Research Article: Low genetic heterogeneity of copy number variations (CNVs) in the genes encoding the human deoxyribonucleases 1-like 3 and II potentially relevant to autoimmunity

Date Published: April 25, 2019

Publisher: Public Library of Science

Author(s): Misuzu Ueki, Junko Fujihara, Kaori Kimura-Kataoka, Kazuo Yamada, Yoshikazu Takinami, Haruo Takeshita, Reiko Iida, Toshihiro Yasuda, Serdar Bozdag.


Deoxyribonucleases (DNases) might play a role in prevention of autoimmune conditions such as systemic lupus erythematosus through clearance of cell debris resulting from apoptosis and/or necrosis. Previous studies have suggested that variations in the in vivo activities of DNases I-like 3(1L3) and II have an impact on autoimmune-related conditions. The genes for these DNases are known to show copy number variations (CNVs) whereby copy loss leads to a reduction of the in vivo activities of the enzymes, thereby possibly affecting the pathophysiological background of autoimmune diseases. Using a simple newly developed quantitative real-time PCR method, we investigated the distributions of the CNVs for DNASE1L3 and DNASE2 in Japanese and German populations. It was found that only 2 diploid copy numbers for all of these DNASE CNVs was distributed in both of the study populations; no copy loss or gain was evident for any of the autoimmune-related DNase genes. Therefore, it was demonstrated that these human autoimmune-related DNase genes show low genetic diversity of CNVs resulting in alterations of the in vivo levels of DNase activity.

Partial Text

It has been suggested that deoxyribonuclease (DNase)-mediated clearance of cell debris resulting from apoptosis and/or necrosis might be primarily involved in the prevention of autoimmune conditions such as systemic lupus erythematosus (SLE) [1,2]. In the context of autoimmunity, it has been postulated that DNase I-like 3 (DNase 1L3) in serum break down chromatin during apoptosis and/or necrosis [3,4], while DNase II in lysosomes is involved in the degradation of endogenous DNA in apoptotic cells that have been engulfed by macrophages [5,6].

Blood samples were collected from healthy unrelated Japanese (n = 265) and German (n = 80) individuals after obtaining written informed consent. DNA was extracted from each blood sample using a QIAamp DNA Mini Kit (Qiagen, Chatsworth, CA, USA). The study was approved by the Human Ethics Committee of Shimane University School of Medicine (the approval number 1024 for the Human Genome and Genetics Analysis Study).

We selected common region as a target CNV regions for amplification by Q-PCR analysis, covering all the CNVs in the gene encoding DNase 1L3 including loss or gain of copy, resulting in alterations in the activity of each enzyme: exon 1 in DNASE1L3 and exon 1 in DNASE2 (Fig 1). The specificity of the products amplified was evaluated by melting curve analysis for both DNASE1L3 and DNASE2 CNVs (S1 Fig). In the Q-PCR analysis, standard curves for each of the target CNV regions, together that for the single-copy ZNF80 gene as a reference, were generated using a 2-fold serially diluted series of each chimeric vector as a template (Fig 2). For DNASE CNV analysis, the PCR efficiencies for the ZNF80 and the target DNASE1L3 CNV were 96.6±1.95% (slope coefficient, -3.41±0.0488) and 97.1±2.95% (slope coefficient, -3.39±0.0759), respectively, while those for the ZNF80 and the target DNASE2 CNV were 97.5±1.87% (slope coefficient, -3.38±0.0472) and 97.5±2.39% (slope coefficient, -3.39±0.0615), respectively. Since the quantitative accuracy of Q-PCR analysis depends on proper normalization, these results demonstrated that copy number estimation using this method was reliable. Furthermore, in order to confirm the validity of copy number determination using this newly developed CNV analysis method, simulated analysis of the DNASE1L3 CNV copy number was performed using mixtures of three vectors–pCRII/DN1L3, pCRII/ZNF or DN1L3-ZNF–in varying proportions as a template. All of the simulated assays yielded the expected number of copies (Fig 3), indicating the reliability of this method. Therefore, this simple, newly developed Q-PCR method for CNV analysis permits reliable determination of copy number in the CNVs located in these DNase genes.

Several CNVs have been found in the genes encoding DNases 1L3 and II which are implicated in autoimmune diseases. In the present study, we focused on the target region common to all of the CNVs in DNASE1L3, together with that for CNV in DNASE2, likely giving rise to alterations in the in vivo levels of enzyme activity through loss or gain of copy in the CNVs (Fig 1). It was anticipated that this would allow evaluation of the distribution of the relevant CNVs in the genes, thus clarifying the overall effect of the CNVs on in vivo enzyme activities. For the CNV analysis, a simple and reliable newly developed Q-PCR method was employed (Figs 2 and 3, and S1 Fig). To our knowledge, the present study is the first to have comprehensively clarified the distribution of the CNVs in the autoimmune-related DNase genes, DNASE1L3 and DNASE2.