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Originally Published IVD Technology March 2004

Assay Development

An image of healthy bone matrix (a) and osteoporotic bone matrix (b). Click to enlarge.

The European Commission has described osteoporosis as a silent epidemic and the World Health Organization (WHO) has identified the disease as a priority health issue.¹ As time goes by, the world population increases, life expectancy expands, and the frequency of fractures due to osteoporosis rises.² As mammals age, their bones undergo formation and resorption. In her youth, the rate of an individual’s bone formation exceeds that of resorption, whereas when she reaches middle age the trend is reversed and overall bone loss gradually occurs. 

The process of bone loss is particularly acute in women because they have smaller, less- dense bones than do men. Their rate of bone loss sharply increases in the five years following menopause, when their ovaries stop producing estrogen. The process of bone loss is gradual. Most cases of osteoporosis remain undetected until an individual appears in the emergency department with fractures, which, in patients suffering from osteoporosis, usually occur in the hip, wrist, or spine. 
Currently, 650,000 hip fractures occur annually in the European Union (EU) and the United States. Due to complications from fracture, the mortality rate in these cases is approximately 20% within 12 months of fracturing. An additional 50% of patients are left with severe long-term disablities.³ Treatment of fractures due to osteoporosis costs the EU alone over U.S. $4 billion annually in hospital healthcare.³ In addition, an estimated 44 million people in the United States, constituting 55% of the population aged over 50, are at risk of osteoporosis and low bone mass.³ A reliable method of detecting osteoporosis at an earlier stage may save both money and lives.

Markers for Detection of Osteoporosis

Currently, physicians use bone mineral density (BMD) and a range of biochemical markers of bone metabolism to determine the rate at which a patient is losing bone mass.⁴ Bone is continually being produced by osteoblasts (bone-forming cells) and degraded by osteoclasts (large cells with many nuclei, found in growing bone, that are responsible for bone resorption). 

During osteoclastic bone resorption, fragments of N- and C-telopeptides and (deoxy)pyridinoline crosslinks from type I collagen molecules in the bone matrix are released and can be detected in urine and serum. Serum tartrate resistant acid phosphatase, urinary calcium, and urinary hydroxyproline are also used to measure osteoclast activity. 

Bone formation can be measured biochemically by bone-specific alkaline phosphatase and osteocalcin. Isoforms of bone-specific alkaline phosphatase differ only by glycosylation. After osteoblasts produce osteocalcin, fragments of it are released into circulation.

Current biochemical markers used for measuring bone formation and bone resorption tend to lack sensitivity and specificity for the diagnosis of osteoporosis. The preferred method of diagnosing osteoporosis, and of identifying individuals at risk for sustaining an osteoporotic fracture is the dual-energy- x-ray absorptiometry (DXA) scan, which uses low-radiation x-ray to determine spine and hipbone mineral density. Other variations of this method for diagnosis include quantitative computed tomography (QCT) to scan the trabecular bones of the lower spine, and quantitative ultrasound (QUS), which uses sound waves to measure bone density at the heel, shin, or finger. 

U.S. guidelines recommend that all women over 65 and younger postmenopausal women with other known risk factors should have a DXA scan to measure their BMD. The WHO classifies a DXA scan that generates a BMD of more than 2.5 standard deviations below the mean for healthy adults as identifying an individual with osteoporosis.⁵ Such individuals are considered to be at risk of osteoporotic fracture. A BMD of 1–2.5 standard deviations below the mean identifies an individual at risk of developing osteoporosis.⁶

For every standard deviation below mean BMD, the risk of fracture increases by 50–100%. Although using DXA scans to determine BMD is the preferred method for diagnosing osteoporosis, this method has some disadvantages. DXA scans require specialized equipment and trained personnel, and are relatively expensive to perform. 

Therefore, an alternative screening approach would be useful to ensure early identification of individuals at increased risk of osteoporosis and osteoporotic fractures prior to the onset of disease. Such an early identification of at-risk individuals would enable closer monitoring and early preventative treatment prior to significant bone loss. 

Osteoporosis Treatment

Figure 1. Polymorphism of an Sp1 binding site in the collagen type 1 alpha 1 gene. Click to enlarge.

Although DXA scans are the current method of choice for diagnosing osteoporosis, biochemical markers are used to monitor treatment and therapy. There is no cure for osteoporosis. However, current treatments aim to prevent further bone loss and development of osteoporosis in those at risk, and to prevent osteoporotic fractures in those with osteoporosis. 

