SEARCH

SEARCH BY CITATION

Keywords:

  • autoimmunity;
  • DNase I;
  • DNase I-like 3 (1L3);
  • functional SNP;
  • genetic distribution

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The objectives of this study were to evaluate all the non-synonymous single nucleotide polymorphisms (SNPs) in the DNase I and DNase I-like 3 (1L3) genes potentially implicated in autoimmune diseases as a functional SNP in terms of alteration of the activity levels. We examined the genotype distributions of the 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, in three ethnic groups, and the effect of these SNPs on the DNase activities. Among a total of 44 and 25 SNPs including those characterized in our previous studies [Yasuda et al., Int J Biochem Cell Biol42 (2010) 1216–1225; Ueki et al. Electrophoresis32 (2012) 1465–1472], only four and one, respectively, exhibited genetic heterozygosity in one or all of the ethnic groups examined. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, 11 activity-abolishing and 11 activity-reducing SNPs in DNASE1 and two activity-abolishing and five activity-reducing SNPs in DNASE1L3 were confirmed as a functional SNP. Phylogenetic analysis showed that all of the amino acid residues in activity-abolishing SNPs were completely or well conserved in animal DNase I and 1L3 proteins. Although almost all non-synonymous SNPs in both genes that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of 13 activity-abolishing SNPs producing a loss-of-function variant in both the DNase genes would be a direct genetic risk factor for autoimmune diseases. These findings may have clinical implications in relation to the prevalence of autoimmune diseases.


Abbreviation
MAF

minor allele frequency

RFLP

restriction fragment length polymorphism

SLE

systemic lupus erythematosus

SNP

single nucleotide polymorphism

SRED

single radial enzyme diffusion

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Endogenous deoxyribonuclease I (DNase I, EC3.1.21.1) and DNase I-like 3 (DNase 1L3) have been noted as candidate endonucleases involved in the breakdown of chromatin during apoptosis and/or necrosis [1-3]. DNase 1L3 is a member of the DNase I family, which also includes DNase I-like 1/DNase X/DNase Xib and DNase I-like 2, whose nucleotide and amino acid sequences resemble those of the originally characterized DNase I [4, 5]. In this context, triggered by nuclear antigens, the clearance of cell debris resulting from cell death through apoptosis and/or necrosis might be primarily involved in the prevention of autoimmune conditions such as systemic lupus erythematosus (SLE); DNase I has been especially highlighted for its possible involvement in the pathogenesis of autoimmune diseases [6-8]. Yasutomo et al. [9] and Dittmar et al. [10] have identified novel nonsense (p.Lys5Ter) and missense (p.Val111Met) mutations that abolished and reduced DNase I activity, respectively, in patients with autoimmune diseases [11]. Recently, in an SLE patient, Al-Mayouf et al. [12] found a null mutation resulting from a homozygous 1-bp deletion (c.643delT; p.Trp215GlyfsX2) and also identified homozygosity for a missense mutation (p.Arg206Cys), the latter producing a loss-of-function variant of DNase 1L3 [13]. It has also been reported that DNase I deficient mice develop an SLE-like syndrome [14], and that lupus-prone MRL and NZB/W F1 mice have impaired DNase 1L3 activity [15]. These findings suggest that mutation and/or single nucleotide polymorphism (SNP) in the genes encoding DNase I and 1L3 (DNASE1 and DNASE1L3, respectively), resulting in variants that are inactive or have low activity, might be substantially responsible for the genetic background determining susceptibility to autoimmune diseases. Furthermore, Napirei et al. have reported that serum DNase I and DNase 1L3 might complement or substitute each other during chromatin degradation [16]. Therefore, in order to clarify the genetic basis of the etiological role of endonucleases in autoimmune diseases, simultaneous evaluation of functional SNPs in both DNASE1 and DNASE1L3 is warranted.

Many SNPs in the DNASE1 and DNASE1L3 have been screened and are available on the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) (Figs 1 and 2). We have been focusing on non-synonymous SNPs that are likely to serve as a functional SNP, and in fact have previously performed genetic and expression analysis of 12 and 5 such SNPs in DNASE1 and DNASE1L3, respectively [11, 13, 17, 18]. Since then, 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, which could potentially affect the activity, have been registered in the database. However, only limited population data are available for these SNPs. Furthermore, it is still unclear whether the amino acid substitutions in the DNase I and 1L3 proteins corresponding to each non-synonymous SNP affect their DNase activities. Therefore, comprehensive data on the biochemical-genetic aspects of these SNPs in DNASE1 and DNASE1L3 potentially affecting the in vivo activity would probably be useful for clarifying their functionality in determining genetic predisposition to disease.

image

Figure 1. Schematic representation of the genomic structure of the human DNASE1 and the position of each SNP examined in this study. SNP nomenclature is based on the recommendations for description of sequence variants (http://www.hgvs.org/mutnomen/examplesDNA.htlm); the sequence GenBank accession no. AB188151 has been used as the genomic and coding DNA reference sequence. Each ID number in the NCBI database is shown. Exons are shown by solid boxes, in which solid and clear boxes correspond to the translated and untranslated regions of the mRNA, respectively. According to the alteration in the levels of DNase I activity resulting from the corresponding amino acid substitution, 44 non-synonymous SNPs in DNASE1 could be classified into 15 SNPs not affecting the activity, 11 abolishing the activity, 11 reducing the activity and seven elevating the activity; each SNP of these four groups is shown at the site of located exons.

