Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia

Abstract

Background: Chronic idiopathic neutropenia is characterized by immune-mediated suppression of neutrophil production. Because patients with immune-mediated bone marrow failure syndromes display age-inappropriate telomere shortening in leukocytes, we investigated telomere lengths in peripheral blood mononuclear cells and granulocytes of patients with chronic idiopathic neutropenia.

Design and methods: We studied 37 patients with chronic idiopathic neutropenia and 68 age- and sex-matched healthy controls. Relative telomere length and telomerase reverse transcriptase expression were assessed by a quantitative real time polymerase chain reaction. Telomerase activity was determined by a polymerase chain reaction-based immunoassay.

Results: The mean relative telomere values of peripheral blood mononuclear cells and granulocytes were significantly lower in patients compared to controls, and significantly lower than expected on the basis of the age-adjusted healthy control distribution. The difference in the relative telomere lengths between patients and controls in both peripheral blood mononuclear cells and granulocytes was prominent in those under the age of 50 years. Contrary to the peripheral blood mononuclear cells, in which an inverse correlation was observed between relative telomere values and age, no significant correlation was noted between granulocyte telomere values and patient age. A significant correlation was observed between individual relative telomere values and absolute neutrophil counts. There was no difference in expression of telomerase reverse transcriptase in peripheral blood mononuclear cells between patients and controls but telomerase activity was identified at a significantly higher frequency in controls than in patients. No correlation was found between telomerase activity or telomerase reverse transcriptase expression and relative telomere lengths of peripheral blood mononuclear cells.

Conclusions: Patients with chronic idiopathic neutropenia display age-inappropriate telomere shortening of peripheral blood cells and low telomerase activity in peripheral blood mononuclear cells. A compensatory increased proliferation of bone marrow hematopoietic progenitor cells in association with lymphocyte replicative exhaustion probably account for these abnormalities.

Figure 1.

Relative telomere length values of PBMCs in CIN patients and age/sex-matched healthy controls. (A-B) Correlation (regression line ±95% confidence limits) between the relative telomere length values of PBMCs and age in the healthy controls and CIN patients, respectively. Analysis performed by Spearman’s correlation test. The equation, coefficient of correlation (r) and degree of significance (P) are indicated. (C) Observed/predicted relative telomere length ratio (O/P ratio) values for patients and controls. Individual predicted values in patient and control groups were estimated from the equation derived from the linear regression analysis of the correlation between the relative telomere length values and age of the controls shown in (A). The dots ± error bars represent the mean O/P ratio±1SD for each group and the floating bars represent the 95% confidence intervals for each distribution. The mean O/P ratio the in the patient group is from the 95% confidence intervals of the normal group suggesting inappropriate telomere loss by age in CIN. The bars (D) show the mean (+1SD) relative telomere length per decade of years in CIN patients and healthy controls. The comparison between patient and control telomere length distributions has been performed by two-way analysis of variance test which compares multiple mean values; the statistically significant difference (P=0.0391) is shown. A subset analysis of telomere length between patients and controls in the younger (≤ 50 years) and older (>50 years) age groups was performed by means of the Man-Whitney U test. A statistically significant difference was demonstrated in the younger but not the older groups and the respective P values are shown with asterisks. PBMCs: peripheral blood mononuclear cells; CIN: chronic idiopathic neutropenia; SD: standard deviation.

Figure 2.

Relative telomere length values of granulocytes in CIN patients and age- and sex-matched healthy controls. (A-B) Correlation (regression line ± 95% confidence limits) between the relative telomere length values of granulocytes and age in the healthy controls and CIN patients, respectively. Analysis has been performed by Spearman’s correlation test. The equation, coefficient of correlation (r) and degree of significance (P) are indicated. (C) Observed/predicted relative telomere length ratio (O/P ratio) values for patients and controls. Individual predicted values in patient and control groups were estimated from the equation derived from the linear regression analysis of the correlation between the relative telomere length values and age of the controls shown in (A). The dots ± error bars represent the mean O/P ratio±1SD for each group and the floating bars represent the 95% confidence intervals for each distribution. The mean O/P ratio in the patient group is from the 95% confidence intervals of the normal distribution suggesting inappropriate telomere loss by age in CIN. The bars in (D) show the mean (+ 1SD) relative telomere length per decade of years in CIN patients and healthy controls. The comparison between patient and control telomere length distributions was performed by the two-way analysis of variance test (which compares multiple mean values) and did not show a statistically significant difference (P=0.0624). A subset analysis of telomere length between patients and controls in the younger (≤ 50 years) and older (> 50 years) age groups was performed by means of the Mann-Whitney U test. A statistically significant difference was demonstrated in the younger but not the older group and the respective P values are shown with asterisks. CIN: chronic idiopathic neutropenia; SD: standard deviation.

Figure 3.

Relative telomere length values of PBMCs and granulocytes in CIN patients and age- and sex-matched healthy individuals. The bars represent the mean (+ 1SD) relative telomere length in PBMCs and granulocytes of healthy individuals (A) and CIN patients (B). The left bars in each graph correspond to the entire group of healthy subjects or CIN patients whereas the bars on the right show the mean relative telomere length in the younger (≤ 50 years) and older (> 50 years) groups of healthy individuals and CIN patients. Comparison between relative telomere lengths of PBMCs and granulocytes was performed by the Mann-Whitney U test and the statistically significant P values are shown. PBMCs: peripheral blood mononuclear cells; CIN: chronic idiopathic neutropenia; SD: standard deviation; N.S.: non-statistically significant differences.

Figure 4.

Correlation between granulocyte relative telomere values and absolute neutrophil counts in CIN. The regression line (± 95% confidence limits) show the correlation between individual relative telomere length values (estimated as relative telomere/single-copy-gene ratio values) and absolute neutrophil counts in the cohort of CIN patients studied. Coefficient of correlation (r) and degree of significance (P value), according to Spearman’s correlation test, are indicated. CIN: chronic idiopathic neutropenia.