The preferred therapy for the prevention of osteoporosis in postmenopausal women already experiencing a loss of bone density is hormone replacement therapy (HRT). This therapy consists of estrogen replacement, sometimes in combination with progestin. HRT substantially slows the rate of postmenopausal bone loss. 

Bisphosphonates (alendronate and risedronate) are the therapy of choice in a variety of bone metabolism disorders, as they inhibit osteoclast-mediated bone resorption. Alendronate increases BMD at all skeletal sites, thereby reducing the incidence of hip and spine fractures by up to 50%. Risedronate increases bone mass in postmenopausal women, thereby reducing the incidence of hip and spine fractures in elderly women with low BMD. 

Alternative therapies to HRT in postmenopausal women are calcitonin or selective estrogen receptor modulators such as raloxifene. Calcitonin can be supplied as a daily nasal spray, and has been shown both to reduce spinal fractures by approximately 30% and to provide additional analgesic properties. Raloxifene reduces bone loss by mimicking estrogen in some tissues, and providing the bone-retaining effects of estrogen without its unwanted side effects (e.g., heightened risk of breast and endometrial cancer). These therapies slow down bone loss, thereby reducing the risk of osteoporotic fractures.⁶ In order for these therapies to be most effective, a rapid test is required to identify at-risk individuals earlier and begin therapy prior to deterioration of bone density. 

Teriparatide, a recombinant parathyroid hormone, has recently entered the marketplace as the first osteoporosis treatment to stimulate bone growth rather than reduce bone loss.^{6, 7} This is a clear step forward in the ability to treat osteoporosis after its diagnosis and prior to the incurrence of cost and debilitating injuries caused by osteoporotic fractures. 

The hormone’s effect on biochemical markers responsible for bone formation and resorption can be detected earlier when using biochemical markers than when using DXA scans, because BMD changes gradually and it can take one to two years for these changes to accumulate and become detectable. Therefore, multiple DXA scans are required over a prolonged period.

Genetic Markers for Osteoporosis

Studies comparing bone density in identical and fraternal twins indicate that up to 85% of variation in bone density is determined genetically.^{4, 8, 9} Genetic markers for diagnosis that encode calcium-regulating hormones, receptors, cytokines, growth factors, and bone-degradative enzymes have been identified previously as potential osteoporosis markers. However, few have been consistently associated with osteoporotic fractures or reduced BMD in multiple populations. The best targets for genetic markers are genes involved in the development of type I collagen, the predominant component of human bone matrix. 

Type I collagen consists of three peptide chains in a triple-helix conformation. Each helix is composed of two µ1 chains from the collagen 1 alpha 1 (COL1A1) gene and one µ2 chain from the collagen 1 alpha 2 (COL1A2) gene. Each chain is 1014 amino acids long, with uninterrupted triplet repeats consisting of glycine-X-Y, where X and Y are often proline and hydroxyproline, respectively.¹⁰
 
Mutations in COL1A1 and COL1A2 genes usually result in substitutions of glycine for bulky, polar, or charged amino acids that are known to cause Osteogenesis imperfecta, (OI), a severe osteoporotic disease found in children.11 This is a heritable connective-tissue disorder associated with fractures and osteopenia (i.e., low bone mass). 

A phenotype similar to human OI has been observed in a null mutant strain of mice (oim) that has a nonlethal, recessively inherited mutation in the COL1A2 gene resulting in an accumulation of µ1(I) homotrimers.¹² The phenotype of the homozygous oim strain of mice includes skeletal fractures, limb deformities, osteopenia, and small body size. Likewise, gene knock-in experiments using the Cre/lox system disrupted the COL1A1 gene, producing a heterozygous mutant mouse strain with bone fragility similar to human OI.¹¹

COL1A1 Poylmorphism

Figure 2. The Axis-Shield assay for the COL1A1 mutation. Click to enlarge.

