Three of 20 patients with juvenile osteoporosis were found to have a heterozygous mutation in the LRP5 gene. No mutations were found in the type I collagen genes. Mutations in the other family members with similar bone phenotype confirmed that LRP5 has a role in both juvenile and adult osteoporosis.
Introduction: The gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5) gene has recently been shown to affect bone mass accrual during growth and to be involved in osteoporosis-pseudoglioma syndrome and a high bone mass phenotype. Mutations in the type I collagen genes (COL1A1 and COL1A2) are known to cause osteogenesis imperfecta, characterized by increased bone fragility.
Materials and Methods: Here we analyzed COL1A1, COL1A2, and LRP5 for mutations in 20 pediatric patients with primary osteoporosis characterized by low BMD, recurrent fractures, and absent extraskeletal manifestations.
Results and Conclusions: No mutations were detected in the type I collagen genes, but two missense mutations (A29T and R1036Q) and one frameshift mutation (C913fs) were found in the LRP5 gene in three of the patients. The frameshift mutation was also seen in the proband's father and brother, who both were found to have significant osteoporosis. R1036Q was observed in the proband's mother and two brothers, who all had osteoporosis. These results indicate that heterozygous mutations in the LRP5 gene can cause osteoporosis in both children and adults.
OSTEOPOROSIS IS A skeletal disorder characterized by parallel loss of bone mineral and matrix, which results in enhanced bone fragility and in increased risk of fractures.(1) The associated fractures, and the morbidity and mortality that ensue, make osteoporosis an enormous public health problem. Bone mass is regarded as the most important determinant of fracture risk.(2) BMD using DXA is now the commonly used standard for measurement of bone mass, and in adults, it is used to define osteoporosis.(3) In adults, each SD decrease in BMD is associated with a doubling or tripling of fracture risk.(2) The etiology of osteoporosis is multifactorial and includes both genetic and environmental factors.(4) Genetic factors that account for 75-85% of the variance in BMD(5) play an important role in the pathogenesis of osteoporosis. The underlying mechanisms are complex involving variations in several genes that regulate BMD, bone geometry, and quality.(6)
Osteoporosis is increasingly recognized in children, usually as a complication of a chronic illness.(7) In addition to the chronic illness itself, several other factors such as medications, nutrient and hormone deficiencies, and decreased physical activity may contribute to either reduced bone mass or bone quality impairment.(7,8) Primary osteoporosis is less commonly recognized and usually diagnosed as either osteogenesis imperfecta (OI) or juvenile idiopathic osteoporosis (JIO). OI results from mutations in the genes encoding the proα1 (COL1A1) and proα2 chains (COL1A2) of type I collagen(9) and is clinically characterized by increased fragility of bones, blue sclerae, and hearing loss. The degree of severity is variable; severe forms of OI result in short stature and deformities.(10,11) JIO usually presents peripubertally as acute symptomatic osteoporosis (bone pain and fractures) in the otherwise healthy child.(12)
The gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5) has recently been shown to affect bone mass accrual during growth.(13)LRP5 is expressed in a wide variety of tissues and cells including osteoblasts, and the protein is involved in the Wnt signaling pathway that alters bone mass through a primary effect on bone formation.(13) Homozygous inactivating mutations in the LRP5 gene cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG), characterized by severe juvenile-onset osteoporosis and congenital or early-onset blindness. Other manifestations include muscular hypotonia, ligamentous laxity, mild mental retardation, and seizures.(14) In OPPG families, obligate carriers of the mutant LRP5 gene have been shown to have reduced bone mass but no other phenotypic features of OPPG.(13,15) Furthermore, in vitro and in vivo studies have shown that heterozygous gain-of-function mutations in LRP5 result in an autosomal dominant high bone mass phenotype because of increased Wnt signaling.(16,17) The Lrp5 gene has been shown to also impact bone mass in mice: Kato et al.(18) showed a low bone mass phenotype in mice with a targeted disruption of the Lrp5 gene, whereas mice expressing the human gain-of-function mutation had a high bone mass phenotype.(19) Taken together, these data suggest that the LRP5 gene impacts bone mass and may thus have a role in the development of osteoporosis.
