The authors have no conflict of interest.
Three Novel Mutations in SQSTM1 Identified in Familial Paget's Disease of Bone†
Version of Record online: 1 OCT 2003
Copyright © 2003 ASBMR
Journal of Bone and Mineral Research
Volume 18, Issue 10, pages 1748–1753, October 2003
How to Cite
Johnson-Pais, T. L., Wisdom, J. H., Weldon, K. S., Cody, J. D., Hansen, M. F., Singer, F. R. and Leach, R. J. (2003), Three Novel Mutations in SQSTM1 Identified in Familial Paget's Disease of Bone. J Bone Miner Res, 18: 1748–1753. doi: 10.1359/jbmr.2003.18.10.1748
- Issue online: 2 DEC 2009
- Version of Record online: 1 OCT 2003
- Manuscript Accepted: 29 APR 2003
- Manuscript Revised: 7 APR 2003
- Manuscript Received: 3 JAN 2003
- Paget's disease of bone;
- sequestosome 1;
- ubiquitin-associated domain;
Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Paget's disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C-terminal ubiquitin associated domain. Disruptions of the C-terminal domain of SQSTM1 seem to be a leading cause of familial PDB.
Introduction: The characteristic features of Paget's disease of bone (PDB) are caused by focal areas of excessive and uncoordinated bone remodeling. A total of seven genetic loci (PDB1-PDB7) have been reported to be associated with the disease. The gene for Sequestosome 1 (p62; SQSTM1) has been identified as the causative gene for PDB3 in numerous French-Canadian families and families predominantly of British descent. To date, a total of three mutations, all affecting the ubiquitin-associated domain of SQSTM1, have been identified: a single 1215 C to T (P392L) transversion in exon 8, a T insertion in exon 8 (E396X), and a G to A mutation at the splice junction of exon 7 (IVS7 + 1).
Materials and Methods: DNA was isolated from blood collected from the members of five U.S. PDB pedigrees. Mutation analysis of the coding sequence of the SQSTM1 gene was performed on the proband and other key individuals in the pedigrees.
Results: Four of the five families had SQSTM1 mutations. Three of these mutations were novel: a single base deletion in exon 8 at position 1210 (1210delT) resulting in a premature stop codon at amino acid 394, a single C deletion in exon 8 at position 1215 (1215delC) also resulting in a premature stop codon at amino acid 394, and a single 1200 C to T (P387L) transversion in exon 7.
Conclusion: Noteworthy is the fact that these three SQSTM1 mutations, in addition to the three previously described mutations, are clustered near the C-terminal of the protein. These mutations may be acting in a dominant-negative fashion to disrupt the ubiquitin-binding function, which could result in abnormal activation of the NF-κB pathway and the subsequent activation of the osteoclasts. These findings imply that SQSTM1 mutations may play a role in the majority of familial PDB in the United States.
Paget's disease of bone (PDB; OMIM 602080) is a nonmalignant disease involving accelerated bone resorption followed by deposition of dense, chaotic bone matrix. These focal areas of excessive uncoordinated bone turnover are caused by an osteoclast defect. Abnormal osteoclasts from pagetic lesions are increased in number and size and contain paramyxovirus-like nuclear inclusions.(1) PDB has a strong genetic component. In familial cases of classic late onset PDB, the disease segregates in an autosomal dominant pattern. However, this disease also exhibits genetic heterogeneity and incomplete penetrance. There are seven susceptibility loci proposed for PDB thus far: PDB1 (6p21.3),(2) PDB2 (18q21),(3,4) PDB3 (5q35),(5) PDB4 (5q31),(5) PDB5 (2q36),(6) PDB6 (10p13),(6) and PDB7 (18q23).(7) In most of the PDB pedigrees studied thus far, linkage to either PDB1 or PDB2 has not been demonstrated. Mutations have been identified in the TNFRSF11A gene, located at 18q21, in one family with early onset PDB, but it is unclear if this same gene is responsible for classical, late onset PDB.(8–10) Recently, the gene for PDB3 has been identified from mutational analyses of sequestosome 1 (SQSTM1; p62), a gene located on 5q35 that codes for a ubiquitin-binding protein, in French-Canadian and British pedigrees with late adult onset PDB. (11,12)
Because of the recent description of the SQSTM1 exon 8 1215 C/T (P392L) mutation that causes PDB in both French-Canadian and British pedigrees(11,12) and because mutations of SQSTM1 have been shown to segregate with PDB in 26.5% of Australian PDB families,(12) we performed mutational analysis of SQSTM1 in five of our PDB pedigrees. Our pedigrees consist of individuals with a phenotype typical of late onset PDB. Our pedigrees differ from many of the previously reported pedigrees exhibiting SQSTM1 mutations because these families are from a multiethnic population found in large metropolitan areas as opposed to being from isolated populations, such as those found in Quebec City, Canada.