To date, the best genetic marker for COL1A1 is a single nucleotide polymorphism (SNP) at G2046T (previous nomenclature referred to this point mutation as G1245T) at the first base of a consensus site for the transcription factor Sp1 in the first intron of the regulatory region for the COL1A1 gene (Chromosome 17q21; GenEMBL accession number J03559) (see Figure 1).^{13, 14}
 
Initially, an association between COL1A1 and bone mass density was observed in two UK studies. Subsequent studies have shown that the presence of a heterozygote or homozygote “s” allele polymorphism has an association with reduced bone mass density and a 50–60% increased risk of developing osteoporosis.^15-19

Gel shift assays have shown that Sp1 transcription factor has an increased affinity for the COL1A1 “s” allele. This affinity increases the ratio of COL1A1 to COL1A2 transcripts to 2.3:1. This altered ratio of COL1A1 transcripts would be expected to produce trimers containing only µ1 chains that would ultimately result in a reduction of bone strength.20 Such production of µ1(I) homotrimers was previously observed in a null mutant oim strain of mice that experienced a reduction in bone strength.¹²

Axis-Shield COL1A1 Genetic Assay Method

The Axis-Shield COL1A1 test is a microtiter plate nucleic acid capture and enzyme-linked immunosorbant assay (ELISA) used in the detection of alleles of the COL1A1 genotype (see Figure 2). The assay uses two allele-specific polymerase chain reaction (PCR) techniques. Each PCR chamber contains a conserved 5' biotinylated oligonucleotide and one of two specific oligonucleotides that are complementary to the COL1A1 sequence. The 3' terminal portions of the oligonucleotides correspond to either the normal (G) or variant (T) nucleotide position of the collagen gene. 

Biotinylated PCR products are dependent on the genotype of the sample, and are captured on a 96-well plate by an oligomer that is complementary to a region within the PCR product. A conjugate of streptavidin horseradish peroxidase (HRP) polymer is bound to the biotin label and detected colorimetrically with HRP substrate. After completing the PCR reactions and nucleic acid hybridization, the Axis-Shield assay uses equipment and technology that is widely available in all laboratories that routinely use ELISAs. The Axis-Shield assay relieves laboratories of the need to purchase additional expensive equipment for alternative COL1A1 sequencing methods.

Axis-Shield COL1A1 Genetic Assay Results

Table I. Comparison of COL1A1 genotyping using Axis-Shield (AS), TaqMan, and restriction fragment length polymorphism (RFLP) assays. Total number of DNA samples analyzed was 55. Click to enlarge.

The Axis-Shield test was initially compared to TaqMan and restriction fragment length polymorphism techniques that are commonly used for COL1A1 genotyping (see Table I). In a study of 55 healthy Caucasian individuals, the COL1A1 genotype was: 60% SS normal (33 individuals), 35% Ss heterozygote (19 individuals) and 5% ss homozygote (3 individuals). 

The Axis-Shield assay showed 100% correlation with both of the other methods. The study of the Axis-Shield assay was extended to test a sample of 678 healthy Caucasian individuals. In this second study, the frequency distributions of COL1A1 genotypes were: 64% SS normal (435 individuals), 33% Ss heterozygote (222 individuals) and 3% ss homozygote (21 individuals) (see Table II). In this sample population, 36% of the cohort possessed the COL1A1 polymorphism and had an increased risk of osteoporosis. 

Conclusion

Early identification of individuals with osteopenia, or at a risk of osteoporosis, may prevent the progression of bone mass loss by revealing the importance of positive lifestyle changes and appropriate therapies. Risk factors for osteoporosis include estrogen deficiency (i.e., early menopause), prolonged corticosteroid therapy, maternal familial history of hip fractures, low body mass index (<19 kg/m2), chronic disorders associated with osteoporosis (e.g., anorexia nervosa, malabsorption syndromes, hyperparathyroidism, posttransplantation, chronic renal failure, hyperthyroidism, prolonged immobilization and Cushing’s syndrome), previous fragility fractures, and loss of height (i.e., widow’s hump). 

Table II. Abundance of COL1A1 polymorphism in 36% of the healthy Caucasian population as determined using an Axis-Shield assay. Results were obtained from testing 678 individuals. Click to enlarge.

If these risk factors are identified early enough, individuals could be presented with techniques that would likely protect or prevent them from developing osteoporosis. These techniques include taking in good nutrition (e.g., foods rich in calcium and vitamin D) to build healthy bones, and the practice of weight-bearing and endurance exercise. For elderly individuals, techniques may include the prevention of falls by proper vision care or by using hip protectors, following exercise programs to improve muscle tone, and regular adjustment of medication. In addition, therapies such as HRT can be used to treat postmenopausal women known to be at risk of developing osteoporosis prior to significant loss of BMD. 

Currently, practitioners perform DXA scans to determine BMD levels after an individual presents with osteoporosis symptoms. Once at-risk individuals experience these symptoms, it is often too late for lifestyle changes to offer a significant impact. If genetic screening becomes more widely available than the current methods of measuring BMD, at-risk individuals are likely to be identified earlier and preventative measures taken sooner than they currently are. 

