genetic influence in its pathogenesis has been described. At least eight different human chromosomal loci have been correlated to PDB.(2–17) In particular, the PDB3 locus in chromosome 5q35-qter hosts the sequestosome1 (SQSTM1) gene, whose mutations account for most of the sporadic and familial forms of PDB reported in the literature.(16,18,19) The SQSTM1 gene encodes the SQSTM1/p62 protein, a component of the NF-kB signaling pathway that mediates intracellular signaling from interleukin (IL)-1/TNFα toward NF-kB,(20–22) crucial for osteoclast differentiation and activity.(23) Exons 7 and 8 DNA sequence account for the ubiquitin protein-binding domain (UBA) and represent a mutational hot spot area.(18) Exon 8 encompasses the stop codon at position 1363 and the entire 3′UTR. An abnormal UBA region could account for the inability to bind to ubiquitin with consequential accumulation of sequestosome protein.(24) Moreover, recently, a reduction of p62 levels by sequestration into aggregates in neuronal cells has been demonstrated to lead to neuronal dysfunction.(25) The first evidence that the SQSTM1 gene is involved in PDB pathogenesis was provided by a study of French Canadian PDB patients identifying the P392L mutation at exon 8.(16) The same mutation has been also reported by Hocking et al.,(18) together with two different mutations of SQSTM1 in 18 PDB families predominantly of British descendent: exon 8 P392L mutation in 13 families (19.1%), exon 8 T insertion at position 396 in 4 families (5.8%), and a splice donor site mutation in intron 7 in 1 family (1.5%). Moreover, the P392L mutation has been reported in 8.9% of the sporadic PDB cases.(18)
More recently, three novel mutations have also been reported in four of five PDB families in the United States:1210delT and 1215delC, both consisting in a premature stop codon at amino acid 394 in exon 8 and a C to T transversion (P387L) at exon 7.(19)
To verify the involvement of this gene in Italian PDB cases, we performed mutational analysis of exons 7 and 8 of the SQSTM1 gene in 62 sporadic PDB patients from Northern, Central, and Southern Italy.
MATERIALS AND METHODS
Thirty-six males (age, 43-89 years) and 26 females (age, 43-82 years) were clinically evaluated by biochemical and imaging tests (the latter consisting of both X-rays and total body bone scintigraphy) in various clinical Centers, following a common protocol. Thirty-seven cases exhibited a monostotic involvement (19 females and 18 males; age, 43-80 years), whereas 25 cases had polyostotic localizations of disease (8 females and 17 males; age, 54-89 years).
After administration of an informed consent form, peripheral blood was obtained, and genomic DNA was extracted from peripheral blood leukocytes using a microvolume extraction method (QIAamp DNA Mini Kit; Qiagen, Hilden, Germany), according to the manufacturer's instructions. The procedures were also performed in 100 healthy control subjects.
Exons 7 and 8 of the SQSTM1 gene were amplified by PCR (I-Cycler; Bio-Rad Laboratories, Milan, Italy) using, respectively, two pairs of primers located in the flanking introns: 5′-GACTGTCTGCCAGGAGCC-3′/5′-CCCTGCAGTGGAGAACATCT-3′ for exon 7 and 5′-CAGTGTGGCCTGTGAGGAC-3′/5′-CAGTGAGCCTTGGGTCTCG-3′ for exon 8. For each patient, we used 0.1 μg of DNA in a final buffer volume of 50 μl [67 mM Tris-HCl, 16.6 mM (NH4)SO4, 0.01% Tween-20, 1.5 mM MgCl2, 0.2 mM deoxyribonucleotides, 0.2 μM of each primer, and 1 U of Polytaq (Polymed, Florence, Italy)]. Thirty PCR cycles were performed: 94°C for 30 s, 58°C for 30 s, and 72°C for 1 minute for exon 7, and 94°C for 30 s, 55°C for 30 s, and 72°C for 1 minute for exon 8. A first denaturing cycle at 94°C for 3 minutes was common for amplification of both exons. A final extension cycle of 5 minutes at 72°C was performed.
PCR products were tested by 2% ethidium bromide-stained agarose gel electrophoresis, purified using the High Pure PCR Product Purification Kit (Roche, Indianapolis, IN, USA), and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing reaction consisted of 25 repeated cycles of denaturation for 10 s at 96°C, annealing for 5 s at 58°C for exon 7 and 55°C for exon 8, and extension for 2 minutes at 60°C. The sequencing products were purified with DyeEx 2.0 Spin Kit (Quiagen) to remove excess dye terminator. Five microliters of each purified sequence was resuspended in 15 μl of formamide and denaturated for 2 minutes at 95°C. Analysis of the forward and reverse sequences was performed on the ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The sequences obtained were compared with reference sequence NM_003900.
The Grantham scale was used to evaluate the difference between amino acids (identifying the chemical factors that individually correlate best with evolutionary exchangeability of protein residues) and for the estimation of the extent to which observed exchanges can be explained by conservation of chemical factors.(26)
No mutation was detected at exon 7 of the SQSTM1 gene in our PDB patients.
The P392L mutation was found in only 1/62 PDB patients, corresponding to a 1.6% rate, which is 5.5 times lower than previously reported in sporadic PDB.(18) Based on a very detailed familial history, we excluded any common ancestor with the French Canadian population for this patient. Interestingly, in two different patients, we found two novel mutations, AG and GA transitions, at exon 8, both consisting of amino acid changes determining, respectively, methionine to valine at codon 404 (M404V) and glycine to arginine at codon 425 (G425R) substitutions (reference sequence, NM_003900; Fig. 1). DNA analysis from 100 healthy control subjects failed to detect such mutations.