Download figure to PowerPoint

image

Figure 2. Schematic representation of the genomic structure of the human DNASE1L3 and the position of each SNP examined in this study. SNP nomenclature is based on the recommendations for description of sequence variants (http://www.hgvs.org/mutnomen/examplesDNA.htlm); the NCBI Reference Sequence NM_004944.3 has been used as the genomic and coding DNA reference sequence. Each ID number in the NCBI database is shown. Exons are shown by solid boxes, in which solid and clear boxes correspond to the translated and untranslated regions of the mRNA, respectively. According to the alteration in the levels of DNase 1L3 activity resulting from the corresponding amino acid substitution, 25 non-synonymous SNPs/mutants in DNASE1L3 could be classified into 18 SNPs not affecting the activity, two abolishing the activity and five reducing the activity; each SNP of these three groups is shown at the site of located exons.

Download figure to PowerPoint

In the present study, we investigated the genotype distribution of the 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, in three ethnic groups (Asian, African and Caucasian), using a novel genotyping method for each SNP, and the effects of all these SNPs on enzymatic activities in order to evaluate the functionality of each SNP. Furthermore, multiple alignment analysis of the amino acid sequences of animal DNase I and 1L3, including phylogenetic analysis, was performed to evaluate the role of each SNP-related amino acid residue in these DNase proteins.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Development of a simple genotyping procedure for each SNP in DNASE1 and DNASE1L3 using a PCR-RFLP method

A simple and novel genotyping procedure was developed using PCR restriction fragment length polymorphism (RFLP) for all of the 32 and 20 SNPs in DNASE1 and DNASE1L3, respectively; those for another 12 and 5 SNPs, respectively, had been developed previously [11, 13, 17, 18]. As the substitution sites corresponding to the 19 and 11 SNPs in DNASE1 and DNASE1L3, respectively, neither suppressed nor created any known restriction enzyme recognition sites, we employed a mismatched PCR amplification method [19] for genotyping; incorporation of a deliberate mismatch close to the 3′-terminus of a PCR primer allows the creation of a recognition site for each enzyme. After digestion of the amplified DNA fragment for each SNP, the appearance of the expected product, as shown in Tables S1 and S2, derived from the respective alleles in each SNP allowed us to determine the genotypes easily; in SNP p.Gly3Asp of DNASE1, the amplified product derived from the C-allele was completely digested with MspI to yield a 115-bp fragment, whereas that from the A-allele did not yield such fragment. The same procedure was employed for the other SNPs. In order to confirm the validity of the genotyping results obtained by these methods, a direct sequencing analysis of the genomic DNA region including the substitution site of each SNP in DNASE1 and DNASE1L3 derived from several representative subjects was performed. The genotyping results for each SNP using a PCR-RFLP method were completely congruent with those using a direct sequencing method, indicating the validity of the novel genotyping methods developed in this study.

Genotype distribution of the 44 non-synonymous SNPs in DNASE1 and 25 non-synonymous SNPs in DNASE1L3 for three ethnic groups

As shown in Tables S3 and S4, among the 44 non-synonymous SNPs in DNASE1, only four (p.Arg2Ser, p.Tyr117Ser, p.Gly127Arg and p.Arg244Gln) exhibited genetic heterozygosity in some or all of the ethnic groups examined, whereas the others were found to be distributed in a mono-allelic manner; all of the subjects were genotyped as homozygous for the predominant allele in the latter SNPs. On the other hand, among 25 non-synonymous SNPs in DNASE1L3, only SNP c.Arg206Cys was found to be polymorphic in the Caucasian populations without a well-balanced distribution; the other 24 SNPs were found to be distributed in a mono-allelic manner in all of the populations examined. In particular, a mutation identified in the patient with SLE, c.643delT (p.Trp215GlyfsX2) [12], was not found in any of the groups, being compatible with its absence from the database.

Effect of amino acid substitution resulting from non-synonymous SNPs in each DNase on the expression of enzyme activity

Forty-four and 25 amino acid substituted constructs in the DNase I and 1L3 protein, respectively, derived from the minor allele in each SNP were prepared separately and transiently expressed in COS-7 cells; the resulting DNase activity in the transfected cells was determined by the single radial enzyme diffusion (SRED) method. Results for the activity-altered construct, corresponding to the activity-reducing or activity-elevating SNPs, of each DNase are presented in Figs 3 and 4.

image

Figure 3. Relative activity of amino acid substituted constructs corresponding to non-synonymous SNPs to that of the wild-type DNase I. Among 44 non-synonymous SNPs in DNASE1, only functional SNPs altering the activity levels are shown. The DNase I activity in the cells transfected with each construct was assayed by the SRED method [11]. The bar gives the SD (n = 4).

Download figure to PowerPoint

image

Figure 4. Relative activity of amino acid substituted constructs corresponding to non-synonymous SNPs to that of the wild-type DNase 1L3. Among 25 non-synonymous SNPs/mutants in DNASE1L3, only functional SNPs altering the activity levels are shown. The DNase 1L3 activity in the cells transfected with each construct was assayed by the SRED method [13]. The bar gives the SD (n = 4).