References

Table II. Abundance of COL1A1 polymorphism in 36% of the healthy Caucasian population as determined using an Axis-Shield assay. Results were obtained from testing 678 individuals. Click to enlarge.

1. P Flynn, “Report on Osteoporosis in the European Community.” (Berne, Switzerland: European Communities/European Foundation for Osteoporosis, 1998) [accessed 29 December 2003] available on the Internet: http://www.connect.ie/effo/index.htm. 

2. P Dolan and DJ Torgerson, “The Cost of Treating Osteoporotic Fractures in the United Kingdom Female Population” Osteoporosis International 8 (1998): 611–617.

3. “The Facts About Osteoporosis and Its Impact” (Nyon, Switzerland: International Osteoporosis Foundation, 2003) [accessed 29 December 2003] available on the Internet: http://www.osteofound.org/press_centre/fact_sheet.html. 

4. TL Stewart and SH Ralston, “Role of Genetic Factors in the Pathogenesis of Osteoporosis,” Journal of Endocrinology 166 (2000): 235–245.

5. P Garnero et al., “Markers of Bone Resorption Predict Hip Fracture in Elderly Women: The EPIDOS Prospective Study,” Journal of Bone Mineral Research 11 (1996): 1531–1538.

6. RC Bonney, “Osteoporosis: Its Prevention and Treatment,” European Clinical Laboratory 22 (2003): 8–10.

7. JF Wilson, “Striving for Perfect Balance,” The Scientist 17 (2003): 33–34.

8. SH Ralston, “The Genetics of Osteoporosis” Quarterly Journal of Medicine 90 (1997): 247–251.

9. NK Arden et al., “Ultrasound of the Calcaneus and Hip Axis Length: A Study of Postmenopausal Twins,” Journal of Bone Mineral Research 11 (1996): 530–534.

10. R Dalgleish, “The Human Type I Collagen Mutation Database,” Nucleic Acid Research 25 (1997): 181–187.

11. A Forlino et al., “Use of the Cre/lox Recombination System to Develop a Nonlethal Knock-in Murine Model for Osteogenesis Imperfecta With an a1(I) G349C Substitution” Journal of Biological Chemistry 274 (1999): 37923–37931.

12. SD Chapman et al., “Defective Proa2(I) Collagen Synthesis in a Recessive Mutation in Mice: A Model Human Osteogenesis Imperfecta,” Proceedings of the National Academy of Sciences USA 90 (1993): 1701–1705.

13. SF Grant et al., “Reduced Bone Density and Osteoporosis Associated With a Polymorphic Sp1 Binding Site in the Collagen Type I Alpha 1 Gene,” Nature Genetics 14 (1996): 203-205.

14. P Bornstein et al., “Regulatory Elements in the First Intron Contribute to Transcriptional Control of the Human Collagen Alpha 1 (I) Collagen Gene,” Proceedings of the National Academy of Sciences USA 84 (1987): 8869–8873.

15. RW Keen et al., “Association of Polymorphism at the Type I Collagen (COL1A1) Locus With Reduced Bone Mineral Density, Increased Fracture Risk, and Increased Collagen Turnover,” Arthritis and Rheumatism 42 (1999): 285–290.

16. M Weichetova et al., “COL1A1 Polymorphism Contributes to Bone Mineral Density to Assess Prevalent Wrist Fractures” Bone 26 (2000): 287–290.

17. AG Uitterlinden et al., “Relation of Alleles of the Collagen Type I a1 Gene to Bone Density and Risk of Osteoporotic Fractures in Postmenopausal Women” New England Journal of Medicine 338 (1998): 1016–1022.

18. BL Langdahl et al., “An Sp1 Binding Site Polymorphism in the COL1A1 Gene Predicts Osteoporotic Fractures in Men and Women” Journal of Bone Mineral Research 13 (1998): 1384–1389.

19. FEA McGuigan, DM Reid, and SH Ralston, “Susceptibility to Osteoporotic Fracture is Determined by Allelic Variation at the Sp1 Site, Rather Than Other Polymorphic Sites, at the COL1A1 Locus” Osteoporosis International 11 (2000): 338–343.

20. V Mann et al., “A COL1A1 Sp1 Binding Site Polymorphism Predisposes to Osteoporotic Fracture by Affecting Bone Density and Quality” Journal of Clinical Investigation 107 (2001): 899–907. 

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