According to the Grantham scale,(26) we estimated that M404V is a conservative substitution, whereas G425R is a radical substitution, although the relevance of amino acid replacements also depends on the location and/or context of the altered amino acid within the protein sequence.
A different degree of likelihood to clinically detect the effect of amino acid substitution has been reported, decreasing from nonsense to radical, moderately radical, and moderately conservative changes.(27) However, the correct interpretation of the estimated likelihood of clinical significance of these results must take into account the degree of evolutionary conservation of an amino acid as an important predictor of a particular substitution.(28)
Patient I (female; age, 73 years) with the P392L mutation, exhibiting a monostotic form of PDB, was originally from central Italy, whereas patients II (female; age, 65 years) and III (male; age, 55 years) were affected by polyostotic PDB and were originally from central and southern Italy (Table 1). No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without mutations.
To study the genetic aspects of hereditary diseases, it is important to have a broader spectrum of genomic DNA from different nationalities. The existence of genetic polymorphisms within and between different communities in the world makes it necessary for gene hunters to investigate several different ethnic groups. The recurrence of mutations at specific protein positions and domains in individuals of widely separated study populations suggests that these are true, background-independent mutations, possibly caused by endogenous mechanisms of mutagenesis or ubiquitous environmental influences. Establishing the association of specific genes with disease phenotypes by mutation screening, particularly for monogenic disorders, provides further assistance in defining the functions of some gene products as well as helping to establish the cause of the disease. A clear example can be represented by mutations at exon 6 of the Pit-1 gene, accounting for pituitary dwarfism; exon 6 represents a “hot spot” mutation region of the Pit-1 gene that is described in affected individuals from different ethnic groups.(29)
Our findings, compared with those of PDB patients of predominantly British descent,(18) suggest a minor involvement (4.8% versus 8.9%) of the SQSTM1 gene in the pathogenesis of sporadic Italian PBD cases, although in a 2.7 times smaller sample size. However, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Interestingly, the Italian population is genetically heterogeneous, as described through HLA polymorphism analysis, with well-defined groups such as Sardinians (central Italy)(30) and Italian subpopulations in the Po valley (northern Italy).(31) Moreover, erythrocyte genetic markers also showed genetic heterogeneity within Puglia and Sicily subpopulations (southern Italy).(32,33)
The fact that amino acid substitutions within the SQSTM1 gene are found in PDB patients strongly supports the causal association of this gene with the disorder. Generally, in-frame amino acid replacements, including changes to nonsense codons, represent the most frequent type of mutation, supporting the notion that Mendelian clinical phenotypes are associated primarily with alterations in the normal coding sequence of proteins.(27) The relevance of amino acid substitutions to the clinical outcome of a disorder can be generally estimated according to two classical criteria: the severity of biochemical damage and the location and/or context of the altered amino acid within the protein sequence and its conservation across species.(27) Indeed, disease-causing mutations are significantly more likely to occur for nonsense substitutions of evolutionary conserved amino acid residues.
The SQSTM1 gene mutations fully satisfied all above-mentioned criteria. As for the originally reported exon 8 P392L mutation,(16,18) the two novel mutations observed in Italian PBD patients, M404V and G425R, consist of a conservative and a nonconservative amino acid substitution at residues M and G, respectively; both are conserved across species.(16) Thus, such evidence strongly supports and enhances the hypothesis that these heterozygous mutations at exon 8 of the SQSTM1 gene are causative of PDB in our patients, according to a dominant negative mode of action, as also recently supported by Johnson-Pais et al.(19)
Several open questions still exist on the pathophysiological role of the SQSTM1/p62 protein in bone metabolism and its clinical application to genetic testing. First, it is not clear how constitutive DNA mutations of exon 8 of the SQSTM1 gene may independently account for the development of both monostotic and polyostotic forms of PDB, considering that all precursors and mature osteoclast cells carry such DNA abnormalities. The role of other gene(s) mutations and/or epigenetic mechanisms should be clarified in future studies. Moreover, the structure of the SQSTM1/p62 protein is not available either by X-ray crystallography or NMR studies, making it difficult to unravel the real effects of its mutations. Finally, should the treatment of asymptomatic carriers exhibiting an increase of circulating levels of bone alkaline phosphatase be appropriate? Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlation and to answer the question on the clinical evaluation of asymptomatic gene carriers. Studies on the structure of the SQSTM1/p62 protein, specific animal models, and cell culture systems may possibly answer these questions.
In conclusion, the presence of two novel mutations at exon 8 of the SQSTM1 gene in sporadic Italian PDB patients confirms evidence of a clustered mutation area at this level in this disorder, supporting the role of the UBA domain in the biological properties of the SQSTM1/p62 protein. This analysis is being extended to other sporadic Italian cases and to first-degree relatives of patients to detect new genetic carriers in potentially familial forms of PDB and to study the co-segregation of such DNA variants with the PDB phenotype. These studies could find new possibilities in the prevention and therapy of PDB and other metabolic bone disorders.
This study was supported by the European Research Program, Fifth Framework Program “Quality of life and management of Living Resources Research and Technological Development Program,” “Genetic Markers for Osteoporosis” to MLB, Cofin M.I.U.R. to MLB, and PNR 2001-2003 (FIRB) to MLB.
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