To elucidate the role of the genes coding for type I collagen and LRP5 in pediatric osteoporosis, we analyzed these genes in 20 children with primary osteoporosis, characterized by reduced BMD and/or increased tendency to fracture and by absence of clinical features suggestive of OI or OPPG. This study was approved by the Research Ethics Board of the Hospital for Sick Children.
MATERIALS AND METHODS
Patients assessed for primary osteoporosis at the Pediatric Osteoporosis Clinic, The Hospital for Sick Children, from January 2002 to July 2003 were eligible for inclusion in the study. Patients with known diagnosis or clinical features suggestive of OI (blue sclerae, dentinogenesis imperfecta, joint hypermobility, hearing impairment) or of OPPG (impaired vision, ligamental laxity, mental retardation, or seizure disorder), as well as patients with an underlying chronic illness or systemic medication, were excluded from the study. None of the patients had joint contractures suggestive of Bruck syndrome(20) or craniosynostosis suggestive of Cole-Carpenter syndrome.(21) Patients were clinically assessed for phenotypic features, height and weight, and pubertal status. The diagnosis of osteoporosis was based on (1) low BMD, defined as a z score (SD score) below −2.0; (2) history of increased bone fragility, defined as at least three peripheral fractures caused by low-impact trauma; and/or (3) compression fracture(s). History of fractures was collected from the hospital records and by parent interview at the clinic visit.
The control group was comprised of 88 patients with an established diagnosis of skeletal dysplasia (Schmid type of metaphyseal dysplasia, multiple epiphyseal dysplasia, or Ehlers-Danlos syndrome) and 35 healthy adults from the same geographical area as the patients. The diagnosis of skeletal dysplasia in the first control group had been clinically and/or radiographically confirmed by one of the authors (WGC) in each case; none of the controls presented with osteoporosis.
Serum calcium, phosphate, alkaline phosphatase (S-ALP), PTH (S-PTH), and 25-hydroxyvitamin D3 [S-25(OH)D3[rsqb] and urine calcium and creatinine were obtained at the time of clinic assessment. Serum calcium, phosphate, and alkaline phosphatase were measured by reflectance spectrophotometry with VITROS 950 Chemistry System (Johnson and Johnson Ortho-Clinical Diagnostics). Reference range for serum calcium was 2.25-2.62 mM. Reference ranges for serum phosphate were age-dependent and for serum alkaline phosphatase were age- and sex-dependent: the measured values were transformed into z scores using normal values to allow for cross-sectional comparison.(22) Serum intact PTH was measured by IMMULITE Intact PTH solid-phase, two-site chemiluminescent immunometric assay with two polyclonal anti-PTH antibodies (44-84 and 1-34; Diagnostic Products). The reference range for intact PTH was 10-65 ng/liter. Serum 25(OH)D3 was measured by radioimmunoassay (25-OH-D RIA kit; DiaSorin) with a reference range of 25-90 ng/ml.