MATERIALS AND METHODS
This study was approved by the Institutional Review Board of St. John's Health Center, Santa Monica, CA, and the Institutional Review Board of the University of Texas Health Science Center, San Antonio, TX. All probands were obtained from the clinical practice of FRS after a positive family history of PDB was reported. Blood samples were collected from members of five PDB families after appropriate informed consent was obtained.
Genomic DNA was isolated from white blood cells.(13) One hundred nanograms of genomic DNA was used for each polymerase chain reaction (PCR) amplification. The primers used to amplify the eight SQSTM1 exons have been previously described (exons 1–8).(11) The PCR products from all eight exons were purified by centrifugation through an ultrafree-MC filtration device (Millipore, Bedford, MA, USA) and sequenced on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA). When sequencing results showed a nucleotide alteration, the PCR products were subcloned using the TOPO TA kit (Invitrogen, Carlsbad, CA, USA) and sequenced using an ABI 3100 genetic analyzer to identify the exact mutation.
Rapid screening for single nucleotide changes
To screen the general population for the single nucleotide changes identified in exon 7 in Family 3 and in the 3′ untranslated region of exon 8 in Family 5, PCR assays were developed. For the exon 7 assay, 100 ng of genomic DNA was amplified with PCR primers that flank exon 7.(11) The resulting 455-bp PCR product was digested with 10 U of AciI restriction endonuclease (New England Biolabs, Beverly, MA, USA) overnight at 37°C. The digested products were electrophoresed in a 2% agarose gel and visualized by staining with ethidium bromide. Digestion of the 455-bp exon 7 PCR product from the wild-type allele (C) results in two fragments of 172 and 283 bp. The presence of a T instead of a C at position 1200 eliminates the AciI restriction site.
To screen the general population for the single nucleotide change 1446 G/A identified in the 3′ untranslated region of exon 8, 100 ng of DNA was PCR-amplified with exon 8-specific primers 5′-CCTTACTGTTTCGGCAGAGG-3′ and 5′-TCAGCACACACACACACAGG-3′. The 367-bp product was digested with 10 U of the restriction endonuclease BsrGI (New England Biolabs) overnight at 37°C. The digested products were electrophoresed in a 2% agarose gel and visualized by staining with ethidium bromide. The PCR product obtained from the wild-type allele does not contain a BsrG1 restriction site. The presence of an A instead of a G at nucleotide position 1446 introduces a BsrGI site. Digestion of the PCR product derived from the variant allele results in two fragments of 114 and 253 bp.
We first clinically evaluated the proband (II-7) of a large multigenerational American pedigree (Family 1; Fig. 1) of mixed European descent with PDB at the age of 59 years. He was discovered to have PDB at the age of 44 years after trauma to his left shoulder led to the discovery of elevated serum alkaline phosphatase activity and X-rays indicating PDB. The following bones were found to be affected by PDB: skull, second cervical vertebrae, eighth, ninth, and eleventh thoracic vertebrae, second to fifth lumbar vertebrae, sacrum, pelvis, left scapula, right clavicle, right humerus, right and left femur, right and left tibia, and right first metatarsal. All affected individuals in this pedigree exhibit one or more skeletal deformities typical of adult onset PDB (Table 1). Members of this pedigree have had tests for serum alkaline phosphatase levels to define disease activity (Table 1).
