Development of a New Biomarker for the Diagnosis of Frailty

Abstract

Frailty is a physical state marked by a lack of physiological reserve and resilience(1). At present the diagnosis of frailty is based on accepted clinical diagnostic tools. Fried et al.’s Frailty Phenotype and Rockwood and Mitnitski’s Frailty Index are the widely accepted clinical diagnostic tools for frailty at the present time(2-6). A biomarker must be reflective of the individual’s biological state or pathological process(es) while also being associated with high validity, reliability and reproducibility(7). Designing a biomarker for frailty has been challenging due to the presence of overlapping age-related syndromes and the lack of sensitivity and specificity demonstrated in previous attempts to identify frailty(8). In addition, the lack of a solid understanding of the pathophysiology of frailty has also been challenging when determining an associated biomarker(9). Previous attempts at detecting a relationship between biomarkers, which include interleukin-6 (IL6), C-reactive protein (CRP), haemoglobin levels, albumin level and frailty have not been promising due to the presence of confounding factors with the ageing process itself or due to the presence of other co-morbidities(9, 10). Individuals with frailty show deficits across multiple physiological systems. The mesenchymal system, which originates from the mesenchymal stem cells (MSCs), has been not only associated with the pathophysiology but also with potential treatments for this condition. We thus hypothesised that frailty could not only constitute a mesenchymal disease but also that biomarkers could be generated from testing the MSCs population. The clinical and phenotypical characteristics associated with frailty are also observed in individuals with Hutchinson Guilford Progeria syndrome (HGPS), a state of accelerated ageing where the affected individuals have defective MSCs biology and lamin A/C processing (11). Patients with HGPS show the typical manifestations of frailty predominantly in the musculoskeletal system. For the purpose of constructing this thesis we propose that alteration in lamin A/C expression in mesenchymal stem cells (MSCs) could also constitute a potential biomarker of frailty. Cells from mesenchymal origin can form various lineage cells, including osteoblasts, adipocytes, and myocytes(12, 13). When these cells with bone-forming capabilities are obtained from blood, they are collectively known as circulating osteogenic progenitor (COP) cells(12). We are therefore, interested in isolating COP cells from the peripheral circulation while also determining their lamin A/C levels to assess whether COP cells and their levels of lamin A/C expression can be used as robust biomarkers of frailty. As the number of COP cells present in the peripheral circulation is low, we developed special flow-cytometry techniques to isolate these cells from the peripheral circulation(14). We also identify the normal reference range for these cells in the circulation which is 0.1%–3.8% of the total peripheral blood mononuclear cell (PBMC) population. Overall, we wish to investigate whether the percentage of COP (%COP) and the lamin A/C levels in those COP cells can serve as a marker of ageing and frailty. To test this hypothesis, we conducted cross-sectional studies. In the Nepean Osteoporosis and Frailty (NOF) study, we recruited healthy volunteers and grouped them according to their frailty status based on the Fried’s Frailty Phenotype (mean age: 82.8 years; N=77; 70% female; 27 non-frail, 23 pre-frail, and 27 frail). We collected the peripheral blood samples, and the %COP cells were quantified using flow cytometry. Logistic regression models showed that older adults have significantly lower %COP values (p < 0.001). Further, the NOF study also demonstrated that low %COP values are associated with disability and poor physical performance (p < 0.001). We also observed that those older adults with %COP in the lower quartile are more likely to be frail (odds ratio: 2.65; 95% confidence interval [CI] : 2.72–3.15; p < 0.001). We, therefore, conclude that a low %COP value is associated with disability and frailty(15). Our next aim is to determine the relationship between %COP with gender and age as well as to define the normal reference range for COP cells in humans. For this purpose we designed a cross-sectional study, ‘Age, gender, and percentage of circulating osteoprogenitor (COP) cell study’(16). In this study we examined 144 healthy participants (20–99-years-old; 10 males and 10 females per age decade). However, in the COP study, we do not go on to observe a significant difference in %COP according to age, gender, height, weight, or body mass index(BMI). We also analysed the lamin A/C expression levels in COP cells and buccal swabs (66 subjects; median age: 74 years; 60% female; 26 non-frail, 23 pre-frail, and 17 frail) of the NOF study participants. Our results do not show a significant relationship between lamin A/C expression in buccal cells with either disability or frailty. Low lamin A/C expression values in COP cells are, however, associated with frailty. Frail individuals show 60% lower levels of lamin A/C levels compared to non-frail individuals (95% CI: −36% to −74%; p < 0.001); frail individuals also have 62% lower levels of COP cells in comparison to pre-frail individuals (95%CI: −40 to −76%; p < 0.001)(16). In summary, in this thesis, we report not only the normal reference range of COP cells in humans, which was unknown, but also report that lower %COP values are associated with frailty and disability. We also found that low lamin A/C expression in COP cells is strongly associated with frailty. However, future longitudinal studies with larger numbers of volunteers are suggested to further clarify this matter and also to bring better understanding of how the COP cells, and the lamin A/C levels, can serve as a risk stratifiers, biomarkers, therapeutic targets, and as predictors of disability in frail older adults.