BMD and spinal radiographs
BMD was measured with a Lunar GE-Lunar Prodigy DXA machine. Reading for the lumbar spine (L2-L4) was used for the analysis; values were transformed into z scores by comparing them with age- and sex-specific reference values for this equipment.(23) No reference data were available for children <5 years of age. For the studied parents, the values were compared with sex-specific reference values(23) and expressed in SD units (T score). Spinal radiographs were obtained within 6 months of the BMD assessment. Standard anterior-posterior and lateral neutral radiographs were taken in the upright position. All thoracic and lumbar vertebrae were reviewed by two pediatric radiologists. Changes in vertebral morphology were assessed and graded by inspection of digitized images (PACs; GE Systems) and classified according to a novel pediatric grading method for vertebral osteoporotic changes.(24)
Genomic DNA was extracted from peripheral blood samples of the 20 study subjects and of the controls by standard procedures. PCR amplification of the 51 exons of COL1A1, 52 exons of COL1A2, and 23 exons of LRP5 was performed as previously described.(13,25) The PCR reactions were performed with a commercial DNA polymerase (AmpliTaq Gold; Applied Biosystems Roche) in a 20-μl volume, with thermal cycling at 95°C for 10 minutes for one cycle, followed by 95°C for 40 s, 54–65°C for 30 s, and 72°C for 50 s for 35 cycles. Conformation-sensitive gel electrophoresis (CSGE) analysis of the LRP5 gene was performed as previously described.(26) PCR products containing heteroduplexes were sequenced by an automated instrument (ABI PRISM 377 or 3100 Sequencers and ABI PRISM Dye Terminator Cycle Sequencing Ready Kit; Applied Biosystems). Before sequencing, the PCR products were treated with exonuclease I to degrade the residual PCR primers and with shrimp alkaline phosphatase to dephosphorylate the residual nucleotides.(27,28)
For RNA studies, the total RNA was extracted from Epstein-Barr virus-transformed lymphoblasts of patient 16 and of a control individual with RNeasy kit (Qiagen). The cDNA synthesis was carried out with the Superscript First Strand Synthesis System for RT-PCR (Invitrogen) and followed by PCR amplification with a pair of primers corresponding to exons 8 and 14 of LRP5 (5′-GCATCGAGCGGGTGCACAAGG-3′ and 5′-GCTCC GCGTTGACGACGATG-3′, respectively). The product was sequenced with a primer corresponding to exon 12 (5′−CTGGACAGACTGGAATCTGC-3′). A single nucleotide polymorphism (SNP) in the exon 15 of the LRP5 gene was analyzed from an RT-PCR product amplified with primers corresponding to exons 13 and 15 (5′-CAGTCGGATGATCCCGGAC-3′ and 5′-CGCTTCAGGTCCGCGTCCAC-3′, respectively). Sequencing of the RT-PCR product was performed with the former primer.
Clinical, biochemical, and radiological findings
Twenty consecutive patients (11 males and 9 females) fulfilling the criteria for primary osteoporosis who lacked phenotypic features of OI or OPPG were included in the study. Their mean age at the time of the study was 10.5 years (range, 4.0-16.0 years). Eighteen of the 20 patients (90%) had one or more peripheral fractures, and 15 patients (75%) had at least three peripheral fractures (median, four fractures). In three patients, the total number of peripheral fractures exceeded eight, and in the others ranged from one to six fractures (Table 1). The fracture mechanism was variable, but all fractures resulted from low- to moderate-impact trauma. Two patients (OP11 and OP13) had a clinical course suggestive of JIO, with peripubertal subacute onset of bone pain and fractures; in the others, bone pain was not a dominant feature, and the first fractures occurred in early years and continued throughout childhood. Spinal radiographs of 15 patients were available for assessment. Seven patients (35% of the total study group) had multiple compression fractures, and five patients had other significant osteoporotic changes in the vertebral body morphology (Table 1). Only five patients had normal spinal radiographs. The BMD z score was available for all 18 patients who were ≥5 years old. Their mean z score was −2.1 (range, −4.7 to +1.0); it was below −2.0 in nine patients and above −1.5 in five patients (Table 1). None of the patients had scoliosis or other deformities. All patients had normal stature (height between the 3rd and 97th percentile for age and sex). None of the patients presented with symptoms suggesting an underlying chronic illness or with significant hypocalcemia, hypophosphatemia, hyperparathyroidism, or hypercalciuria, which could result in impaired bone health. All had normal 25(OH)vitamin D serum concentrations. The mean S-ALP concentration was −1.7 SD score (range, −2.6 to −0.1 SD score); S-ALP was below the age- and sex-specific reference values in 6 of the 20 patients (Table 1). Impaired vision or other significant ophthalmological problems typical for OPPG were not reported in any of the patients.
Table Table 1.. Clinical and Radiographic Findings in the 20 Patients With Primary Osteoporosis
Analysis of the COL1A1, COL1A2, and LRP5 genes for sequence variations
Mutation analysis of all COL1A1 and COL1A2 exons and the corresponding exon boundaries by CSGE and sequencing did not reveal putatively disease-associated mutations. An SNP G/T in the Sp1 binding site in the first intron of the COL1A1 reported to associate with osteoporosis(29) was analyzed by sequencing. Five patients had the G/T, and 15 patients had the G/G genotype. None of our patients were homozygous for the T allele, which has been associated with low BMD.