The only SQSTM1 sequence alteration in Family 1 was found in exon 8 (Table 1). PCR products for SQSTM1 exon 8 from family members that showed a mutation were subcloned and the individual alleles were sequenced to identify the mutation. The SQSTM1 mutation identified in Family 1 was a single nucleotide deletion of a T at position 1210 (1210delT; Fig. 2) that results in the introduction of a stop codon at amino acid 394. The ubiquitin-associated (UBA) domain is comprised of amino acids 394–440, and introduction of this stop codon entirely eliminates this domain. With the exception of members in the third generation (III-2, -3, and -8), all individuals with the mutation had PDB. Individual II-9, however, was the only individual with PDB who was not heterozygous for the allele with the deletion. The presence of the mutated allele in three unaffected individuals in the third generation implies that even in pedigrees with a strong family history there is evidence of incomplete penetrance. It is interesting to note that individuals III-3 and -8 exhibit elevated or high normal serum alkaline phosphatase levels, although all three individuals (III-2, -3, and -8) have had bone scans that are negative for PDB (Table 1).
The second PDB family (Family 2; pedigree not shown) is of Italian descent. The proband of this pedigree had PDB in numerous bones. The mother and father of the proband had no known PDB, although the mother died at a very young age (38 years of age). A paternal uncle of the proband had difficulty walking, although there was never a diagnosis of PDB. A total of eight offspring were born to this couple, and six have been diagnosed with PDB. PDB was found in various sites in these individuals including the legs, hips, spine, and skull. We performed mutational analyses on DNA from the proband, an affected sibling of the proband, and the proband's two offspring.
In Family 2, the only nucleotide alteration found in the SQSTM1 coding region of the proband was an exon 7 single nucleotide C to T transversion at position 1200, which causes the nonconservative substitution of proline 389 to a leucine (P389L; Fig. 2). This C to T transversion in exon 7 results in the disruption of a recognition site for the restriction endonuclease AciI, thus creating an easy screening tool for this mutation (Fig. 3A). The two sons of the proband carry the mutant P389L allele but do not have PDB. At 63 and 56 years of age, these individuals had normal serum alkaline phosphatase levels (85 and 102 U/liter, respectively; normal < 135 U/liter) and no detectable skeletal deformities. The 63-year-old son has also had a negative bone scan. The affected sibling of the proband does not carry this P389L mutation.
To determine if this nucleotide alteration was indeed a mutation or a common polymorphism, 67 unrelated white individuals from the general population were genotyped for this C to T transversion in exon 7. Genotyping was performed by AciI digestion of exon 7-derived PCR products. This analysis revealed no evidence for the variant allele in the 134 chromosomes studied, which indicates that the nucleotide change at this position is not likely to be caused by a single nucleotide polymorphism.
Our third PDB family (Family 3) is of Lithuanian descent, with many family members residing in Australia and the United States. The American proband in this pedigree has PDB in the skull, thoracic and lumbar spine, pelvis, and right clavicle. At the age of 60, her alkaline phosphatase levels were 140 U/liter (normal < 120 U/liter). Affected members of this pedigree include the mother, 12 maternal aunts and uncles, and 2 siblings of the proband. Mutational analysis was performed on DNA isolated from the affected proband.
In Family 3, a deletion mutation that consists of the deletion of a C at position 1215 in exon 8 was identified in the affected proband (Fig. 2). This is the same nucleotide that is involved in the P392L mutation found in French-Canadian and Australian families. This mutation is similar to the mutation observed in Family 1 (1210delT) in that the mutation also introduces a premature stop at amino acid 394 with the subsequent loss of the UBA domain.
In the fourth pedigree with classic late-onset PDB (Family 4), we identified the exon 8 P392L mutation (data not shown), which is the most common mutation observed in PDB families thus far. Family 4 is a large kindred of Mayan, Spanish, African, and Scottish descent, which was previously identified as being linked to chromosome 18q.(3) We studied 16 individuals older than 35 years of age; 8 of these individuals have PDB. All but one of the affected individuals carried the P392L mutation.
In addition to the four mutations that were observed in the coding region of SQSTM1 in Families 1–4, a unique single nucleotide polymorphism 1446 G/A was identified in the 3′ untranslated region of exon 8 in the affected proband from Family 5. This alteration has unknown clinical relevance. The G to A change at this position creates a recognition site for the restriction endonuclease BsrGI, which can be used as a rapid screening tool for this allele (Fig. 3B). To determine if this alteration represents a common polymorphism in the gene, 50 unrelated control individuals from the general population were genotyped for this allele by restriction enzyme analysis of exon 8 PCR products. The variant allele was not present in any of the 100 chromosomes analyzed, which indicates that the G to A alteration at this position is not a common polymorphism.
