Genomic alterations in abnormal neutrophils isolated from adult patients with systemic lupus erythematosus.

Namrata Singh,Pamela Traisak,Kayla A Martin,Michael F Denny,Philip L Cohen,Mariana J Kaplan

doi:10.1186/ar4681

Abstract

IntroductionPatients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell. Because a similar discrepancy in maturation status is observed in myelodysplasias, and disruption of neutrophil development is frequently associated with genomic alterations, genomic DNA isolated from autologous pairs of LDGs and normal-density neutrophils was compared for genomic changes.MethodsAlterations in copy number and losses of heterozygosity (LOH) were detected by cytogenetic microarray analysis. Microsatellite instability (MSI) was detected by capillary gel electrophoresis of fluorescently labeled PCR products.ResultsControl neutrophils and normal-density SLE neutrophils had similar levels of copy number variations, while the autologous SLE LDGs had an over twofold greater number of copy number alterations per genome. The additional copy number alterations found in LDGs were prevalent in six of the thirteen SLE patients, and occurred preferentially on chromosome 19, 17, 8, and X. These same SLE patients also displayed an increase in LOH. Several SLE patients had a common LOH on chromosome 5q that includes several cytokine genes and a DNA repair enzyme. In addition, three SLE patients displayed MSI. Two patients displayed MSI in greater than one marker, and one patient had MSI and increased copy number alterations. No correlations between genomic instability and immunosuppressive drugs, disease activity or disease manifestations were apparent.ConclusionsThe increased level of copy number alterations and LOH in the LDG samples relative to autologous normal-density SLE neutrophils suggests somatic alterations that are consistent with DNA strand break repair, while MSI suggests a replication error-prone status. Thus, the LDGs isolated have elevated levels of somatic alterations that are consistent with genetic damage or genomic instability. This suggests that the LDGs in adult SLE patients are derived from cell progenitors that are distinct from the autologous normal-density neutrophils, and may reflect a role for genomic instability in the disease.Electronic supplementary materialThe online version of this article (doi:10.1186/ar4681) contains supplementary material, which is available to authorized users.

Highlights

Patients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell
Copy number alterations are present in LDGs isolated from human SLE patients To determine whether the LDGs isolated from human SLE patients have evidence of genomic instability, we applied diagnostic techniques commonly utilized in cancer research
Cytogenetic microarray analysis was used to identify copy number alterations, as well as copy number-neutral losses of heterozygosity (LOH), in genomic DNA isolated from autologous pairs of LDGs and normal-density neutrophils

Summary

Introduction

Patients with systemic lupus erythematosus (SLE) have an abnormal population of neutrophils, called low-density granulocytes (LDGs), that express the surface markers of mature neutrophils, yet their nuclear morphology resembles an immature cell. Research toward defining the importance of additional cell lineages in the development of SLE is ongoing, and alterations in granulocyte function have been identified in both pediatric and adult SLE [12,13,14,15] In this regard, an intriguing population of abnormal granulocytes has been identified and isolated from SLE patients [12,13,14,16,17]. LDGs mediate enhanced proinflammatory and cytotoxic responses compared to those of autologous normal-density neutrophils [12,13] These LDGs readily induce endothelial cell death and spontaneously form neutrophil extracellular traps (NETs) [12,13]. Mechanisms other than in situ activation may contribute to the development of LDGs in SLE patients

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