We screened the 23 exons and exon boundaries of the LRP5 gene in the 20 patients. CSGE and subsequent sequencing revealed three novel heterozygous mutations. Two of them were missense mutations: A29T (c.85G>A) and R1036Q (c.3107G>A) in exons 1 and 14, respectively, and one was a frameshift mutation C913fs (c.2737_2738insT) in exon 12, which predictably results in premature termination codon in exon 13. Similar sequence variations were not observed in any of the controls or the patients' healthy family members, suggesting that these changes were associated with the phenotype.
Patient OP8 with the missense mutation A29T had, at the age of 16 years, a history of four peripheral fractures (at ages 4, 7, 8, and 14 years), of which three resulted from low-impact trauma and one from a sport-related injury. Her height was 165 cm, and she was postpubertal. Her BMD z score was −1.9 for the lumbar spine. Spinal radiographs showed multiple compression fractures at vertebral bodies T6-T9. Her father had a history of one sport-related fracture; his BMD was normal (T score, −0.7). The proband's mother had no fractures, and her BMD was normal (T score, 0). Thus, the parents had no evidence of osteoporosis, and neither carried the same sequence change in the LRP5 gene as the proband, suggesting that the patient had a de novo missense mutation.
Patient OP5 with the missense mutation R1036Q had a history of five peripheral fractures between 14 months and 5 years of age; all fractures were associated with low-impact trauma. His height at the age of 9 years was 144 cm (95th percentile). His BMD z score was −0.1. Spinal radiographs were not available for assessment. The proband's mother had a history of three peripheral fractures, resulting from low- to moderate-impact trauma; the father's fracture history was negative. The mother was found to have the same mutation as the proband; the father's LRP5 sequence was normal. No BMDs were available for the parents because they did not consent to further studies.
Patient OP16 had a frameshift mutation C913fs (c.2737_2738insT) in exon 12, which was predicted to result in a premature termination codon in exon 13. He had a humerus fracture at 2 years of age. At 16 years of age, he was found to have a compression fracture after a sport-related injury. Assessment at the Osteoporosis Clinic revealed a healthy postpubertal adolescent with normal height (175.6 cm) and body proportions, absence of limb deformities, and normal vision. His BMD was markedly reduced (z score, −2.9), and review of the spinal radiographs showed an osteopenic appearance and compression deformities of variable severity in the vertebrae. His 18-year-old brother was subsequently found to have significantly reduced BMD (z score, −3.3) and spinal changes. He was asymptomatic and had not experienced any peripheral fractures. Based on these findings, the parents were also assessed for osteoporosis. The proband's father, 54 years of age, was asymptomatic and had a negative fracture history; his BMD T score was −1.9. He had multiple compression fractures. The mother had normal BMD. Both the father and the brother were found to have the same mutation as the proband.
Several additional SNPs were identified in LRP5 in the patients (Table 2). However, these changes are unlikely to associate with significant osteoporosis in our study subjects because many of them were found in equal frequencies in the cases and controls (Table 2).
Table Table 2.. Observed Single Nucleotide Polymorphisms in the LRP5 Gene in the Patients and Controls
RT-PCR analysis was performed to understand the effects of the frameshift mutation C913fs in patient OP16. Total RNA was isolated from Epstein-Barr virus (EBV)-transformed lymphoblasts of the patient and a control and used as a template for cDNA synthesis. This was followed by PCR amplification of a region corresponding to exons 8-14. A product of 1534 bp was obtained from both samples (data not shown). Sequencing of the patient's RT-PCR product indicated, however, only the presence of the normal allele. Both the cDNA and genomic DNA of patient OP16 were analyzed for the presence of a synonymous SNP, c.3297C>T (D1099D), in exon 15. CSGE analysis and sequencing of the genomic DNA showed that the patient was heterozygous for the SNP, but sequence analysis of the cDNA revealed the presence of only one allele. Thus, it was likely that the frameshift mutation in the LRP5 gene resulted in haploinsufficiency through nonsense-mediated mRNA decay.(30)
We performed a candidate gene study to analyze the contribution of LRP5, COL1A1, and COL1A2 genes in the development of primary osteoporosis. In our cohort of 20 consecutive patients with tendency to fracture and/or a low BMD but no features of OI or OPPG, 3 patients were found to have novel heterozygous mutations, A29T in exon 1, C913fs in exon 12, and R1036Q in exon 14, in the LRP5 gene.