Download figure to PowerPoint

The levels of DNase I activity derived from the Q31E, R53C, Y54C, Q60H, V111M, R139G, P159L, D167H, R207C, Q244R and A246P constructs were significantly lower than that of the wild-type enzyme. Obviously, substitution of the Gln, Tyr, Gln, Val, Arg, Pro and Gln residues at positions 31, 54, 60, 111, 139, 159 and 244, respectively, in the DNase I protein reduced the activity to about 20–50% of that of the wild-type enzyme, and furthermore substitution of Arg, Arg and Ala residues at positions 53, 207 and 246, respectively, reduced the activity markedly to less than 20%. On the other hand, compared with the activity level of the wild type, the activity levels of Y46H, V70L, D120N, G127R, R143Q, P154A and A168V were significantly high; substitution of Val, Arg, Pro and Ala residues at positions 70, 143, 154 and 168, respectively, in the protein elevated the DNase I activity to about 1.5-fold that of the wild-type enzyme, and furthermore substitution of Tyr, Asp and Gly residues at positions 46, 120 and 127 by His, Asn and Arg, respectively, elevated the activity greatly to about 200%. It is noteworthy that the 11 constructs, K5Ter, Q60R, R107G, R133Q, R133L, F140C, D190H, N192I, C231Y, R235W and R244Ter, exhibited no DNase I activity under our assay conditions. Therefore, among the SNPs affecting the enzyme activity, DNase I activity was found to be abolished by amino acid substitutions resulting from p.Gln60Arg, p.Arg107Gly, p.Arg133Gln, p.Arg133Leu, p.Phe140Cys, p.Asp190His, p.Asn192Ile, p.Cys231Tyr and p.Arg235Trp and nonsense substitutions from p.Lys5Ter and p.Arg244Ter. However, the levels of DNase I activity derived from the other amino acid substituted constructs were virtually similar to that of the wild type, indicating that substitutions of Arg, Gly, Lys, Ala, Ala, Glu, Arg, Val, Tyr, Asp, Asn, Val, Val, Pro and Gly residues at positions 2, 3, 5, 14, 26, 35, 95, 114, 117, 129, 132, 172, 185, 219 and 262, respectively, in the DNase I protein exert little effect on the activity. It was demonstrated that, among the amino acid residues related to non-synonymous SNPs in the DNase I protein, each amino acid residue at positions 31, 46, 54, 70, 111, 120, 127, 139, 143, 154, 159, 168 and 244 might be involved in expression of the activity and that, furthermore, those at positions 60, 107, 133, 140, 190, 192, 231 and 235 might be indispensable. Also, since the C-terminal 13 amino acid residues in the DNase I protein, which partly form an essential part of the active site, were deleted, it is likely that SNP p.Arg244Ter might produce a loss-of-function variant, in the same manner as p.Trp215GlyfsX2 in DNASE1L3 [12].

Next, among 25 non-synonymous SNPs in DNASE1L3, the levels of DNase 1L3 activity derived from the V46M, R92W, V150M, T156P and F255S constructs were significantly lower than that of the wild-type enzyme. Obviously, substitution of a Val residue at positions 46 and 150 in the DNase 1L3 protein by Met reduced the DNase I activity to about 60% of that of the wild-type enzyme, and also substitution of Arg, Thr and Phe residues at positions 92, 156 and 255 by Trp, Pro and Ser, respectively, reduced the activity markedly to less than 30%. Furthermore, the R206C and p.Trp215GlyFsX2 constructs exhibited no DNase 1L3 activity under the assay conditions we employed. However, the other amino acid substituted constructs, S14Y, R22K, I23V, D38H, A41V, C52G, E73K, G82R, V88M, N96K, I165M, V175L, R178H, T209I, I243M, Y261C, A268G and R285K, exhibited a level of DNase 1L3 activity similar to that of the wild-type enzyme, indicating that the substitutions of the corresponding amino acid residues at positions 14, 22, 23, 38, 41, 52, 73, 82, 88, 96, 165, 175, 178, 209, 243, 261, 268 and 285 in the DNase 1L3 protein had little influence on the activity. Among the amino acid residues related to non-synonymous SNPs in the DNase 1L3 protein, each amino acid residue at positions 46, 92, 150, 156, 206 and 255 might play a role in the expression of enzyme activity. On the other hand, no construct exhibited significantly higher levels of activity than that of the wild-type enzyme. Therefore, in contrast to the SNPs in DNASE1, it is obvious that no SNP in DNASE1L3 elevated the level of activity.

Multiple alignment analysis of the amino acid sequences of animal DNases I and 1L3

Multiple alignment analysis of the amino acid sequences of animal DNase I and 1L3 is useful for assessing the role of each SNP-related amino acid residue in these DNase proteins. Therefore, in order to survey orthologs of DNases 1 and 1L3 in Animalia organisms available in the KEGG database, a blast search was performed in each genome database using the amino acid sequence of human DNases I or 1L3 as a query sequence. Each ortholog of human DNase I and 1L3 with a low E value was revealed in 26 organisms, demonstrating that both the DNases of Metazoa are present in not only vertebrates including mammals, birds, reptiles, amphibians and fish, but also lancelets, ascidians, echinoderms, cnidarians and placozoans; in particular, an ortholog of human DNase 1L3 was found in a poriferan sponge (Amphimedon queenslandia), representing the lowest class in Metazoa, whereas no such ortholog of DNase I was found. The placozoan Trichoplax adhaerens, probably situated in an intermediate molecular-phylogenetic position between Porifera and Animalia, was found to possess both of these DNases. From these findings, it seems plausible to assume that a common ancestor of the DNase I family may be DNase 1L3, from which other members of the family might have arisen. Furthermore, neither of the DNases could be found in any of the genome databases available for Protostomia, including insects and nematodes, among the Animalia. The phylogenetic distribution of DNase I and 1L3 in animals based on these findings is summarized in Fig. 5, allowing us to demonstrate that both DNase I and 1L3 are not entirely distributed among Animalia, in contrast to DNase II, which has been identified even in non-metazoan organisms [20]; both DNase I and 1L3 are distributed extensively in Deuterostomia but not in Protostomia. However, since DNases are found in Poriferans and Placozoans, it seems plausible that a final common-ancestral species, from which Protostomia and Deuterostomia developed, possessed both of the DNases. It remains unknown why both DNases disappeared during the evolution of Protostomia from the predicted ancestral species of Eumatazoa, in contrast to Deuterostomia.

