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. 2014 Aug 8;16(4):R165.
doi: 10.1186/ar4681.

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

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

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

Namrata Singh et al. Arthritis Res Ther. .

Abstract

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. 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.

Methods: Alterations 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.

Results: Control 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.

Conclusions: The 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.

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Figures

Figure 1
Figure 1
LDGs isolated from SLE patients have greater levels of copy number alterations relative to control neutrophils and autologous neutrophils. (A) Genomic DNA from nine healthy female donors and thirteen SLE patients was analyzed by cytogenetic microarray analysis. Values are mean ± SEM. *Distribution differs significantly from autologous normal-density SLE neutrophils, Wilcoxon signed-rank test, one-tailed, P <0.01. (B) The incidence of genomic duplications and deletions was similar in the neutrophil samples isolated from the healthy controls and SLE patients, whereas the autologous LDGs had a significant increases in the number of deletions (a) Wilcoxon signed-rank test, one-tailed, P <0.01) and duplications (b) Wilcoxon signed-rank test, one-tailed, P <0.01). Values are mean ± SEM. (C) Copy number variations for each healthy control (open blue diamonds, n = 9), and autologous pairs of SLE neutrophils (open red squares) and LDGs (filled red squares) are indicated. LDG samples with 18 or more copy number variations were considered ∆CNVhi. Autologous sample pairs for each SLE patient are indicated by a solid black line for ∆CNVhi, and a dashed gray line for the ∆CNVneg. CNV, copy number variation; LDG, low-density granulocyte; SEM, standard error of the mean; SLE, systemic lupus erythematosus.
Figure 2
Figure 2
Selective distribution of acquired copy number variations in LDGs to specific chromosomes. The frequency of copy number variations in healthy controls (open blue bar), SLE neutrophils (open red bar) and autologous LDGs (filled red bar) are displayed. *Significant increase in copy number alterations located on chromosome 19 were detected in LDGs relative to control neutrophils (Kruskal-Wallis test with Mann-Whitney post hoc analysis, one-tailed, P <0.01). Trends for increases levels of CNVs in the LDGs were also suggested for chromosomes 8, 17, and X. CNV, copy number variation; LDG, low-density granulocyte; SLE, systemic lupus erythematosus.
Figure 3
Figure 3
The copy number alterations on chromosome 19 are localized to particular intervals. Chromosome 19 is shown in detail, and the relative locations of individual duplications (upward filled arrowheads) and deletions (downward open arrowheads) are indicated for each healthy control sample (dashed blue line adjacent to ideogram), SLE neutrophil (dashed red line) and autologous LDG sample (solid red line). CNVs in the LDGs were predominantly within regions in 19p13 encoding the MUC16 gene, intergenic intervals in 19p12 within a ZNF gene cluster, 19q12-q13 containing several Pregnancy-specific glycoproteins, and 19q13.4 amid an miRNA cluster. CNV, copy number variation; LDG, low-density granulocyte; miRNA, microRNA; SLE, systemic lupus erythematosus.
Figure 4
Figure 4
Losses of heterozygosity (LOH) in LDGs from SLE patients. (A) LOH are prevalent in the ∆CNVhi SLE patients. The mean ± SEM of LOH >2 Mb for healthy controls (open blue bar, n = 9), SLE neutrophils (red open bar) and SLE LDGs (red filled bar). ∆CNVneg (n = 7) and ∆CNVhi (n = 6) SLE patients are indicated by unhatched and black-hatched bars, respectively. *Differs significantly from autologous normal-density SLE neutrophils, Wilcoxon signed-rank test, one-tailed, P <0.01. (B) A LOH at 5q23-q31 is observed in four SLE patient samples. The autologous pairs of neutrophils and LDGs are indicated by the dashed red line and solid red line, respectively. The affected 3.5 Mb interval is expanded to show the relative positions of genes indicated in the text, additional genes within the interval are not shown for the purposes of clarity. SLE patient 11 has a loss of heterozygosity that is restricted to the LDG sample. Refer to Additional file 5 information regarding SLE patients. Genomic positions based upon Human Genome Assembly Build 36.3. CNV, copy number variation; LDG, low-density granulocyte; SEM, standard error of the mean; SLE, systemic lupus erythematosus.
Figure 5
Figure 5
Microsatellite instability (MSI) in SLE LDGs. Representative chromatographic traces for capillary gel electrophoresis analysis of duplex PCR reactions for the quasimonomorphic BAT25 and NR22 microsatellites. The size of the BAT25 amplicon differs between an autologous pair of SLE neutrophils (red line) and LDGs (blue line). The NR22 product was scored as stable in this SLE patient since the primary peak in the LDGs is the same size as the autologous normal-density neutrophils. LDG, low-density granulocyte; SLE, systemic lupus erythematosus.
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