Osteoporosis is a highly prevalent illness, especially among postmenopausal women. Left untreated, it can lead to fragility and compression fractures; in turn, associated with increased mortality and morbidity. Diagnosis of osteoporosis is currently made by bone density scan (DEXA) or when the patient experiences symptoms (fragility or compression fractures).
This scientific report published in Nature is of great significance. For the first-time its authors identify a potential blood test to diagnose osteoporosis, without having patients undergo bone scanning or present with symptoms. The test is Cathepsin Z mRNA and is measured in human peripheral blood mononuclear cells. Cathepsin Z is a protease synthesized by both bone remodeling cells - osteoclast and osteoblasts.
The test is not influenced by acute or chronic inflammation. Its diagnostic positive predictive value (PPV) is 95% with a negative predictive value (NPV) of 80%. Authors found a strong correlation of elevated levels of Cathepsin Z mRNA in patients with osteopenia in addition to those with osteoporosis.
Although study findings need to be fully validated, the results are exciting. Early diagnosis and treatment of osteoporosis are crucial to preventing fractures and its complications.
Osteoporosis, one of the most prevalent chronic age-related bone diseases, often goes undetected until the first fragility fracture occurs, causing patient suffering and cost to health/social care services.
Osteoporosis arises from imbalanced activity of osteoClasts and osteoBlasts. Since these cell lineages produce the protease, cathepsin Z, the aim of this study was to investigate whether altered cathepsin Z mRNA levels are associated with osteoporosis in clinical samples.
Cathepsin Z mRNA in human peripheral blood mononuclear cells was significantly differentially-expressed among non-osteoporotic controls, osteopenia and osteoporosis patients (p < 0.0001) and in female osteoporosis patients over the age of 50 years (P = 0.0016).
Cathepsin Z mRNA level strongly correlated with:
Low bone mineral density (BMD, g/cm2) (p = 0.0149)
Lumbar spine L2-L4 (T-scores) (p = 0.0002)
Femoral neck (T-scores) (p = 0.0139).
Importantly, cathepsin Z mRNA was significantly associated with fragility fracture in osteoporosis patients (P = 0.0018).
The levels of cathepsin Z mRNA were not significantly higher in patients with chronic inflammatory disorders in these two groups compared to those without (P = 0.774 and 0.666, respectively).
ROC analysis showed that cathepsin Z mRNA has strong diagnostic value for osteoporosis and osteoporotic fracture.
The results show for the first time that cathepsin Z could be a future diagnostic biomarker for osteoporosis including female osteoporosis patients over the age of 50 years.
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Osteoporosis is one of most common chronic age-related bone diseases in the world, and is often, but not exclusively, associated with the reduced bone density in postmenopausal women, arising from reduced estrogen levels. Osteoporosis often goes undetected until the first fragility fracture occurs, which causes unnecessary suffering to the patient and cost to the health service. Thus, there is a need to identify biomarkers that could be exploited, in the future, for a clinical test that could indicate the presence of osteoporosis before fragility fracture occurs.
Osteoporosis is thought to arise from an imbalance between the activity of bone degrading osteoClasts (derived from monocyte lineages of immune cells) and bone-synthesising osteoBlasts (derived from mesenchymal stem cells).
The lysosomal cysteine protease, cathepsin Z, believed to be encoded by the same mRNA sequence as cathepsin X, is highly expressed in cells of the immune system.
In particular, it is expressed in T-lymphocytes and monocytes the latter being precursors of osteoClasts, which produce the related protease, cathepsin K that has been shown to be required for bone resorption.
Both T-lymphocytes and monocytes are components of the peripheral blood mononuclear cell (PBMC) fraction that can be isolated from whole blood.
In addition, cathepsin Z protein has been reported to be secreted by human osteoBlasts and thus cathepsin Z appears to be produced in cells of BOTH osteoclast and osteoBlast lineages.
Alterations in the expression of the cathepsin Z gene have been linked, bioinformatically, to fragility fractures in cancer patients in association with reduced levels of circulating estrogen.
A genome wide association study into the genetic factors that may be associated with fragility fractures arising from the treatment of estrogen-receptor-positive breast cancer patients with aromatase inhibitor, identified a single-nucleotide polymorphism in or near a 3-gene cluster on chromosome 20 containing the cathepsin Z gene.
This polymorphism was shown to be associated with the occurrence of fragility fractures. Using an osteoBlast cell culture model system, the estrogen-receptor dependence of cathepsin Z gene expression was demonstrated in these cells. The polymorphism not only affected the expression of the cathepsin Z gene, but experimental knockdown of cathepsin Z levels affected the expression of osteoporosis-associated genes, suggesting an association between cathepsin Z expression, fragility fracture and the expression of osteoporosis-related genes in an estrogen-reduced, clinical situation. However, the direct effect of cathepsin Z on other cell activities was not studied.
That cathepsin Z is found in BOTH osteoBlast and osteoClast lineages of cells, the two cell types predominantly dysregulated in osteoporosis, raises the possibility that the occurrence of osteoporosis in human subjects might be associated with changes in cathepsin Z expression in these cells.