image

Figure 5. Molecular-phylogenetic distribution of DNase I and 1L3 in animals. blast search was performed in each genome database is available in the KEGG database (http://www.genome.jp/kegg/) using the amino acid sequence of human DNases I or 1L3 as a query sequence to survey orthologs of DNases 1 and 1L3 in Animalia organisms. Each ortholog of human DNase I and 1L3 with low E value could be found (+) or not (−).n.a., genome database is unavailable.

Download figure to PowerPoint

Next, multiple alignment analysis of the amino acid sequences of DNase I and 1L3 from Animalia organisms available on the genome database, in addition to vertebrate DNases I determined previously [21-24], was performed; DNase I and 1L3 derived from 40 and 26 species, respectively, were analyzed (Figs S1 and S2). Among the amino acid sequences of animal DNases I, all the organisms fully conserved 27 amino acid residues, which included the four amino acid residues responsible for the active site [25] and two Cys residues that form the disulfide bond responsible for structural stability of the enzyme [26], corresponding to Glu100, His156, Asp234 and His274, and the Cys residues at positions 195 and 231 in human DNase I, respectively. It was clarified that, on the whole, all the amino acid residues related to SNPs abolishing the enzyme activity were completely or well conserved in animal DNase I, whereas many of those corresponding to SNPs not affecting the activity were not conserved. On the other hand, each of the SNPs reducing and elevating the activity weaved the corresponding amino acid residues well conserved with those not conserved in the amino acid sequence of animal DNases I, suggesting that the effect of the amino acid substitutions resulting from these SNPs on the enzyme activity may have been attributable to the properties of individual amino acids. Next, all of the organisms fully conserved 35 residues in the amino acid sequences of animal DNase 1L3, which included the four amino acid and two Cys residues responsible for the active site and structural stability of the enzyme, respectively, deduced from DNase I; exceptionally, the residue corresponding to His275 in human DNase I was replaced by Gln in the Florida lancelet. When the amino acid residues involved in SNPs reducing or abolishing the enzyme activity were examined to determine whether they were conserved in the amino acid sequences of animal DNase 1L3, all of the residues other than that corresponding to p.Val46Met were well conserved.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, we have extensively continued our previous studies on the genetic and functional characterization of non-synonymous SNPs in DNASE1 and DNASE1L3 potentially relevant to autoimmune diseases [11, 13, 17, 18]; thereby, all of the non-synonymous SNPs (44 in DNASE1 and 25 in DNASE1L3) registered in the database or reported, which probably give rise to an alteration in the levels of in vivo DNase activity through amino acid substitution, could be evaluated as a functional SNP [27] (Tables 1 and 2). To our knowledge, this study is the first to have comprehensively demonstrated the genetic distribution of all the non-synonymous SNPs in both the DNase genes, and also the effect of each SNP on the enzyme activity.

Table 1. Summary of the evaluation of all the non-synonymous SNPs in DNASE1 as a functional SNP; genetic heterozygosity in our study populations and effect of the corresponding amino acid substitution on the DNase I activity. The minor allele frequency (MAF) and heterozygosity of each SNP were calculated based on the total subjects (= 1752) examined in this study. The values of activity are expressed as relative activity of each amino acid substituted construct to that of the wild type, representing the mean ± SD (= 4). P values in parentheses were calculated on differences between the activities of the substituted and wild-type enzyme by means of the unpaired Student's t test. The amino acid multiple alignment of 40 vertebrate DNase I is taken from Fig. S1. The numbers in parentheses are the number of species in which the corresponding amino acid is substituted. n.d., the activity derived from the corresponding amino acid substituted construct could not be detected under our assay conditions
SNPPolymorphismMAFHeterozygosityActivityAmino acid multiple alignment of 40 vertebrate DNase I
  1. a

    Taken from our previous study [11].

  2. b

    Taken from our previous study [45].

  3. c

    Taken from [9].

SNPs not affecting the activity

rs8176927

p.Arg2Ser; c.6G>T

Africana0.02420.0471.17 ± 0.17In the signal sequence

rs61741279

p.Gly3Asp; c.8G>A

Mono-allelic< 0.00030.0001.17 ± 0.17In the signal sequence

rs145239050

p.Lys5Arg; c.14A>G

Mono-allelic< 0.00030.0001.63 ± 0.50In the signal peptide

rs148015097

p.Ala14Val; c.41C>T

Mono-allelic< 0.00030.0001.10 ± 0.35In the signal peptide

rs141673463

p.Ala26Thr; c.76G>A

Mono-allelic< 0.00030.0001.14 ± 0.50Not conserved; substituted by Gly (11), Cys or Ser (4)

rs34907394

p.Glu35Asp; c.105G>C

Mono-allelic< 0.00030.0001.23 ± 0.28Not conserved; substituted by Asp (14), Met (2), Thr (2), Ala, Asn, Gln, Arg or Val

rs190768401

p.Arg95Gln; c.284G>A

Mono-allelic< 0.00030.0001.14 ± 0.39Well conserved in 35 species; substituted by His (3), Thr or Ala