In three of our pedigrees, we discovered unique mutations within the coding region of SQSTM1. Interestingly, the mutations do not consistently segregate with disease in the youngest generations of the pedigrees. In Family 1, the 1210delT mutation segregated with the disease in all but three of the third generation; in Family 2, the mutation did not segregate with disease in the two offspring of the proband.
One explanation for the incomplete penetrance observed in the younger members of Family 1 may be the fact that these individuals have not yet reached the age at which PDB is normally diagnosed. There is also the possibility that the incomplete penetrance may be related to a lack of exposure to the measles virus, which is one of the cofactors believed to play a role in PDB.(1) Ultrastructural studies of osteoclasts from PDB patients have shown nuclear inclusions that resemble the paramyxovirus nucleocapsid, and measles virus mRNA transcripts have been detected in pagetic bone marrow.(14) Recently, the measles virus nucleocapsid gene has been isolated from pagetic lesions of four patients with PDB showing that chronic measles virus infection is present in areas of active disease.(15) Since the advent of wide-spread measles vaccination in the United States in 1963,(16) the incidence of measles has been drastically reduced, and the chance of exposure to the virus has been virtually eliminated. Although lack of exposure to measles virus may explain why the younger individuals did not have PDB, this does not explain all the inconsistencies observed in the pedigrees. Other inconsistencies include an absence of SQSTM1 mutations in an affected sibling of the proband in both Families 1 and 2. This is not a unique observation as Hocking et al.(12) described a family in which two of four affected individuals did not carry a SQSTM1 mutation.
The identification of the P392L mutation in Family 4, which had previously been linked to chromosome 18q, was unexpected. Although the linkage to 18q may have been a spurious result, we propose the possibility that PDB may be an example of a Mendelian disorder with digenic inheritance.(17) In this type of inheritance, an affected individual has mutations in each of two unlinked genes that together cause a disease phenotype. The disease phenotype may or may not be observed with one or the other mutation. We hypothesize that PDB2 may be a modifier of expressivity for SQSTM1. Alternatively, SQSTM1 may be a modifier of PDB2. Presumably these two loci function directly or indirectly in the same signaling pathway, ultimately leading to activation of the osteoclast. PDB is known to be a genetically heterogeneous disease, and many pedigrees exhibit incomplete penetrance and variable expressivity. The reduced penetrance and phenotypic differences observed in PDB families may be caused by more than just a single gene mutation and is more likely caused by multiple genetic mutations. Several diseases that have been considered to be single gene disorders, such as retinitis pigmentosa, Bardet-Biedl syndrome, and Hirschsprung disease, are in fact caused by more than one genetic hit.(17) The second genetic hit may be in a modifier locus that controls the development of PDB in individuals carrying the mutant SQSTM1 allele; this may explain, in part, the lack of penetrance observed in the younger generations of our pedigrees.
Sequestosome 1 has the ability to bind ubiquitin through amino acids 386–434 in the C terminus of the protein, a region that is referred to as a UBA domain.(18) Sequestosome 1 has the ability to bind multi-ubiquinated chains and may act to store ubiquinated proteins. Sequestosome 1 is known to function as a protein scaffold in both the TNFα and interleukin 1 pathway for selective activation of NF-κB. The three novel mutations of SQSTM1 we describe here and the previously described mutations are all clustered around the UBA domain. The mechanisms by which these truncating mutations in the C-terminal region of the protein cause a susceptibility to PDB still are unknown, but it has been speculated that loss of the SQSTM1 UBA domain causes accumulation of the protein and the subsequent activation of NF-κB pathway.(12)
Although recent data suggest that mutations in SQSTM1 account for approximately 26% of familial adult late onset PBD cases,(12) we found the percentage to be much higher. In four of our pedigrees, we discovered significant nucleotide alterations in SQSTM1 surrounding the C-terminal region, implying that mutations in this gene may be a frequent cause of adult late onset PDB in the general U.S. population.
This work was supported by interactive R01s from the National Institute of Arthritis and Musculoskeletal and Skin Diseases AR044919 (RJL) and AR044904 (MFH), and funds obtained from the Eli and Edythe L. Broad Foundation (FRS) and the Cynthia and Edward Lasker Foundation (FRS).
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