In the present experiments, it is shown that levels of cathepsin Z mRNA in clinical samples of peripheral blood mononuclear cells are significantly associated with:
Low bone mineral density, and
Occurrence of fragility fractures.
Cathepsin Z mRNA has been shown for the first time to be significantly elevated in the PBMCs of patients with either osteopenia or osteoporosis compared to non-osteoporotic controls.
For all participants, the mean ages of the non-osteoporotic control, osteopenia and osteoporosis groups were significantly different at 56, 65 and 69 years with the ages of the osteopenia and osteoporosis groups being significantly different from the non-osteoporotic control group (P = 0.025 and 0.0045 respectively, ANOVA post hoc Dunnett multiple comparison with a control).
Thus, we cannot rule out entirely the possibility that cathepsin Z levels are, in part, a function of age for all participants. However, the increase in cathepsin Z mRNA level in osteopenia/osteoporosis was also observed when male and female participants over the age of 50, or female participants over the age of 50 were analysed.
In these cases, the ages of the osteopenia and osteoporosis groups were not significantly different from the non-osteoporotic controls (male and female participants over the age of 50, osteopenia, P = 0.23 and osteoporosis P = 0.97; females over the age of 50, osteopenia, P = 0.185 and osteoporosis, P = 0.727, ANOVA post hoc Dunnett multiple comparison with a control). These observations suggest that at least for participants over the age of 50, age is not an important factor in the increase of cathepsin Z mRNA in osteopenia/osteoporosis.
That the PBMC preparations consist predominantly of immune cells raises the possibility that the observed osteoporosis-related increase in cathepsin Z mRNA reflects a change in the immune state of the osteopenia/osteoporosis patients relative to the normal controls. For example, osteoporosis could be a comorbidity with chronic inflammatory disorders, such as arthritis, COPD, coeliac disease.
In the present group of participants, only 17% were recorded as suffering from chronic inflammatory conditions. However, in patients with osteopenia and osteoporosis, the cathepsin Z mRNA levels were not significantly different between those with and those without inflammatory disorders (P = 0.774).
The results suggest that the increase in cathepsin Z mRNA in patients’ PBMCs was independent of chronic inflammation, at least in this group of patients.
Similarly, no evidence was found for cathepsin Z mRNA levels being associated with virally-induced inflammation via the γ interferon pathway, although a more direct assessment of chronic inflammation, by directly assaying the blood levels of inflammatory cytokines, such as tumour necrosis factor (TNF alpha) would help to confirm these findings.
Cathepsin Z mRNA was measured in PBMC preparations from human subjects. Such preparations contain many types of circulating cells of the immune system, including both B and T lymphocytes, dendritic cells and CD14+, CD16− monocytes, some of the latter being precursors of osteoClasts.
Cathepsin Z mRNA/protein has been reported to be present at varying levels in all these cell types. Thus, in the present study, the precise cell type that is the source of the elevated cathepsin Z mRNA is unknown. It has been reported, using PBMC preparations, that the mature form of cathepsin Z interacts with lymphocyte function-associated antigen-1 (LFA-1), a β-2-integrin on the surface of T lymphocytes.
Using specific cathepsin Z inhibitors, this interaction has been shown to alter T lymphocyte behaviour, for example, increasing activated proliferation rates in PBMCs and migration, invasion and aggregation using a cultured T cell line.
Cathepsin Z can also alter the behaviour of differentiated monocytes, by interacting with the Mac-1 β2-integrin. Thus, the presence of elevated levels of cathepsin Z mRNA/protein in the PBMC cells of osteoporosis patients, might be associated with altered behaviour of monocytes and T lymphocytes. Whilst monocytes are the precursors of osteoClasts, the role of T lymphocytes and the various subtypes in osteoporosis has been recognised, but is not yet fully understood.
Irrespective of the biological effects of cathepsin Z, it has now been shown for the FIRST TIME here that the increase in cathepsin Z mRNA in isolated PBMC fractions is very strongly diagnostic for osteoporosis, at least in the patient group studied.
Overall, the predictive value of a positive test (PPV) was 95%, the post-test likelihood of no disease following a negative test (NPV) was 80%, whilst the post-test likelihood of disease following a negative test was thus only 20%.
In those osteoporosis patients who had experienced a previous fracture, the predictive value of a positive test was similar at PPV 92%, with the post-test likelihood of no disease following a negative test rising to NPV 100%.
These results for cathepsin Z mRNA as a single biomarker are comparable to a peripheral blood biomarker panel consisting of 10 mRNAs including cathepsin Z mRNA for diagnosis of ischaemic stroke. Furthermore, the observation that subjects with osteopenia also showed a significant increase in cathepsin Z mRNA compared to non-osteoporotic controls, strongly suggests that if replicated in a larger study, the cathepsin Z mRNA in patients’ PBMC preparations could form the basis of a test for osteoporosis, which could aid in the detection of osteoporosis before a critical and expensive fragility fracture occurs.