Not registered

p.Val114Met; c.340G>A

Japaneseb< 0.00030.0001.10 ± 0.35Not conserved; substituted by Leu (15), Arg, Val (9), Thr (5), Ser, Ala (2), Met or Gln

rs34923865

p.Tyr117Ser; c.590A>C

Caucasiana0.00260.0061.29 ± 0.37Well conserved in 35 species; substituted by Phe (4) or Glu

rs144059899

p.Asp129Asn; c.385G>A

Mono-allelic< 0.00030.0001.92 ± 0.81Well conserved except reptiles (Gly or Thr)

rs76397583

p.Asn132Ser; c.395A>G

Mono-allelic< 0.00030.0001.45 ± 0.66Not conserved; substituted by Ser (20), Ile (3), Pro, Met (2) or Glu (4)

rs140745748

p.Val172Ile; c.514G>A

Mono-allelic< 0.00030.0001.21 ± 0.71Well conserved in 39 species; substituted by Ala

rs74892550

p.Val185Ile; c.514G>A

Mono-allelic< 0.00030.0001.49 ± 0.36Not conserved; substituted by Ile (23), Met (3) or Ala (4)

rs34186031

p.Pro219Ser; c.655C>T

Mono-allelic< 0.00030.0001.13 ± 0.29Not conserved; substituted by Ser (3), Gly (7) or Thr (3)

rs8176924

p.Gly262Glu; c.785G>A

Mono-allelic< 0.00030.0001.16 ± 0.54Not conserved; substituted by Arg (2), Asn (7), Lys (5), His (4) or Asp (2)
SNPs abolishing the activity

Rs121912990

p.Lys5Ter; c.13A>T

Japanesec< 0.00030.000n.d.Nonsense substitution in the signal sequence

rs142318540

p.Gln60Arg; c.179A>G

Mono-allelic< 0.00030.000n.d.Well conserved in 38 species; substituted by Asp or Glu

rs8176928

p.Arg107Gly; c.319A>G

Mono-allelic< 0.00030.000n.d.Well conserved in 38 species; substituted by Ile or Lys

rs150621329

p.Arg133Gln; c.398G>A

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs150621329

p.Arg133Leu; c.398G>T

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs149379211

p.Phe140Cys; c.419T>G

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs142644209

p.Asp190His; c.568G>C

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs146236198

p.Asn192Ile; c.575A>T

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs8176940

p.Cys231Tyr; c.692G>A

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs139254891

p.Arg235Trp; c.703A>T

Mono-allelic< 0.00030.000n.d.Conserved in all the species

rs201571412

p.Arg244Ter; c.730C>T

Mono-allelic< 0.00030.000n.d.Nonsense substitution
SNPs reducing the activity

rs77254040

p.Gln31Glu; c.91C>G

Japaneseb< 0.00030.0000.43 ± 0.10 (< 0.001)Not conserved; substituted by Arg (20), Lys (5), Glu (2), Ala, Asp or Phe

rs143865851

p.Arg53Cys; c.157C>T

Mono-allelic< 0.00030.0000.20 ± 0.050 (< 0.001)Well conserved in the 32 species; substituted by Gln (2), Glu (2), Thr, Ala, Gly or Met

rs144227093

p.Tyr54Cys; c.161A>T

Mono-allelic< 0.00030.0000.39 ± 0.10 (< 0.001)Conserved in 39 species; substituted by His

rs45545238

p.Gln60His; c.180G>C

Mono-allelic< 0.00030.0000.41 ± 0.081 (< 0.001)Well conserved in 38 species; substituted by Asp or Glu

rs143058517

p.Val111Met; c.331G>A

Mono-allelic< 0.00030.0000.24 ± 0.019 (< 0.001)Well conserved in all the species above reptile; substituted by Ala, Leu (4), Met or Phe

rs138676148

p.Arg139Gly; c.415A>G

Mono-allelic< 0.00030.0000.31 ± 0.040 (< 0.001)Not conserved; substituted by Lys (17), Trp, Met (7) or His

rs146238243

p.Pro159Leu; c.476C>T

Mono-allelic< 0.00030.0000.27 ± 0.10 (< 0.001)Conserved in all the species

rs139424576

p.Asp167His; c.499G>C

Mono-allelic< 0.00030.0000.52 ± 0.073 (< 0.001)Not conserved; substituted by Asn (7), Ser (2) or Gln

rs148373909

p.Arg207Cys; c.619C>T

Japaneseb< 0.00030.0000.12 ± 0.022 (< 0.001)Well conserved in the higher species above chondrichthyes; substituted by Ser (2) or Glu

rs1053874

p.Arg244Gln; c.731A>G

Polymorphica0.43550.4920.48 ± 0.015 (< 0.001)Not conserved; substituted by Arg (7), Leu (2), Met (5), Lys (4) or Thr

rs8176939

p.Ala246; c.736G>C

Mono-allelic< 0.00030.0000.16 ± 0.060 (< 0.001)Not conserved; substituted by Ser (7), Gly (8), Ile or Asp
SNPs elevating the activity

rs147546841

p.Tyr46His; c.136T>C

Mono-allelic< 0.00030.0001.85 ± 0.63 (< 0.05)Not conserved; substituted by Val, Phe (3), Ile (10), Ser, Leu (3), Arg (2), Met or Thr (2)

rs143363152

p.Val70Leu; c.208G>T

Mono-allelic< 0.00030.0001.57 ± 0.22 (< 0.005)Well conserved in the higher species above reptiles; substituted by Ile (7), Thr (6), Leu or Met