Interestingly, all LRP5 mutations that have been identified in high bone mass phenotypes(16,17,31) involve conserved amino acids in exons 2, 3, and 4 of LRP5 gene, which are all situated in the first YWTD/EGF domain of the LRP5 protein (Fig. 1). In contrast, the mutations identified in OPPG are primarily located in the second and third YWTD/EGF domains (consist of an epidermal growth factor [EGF] repeat and six YWTD repeats; Fig. 1). In the present cohort, three mutations, A29T, C913fs, and R1036Q, were situated in the first, third, and fourth YWTD/EGF domains, respectively. The frameshift mutation in exon 12 likely results in haploinsufficiency through nonsense-mediated mRNA decay. The mechanisms through which the two missense mutations result in osteoporosis is most likely formation of a mutant LRP5 protein, which is functionally abnormal because of a disrupted protein-binding site. Apparently, the specific location of a mutation within the functionally different LRP5 domains impacts the LRP5 activity and/or Wnt signaling by variable mechanisms, thus resulting in variable bone phenotypes. Further studies on the role and function of LRP5-mediated Wnt signaling are needed to enhance understanding of the mechanisms through which the variable LRP5-associated skeletal phenotypes arise.
In addition to the three mutations, eight SNPs were observed within the coding region of the LRP5 gene in the 20 patients (Table 2). Because four of the SNPs were synonymous (E644E, D1099D, V1119V, and P1241P), they are unlikely to affect the protein function. Seven of the SNPs were also found in the control samples and therefore cannot be regarded as the cause of significant osteoporosis and fractures seen in our study subjects. However, it is possible that some of them may impact bone mass accrual and contribute to the variation in bone mass seen even in general population. Indeed, this is supported by the recent findings that indicate that LRP5 allelic variation contributes significantly to the determination of vertebral bone mass and size in men by influencing vertebral bone growth during childhood.(32)
No mutations in the LRP5,COL1A1, and COL1A2 genes were found in 17 of the 20 patients. Some of them had a positive family history for osteoporosis and/or fractures, suggesting a dominantly inherited susceptibility. As previous studies have identified several potential loci as determinants of peak bone mass and susceptibility for osteoporosis,(33,34) it is probable that future research will identify new genes involved in bone mass accrual and juvenile-onset primary osteoporosis.
Whereas secondary osteoporosis has been increasingly recognized in chronically ill children, primary osteoporosis seems to be a relatively rare diagnosis in pediatric patients. Most such patients have phenotypic features suggestive of OI, in addition to increased bone fragility. No responsible genes have thus far been identified in IJO or in other forms of primary osteoporosis in children. This study identified three novel heterozygous mutations in the LRP5 gene that resulted in primary osteoporosis characterized by low BMD and increased bone fragility at an early age without associated ocular manifestations. In pediatric patients with primary osteoporosis and no features suggestive of OI or OPPG, screening of LRP5 is recommended for early detection of heterozygous carriers of mutations and for early treatment to prevent permanent complications of osteoporosis. Our observations also suggest that LRP5 mutations have a role in the general adult population with osteoporosis. This, however, needs to be elucidated in future studies.
The authors thank Satu Koljonen and Irma Vuoti for expert technical assistance. This work was supported by grants from the Foundation for Paediatric Research and the Finnish Medical Association Duodecim, Finland and by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S (to OM); the Canadian Institutes of Health Research and the Canadian Arthritis Network (to WGC); and the Academy of Finland, the Louisiana Gene Therapy Research Consortium (New Orleans, LA), and HCA-The Health Care Company (Nashville, TN) (to LA-K).