rs141801594

p.Asp120Asn; c.358G>A

Mono-allelic< 0.00030.0001.87 ± 0.40 (< 0.05)Well conserved in the 34 species; substituted by Asn, Lys, Ser (2) or Pro (2)

rs8176919

p.Gly127Arg; c.379G>A

Africana0.00940.0182.11 ± 0.52 (< 0.01)Conserved except reptile (Ser)

rs139615062

p.Arg143Gln; c.428G>A

Mono-allelic< 0.00030.0001.76 ± 0.43 (< 0.05)Not conserved; substituted by Pro (21), His, Leu (4), Lys (3), Asn, Ser (4) or Ala

rs1799891

p.Pro154Ala; c.460C>G

Japaneseb< 0.00030.0001.43 ± 0.25 (< 0.05)Conserved in the 31 species; substituted by Ala (3), Ser (4), Val or Gly

rs150933932

p.Ala168Val; c.503C>T

Mono-allelic< 0.00030.0001.64 ± 0.31 (< 0.05)Well conserved in the 29 species; substituted by Ser (5), Gly, Ile or Asn
Table 2. Summary of the evaluation of all the non-synonymous SNPs in DNASE1L3 as a functional SNP; genetic heterozygosity in our study populations and effect of the corresponding amino acid substitution on the DNase 1L3 activity. The minor allele frequency (MAF) and heterozygosity of each SNP were calculated based on the total subjects (= 1752) examined in this study. The values of activity are expressed as relative activity of each amino acid substituted construct to that of the wild type, representing the mean ± SD (= 4). P values in parentheses were calculated on differences between the activities of the substituted and wild-type enzyme by means of the unpaired Student's t-test. The amino acid multiple alignment of 26 vertebrate DNase 1L3 was taken from Fig. S2. The numbers in parentheses are the number of species in which the corresponding amino acid is substituted. n.d., the activity derived from the corresponding amino acid substituted construct could not be detected under our assay conditions
SNPPolymorphismMAFHeterozygosityActivityAmino acid multiple alignment of 26 vertebrate DNase 1L3
  1. a

    Taken from our previous study [13].

  2. b

    Taken from [12].

SNPs not affecting the activity

rs142751723

p.Ser14Tyr; c.41C>A

Mono-allelic< 0.00030.0000.73 ± 0.30In the signal sequence

rs139865883

p.Arg22Lys; c.65G>A

Mono-allelic< 0.00030.0000.90 ± 0.22Not conserved; substituted by Lys (7), Asn, Gln or Leu

rs188529894

p.Ile23Val; c.67A>G

Mono-allelic< 0.00030.0001.0 ± 0.29Well conserved in 20 species; substituted by Leu (5) or Val

rs148208826

p.Asp38His; c.112G>C

Mono-allelic< 0.00030.0001.3 ± 0.25Not conserved; substituted by Asn (7), Arg (5), Lys (6) or Thr

rs145973332

p.Ala41Val; c.122C>T

Mono-allelic< 0.00030.0000.90 ± 0.20Not conserved; substituted by Thr, Val (13) or Ile (2)

rs148406554

p.Cys52Gly; c.154T>G

Mono-allelic< 0.00030.0001.1 ± 0.25Well conserved in the higher species above fish; substituted by Tyr (4)

rs149330798

p.Glu73Lys; c.217G>A

Mono-allelic< 0.00030.0000.82 ± 0.16Not conserved; substituted by Gln (3), Lys, Asp, Ala, Thr or Ser

rs74350392

p.Gly82Arg; c.244G>C

Mono-allelic< 0.00030.0000.68 ± 0.21Not conserved; substituted by Ser (3), Arg (2), Asn (2), Pro (2), Lys or Gln

rs143383107

p.Val88Met; c.262G>A

Mono-allelic< 0.00030.0001.0 ± 0.17Not conserved; substituted by Thr (2), Phe, Ile, Ser, Leu (3) or Ala

rs12491947

p.Asn96Lys; c.288C>A

Mono-allelic< 0.00030.0000.82 ± 0.23Not conserved; substituted by Lys (5), Thr, Ser (5) or Gln

rs142361820

p.Ile165Met; c.495C>G

Mono-allelic< 0.00030.0000.90 ± 0.20Well conserved in 22 species; substituted by Met, Asn (2) or Leu

rs140654958

p.Val175Leu; c.523G>T

Mono-allelic< 0.00030.0001.0 ± 0.050Not conserved; substituted by Ala (3), Ile (3) or Thr (2)

rs3772986

p.Arg178His; c.533G>A

Mono-allelic< 0.00030.0000.82 ± 0.31Not conserved; substituted by Gln (3), His (3), Ala, Val or Lys (2)

rs147210152

p.Thr209Ile; c.626C>T

Mono-allelic< 0.00030.0001.0 ± 0.26Well conserved in 21 species; substituted by Asn (2), Ser, Val or Met

rs76440799

p.Ile243Met; c.729C>G

Mono-allelic< 0.00030.0000.82 ± 0.10Not conserved; substituted by Leu (10) or Met (2)

rs146444966

p.Tyr261Cys; c.533G>A

Mono-allelic< 0.00030.0001.5 ± 0.51Not conserved; substituted by Phe (6) or Leu (2)

rs113005222

p.Ala268Gly; c.803C>G

Mono-allelic< 0.00030.0000.80 ± 0.10Well conserved in 22 species; substituted by Thr (3) or Asp

rs151161986

p.Arg285Lys; c.854G>A

Mono-allelic< 0.00030.0001.1 ± 0.076Not conserved; substituted by Gly (4), Ser (2), Val or Lys (3)
SNPs abolishing the activity

rs35677470

p.Arg206Cys; c.602C>T

Caucasiana0.00090.018n.d.Conserved in 22 species; substituted by Val, Asp, Ser or Lys

Not registered

p.Trp215GlyfsX2;c.643delT

Caucasianb< 0.00030.000n.d.Well conserved in all the species
SNPs reducing the activity

rs145888358

p.Val46Met; c.136G>A

Mono-allelic< 0.00030.0000.60 ± 0.15 (< 0.05)Not conserved; substituted by Arg (2), Thr, Ile (2), Ser, Leu or Lys

rs141477807

p.Arg92Trp; c.274G>C

Mono-allelic< 0.00030.0000.19 ± 0.11 (< 0.001)Conserved in all the species

rs146805633

p.Val150Met; c.447G>A

Mono-allelic< 0.00030.0000.63 ± 0.17 (< 0.05)Well conserved in the mammals; substituted by Ala (4), Phe, Leu or Thr

rs147219402

p.Thr156Pro; c.466A>C

Mono-allelic< 0.00030.0000.30 ± 0.18 (< 0.001)Well conserved in 21 species; substituted by Ala (5)

rs148314913

p.Phe255Ser; c.764T>C

Mono-allelic< 0.00030.0000.21 ± 0.020 (< 0.001)Well conserved in 23 species; substituted by Tyr (2) or Val

Almost all of the non-synonymous SNPs in the genes encoding other members of the human DNase family, DNase I-like 1 [28], I-like 2 [29] and II [30], exhibited a mono-allelic distribution in the same study populations, similarly to those of DNase I and 1L3. Analysis of the genotype distribution of all these SNPs demonstrated that not only the two mutants, p.Val114Met in DNASE1 [31] and p.Trp215GlyfsX2 in DNASE1L3 [12], but also SNPs p.Lys5Ter [9] and p.Val111Met [10] in DNASE1, related to autoimmune diseases reported previously, were not distributed in any of the study populations. With regard to the non-synonymous SNPs potentially resulting in alterations of in vivo DNase activity, other than p.Arg244Gln in DNASE1, the human DNase family genes showed remarkably low genetic diversity. These facts allow us to conclude that the human DNase family has, on the whole, been well conserved at the protein level during the evolution of human populations.

From expression analysis of the amino acid substituted DNase I and 1L3 corresponding to each of the non-synonymous SNPs in the two genes, we were able to classify these SNPs into four classes according to alterations in the enzyme activity levels brought about by the corresponding amino acid substitution: those not affecting the activity, those elevating it, those abolishing it and those reducing it. Forty-four non-synonymous SNPs in DNASE1 were sorted into 15 SNPs not affecting the activity, 11 that abolished it, 11 that reduced it and seven that increased it, whereas 25 non-synonymous SNPs in DNASE1L3 included 18 that did not affect the activity, two that abolished it and five that reduced it (Figs 1 and 2). It is worth noting that all the amino acid residues involved in activity-abolishing SNPs were completely or well conserved in animal DNase I and 1L3 proteins (Figs S1 and S2). The DNase I protein has two disulfide bonds, Cys123–Cys126 and Cys195–Cys231, the latter of which has been shown to contribute to the stability of the enzyme [32]. Baron et al. demonstrated that human DNase 1L3 contains five Cys residues, two (Cys194 and Cys231) of which are conserved with a pair that forms a disulfide bond [33]. This reflects the fact that replacement of Cys231 by Tyr derived from SNP p.Cys231Tyr in DNASE1 abolished its activity. Furthermore, SNP p.Arg207Cys in DNASE1 and p.Arg206Cys in DNASE1L3 give rise to an additional Cys residue between the two essential Cys residues, assuming that the new Cys residue at position 207 (206) might form an aberrant disulfide bond with Cys 195 (194) or Cys 231, inducing structural instability through loss of the essential disulfide bond, thereby drastically reducing or abolishing the activity [13, 34]. In contrast, the amino acid substituted DNase 1L3 corresponding to p.Tyr261Cys showed an activity level similar to that of the wild-type enzyme. It seems plausible that the appearance of the new Cys residue distal from the essential Cys residues at the C-terminal end of the protein might not affect formation of the normal disulfide bond. Pan et al. [35] have proposed that the amino acid residues in the DNase I protein be classified into four groups based on their different function roles; the first, second, third and fourth classes are composed of the four residues absolutely essential for catalysis, the three crucial acidic residues serving as ligands for metal ion chelation, the seven residues critically involved in DNA-interacting positions encircling the active site, and the 13 residues interacting with DNA distal from the active site, respectively. Among the non-synonymous SNPs in DNASE1 examined, the amino acid residues corresponding to p.Asp190His, and to p.Arg133Gln, p.Arg133Leu and p.Asn192Ile, belong to the second and the third classes, respectively. These amino acid substitutions resulting from each SNP abolished the DNase I activity, consistent with the findings that Ala substitution of Arg or Asn at position 133 and 192, respectively, in the DNase I protein resulted in a drastic decrease of enzyme activity [35]. Furthermore, polymorphic p.Gln31Glu in a Japanese population [11], belonging to the third class, reduced the activity to about 40% of that of the wild-type enzyme, perhaps due to the introduction of a negatively charged residue. On the other hand, although p.Glu35Asp, p.Arg95Gln, p.Asp129Asn and p.Asn132Ser belong to the fourth class, the corresponding amino acid substitutions had little effect on expression of the activity. However, amino acid residues in the fourth class are considered to make only a minor contribution to the activity of DNase I [35]. In this context, when the possible roles of the amino acid residues in DNase 1L3 protein were deduced from those of human DNase I, only p.Asn96Lys among the non-synonymous SNPs in DNASE1L3 belonged to the fourth class. In the same manner as the four SNPs of the fourth class in DNASE1, no alteration of the DNase 1L3 activity resulting from the corresponding amino acid substitution was observed. DNASE1 contains many more non-synonymous SNPs (44 sites) than DNASE1L3 (25 sites), as shown in Figs 1 and 2. Furthermore, 66% of amino acid substitutions in the former result in alterations in the level of enzyme activity, whereas only 28% of amino acid substitutions in the latter do so. These findings allow us to conclude that DNASE1 is able to tolerate the generation of non-synonymous SNPs, and furthermore that the amino acid substitutions resulting from the SNPs easily create alterations in the levels of enzyme activity in comparison with DNASE1L3.

It has been reported that, in comparison with healthy subjects, levels of serum DNase I activity are reduced in patients with SLE [36-39]. Recently, Rekvig and colleagues have demonstrated that in lupus nephritis, which is the most serious complication of SLE, downregulation of renal DNase I results in reduced chromatin fragmentation and deposition of extracellular chromatin–IgG complexes in the glomerular basement membranes in subjects that produce IgG antibodies against chromatin [8, 40]. On the other hand, another member of the DNase I family, DNase 1L3, is present in serum in addition to DNase I, and it was assumed that these DNases may be concerned with each other during DNA degradation, providing effective clearance after exposure or release from dying cells [2, 16, 41]. Furthermore, a lack or reduction of these DNase activities was attributed to mutations identified in the patients [9, 10, 12, 13], and knockout of DNASE1 [14] was associated with the development of SLE. Considering these findings, it is likely that since DNase I and 1L3 are both implicated in the clearance of apoptotic and/or necrotic cell debris, loss and/or functional deficiency of either enzyme could result in failure to clear debris as the origin of nucleosomes, against which an immune response can be induced, resulting in autoimmune dysfunction. Thus it has been suggested that the serum levels of both DNases might be related to disease pathogenesis. Therefore, functional SNPs in both of the genes producing genetic isoforms with reduced and/or abolished activity should be considered as notable genetic factors involved in the abolishment or reduction of in vivo DNase I and 1L3 activity, thereby leading to autoimmune dysfunction. However, there is little information about functional SNPs in both of the genes that might affect enzyme activity. Indeed, previous findings [42] have indicated that the frequency of the homozygote for the G-allele in SNP p.Arg244Gln of DNASE1 is much higher in patients with SLE who have the corresponding autoantibodies than in patients who do not have them, for which production of the low activity-harboring DNase I isoform by the G-allele could partly account [11]. Furthermore, the T-allele in SNP p.Arg206Cys of DNASE1L3 producing an inactive DNase 1L3 isoform [13] has been identified in Caucasian patients with SLE [12]. In the present study of DNASE1 and DNASE1L3, we were able to identify many of the functional SNPs affecting the activity of each DNase, irrespective of whether or not they showed polymorphic distribution worldwide; in particular, 11 activity-reducing and 11 activity-abolishing SNPs in the former and five activity-reducing and two activity-abolishing SNPs in the latter were confirmed. It seems plausible that a minor allele of p.Gln60Arg, p.Arg107Gly, p.Arg133Gln, p.Arg133Leu, p.Phe140Cys, p.Asp190His, p.Cys231Tyr, p.Arg235Trp and p.Arg244Ter in DNASE1 and p.Arg206Cys and p.Trp215GlyfsXs in DNASE1L3 found in patients producing a loss-of-function DNase variant might be a genetic risk factor for autoimmune diseases. Among the functional SNPs revealed, only p.Arg244Gln in DNASE1 exhibited a commonly polymorphic distribution in all the study populations examined with well-balanced allele frequencies, the DNase I isoform derived from the G-allele having about half the activity of the isoform from its counter allele, the A-allele. On the other hand, the T-allele in SNP p.Arg206Cys in DNASE1L3 producing an inactive form of the DNase 1L3 is specifically distributed in Caucasian populations. In our present study of Caucasian populations, subjects (occurrence rate 3.5–8.7%) who have a simultaneously homozygotic genotype for the G-allele in DNASE1 and a homo/heterozygotic genotype for the T-allele in DNASE1L3 exhibited much lower overall levels of in vivo DNase activity than those with other genotypes. Therefore, it could be assumed that, in comparison with the latter Caucasian subjects, the former may be more prone to the development of autoimmune diseases such as SLE through a defect in the clearance of apoptotic/necrotic cell debris as the origin of nucleosomes, which can induce an immune response. In this context, in order to clarify any clinical association of these functional SNPs in DNASE1 and DNASE1L3 with the incidence of autoimmune diseases, the distribution of each SNP in various patient groups should be examined. Therefore, further studies will be required to examine the correlation between the levels of DNase activity in serum and the prevalence of autoimmune diseases for each SNP genotype.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Samples from subjects

Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Chatsworth, CA, USA) from blood or bloodstain samples randomly collected from healthy subjects (= 1752) derived from three ethnic groups; the Asian, Caucasian and African populations were composed of 1043, 403 and 306 subjects, respectively. Written informed consent was obtained from each participant. The study was approved by the Human Ethical Committees of the Institute.

Genotyping of non-synonymous SNPs in the DNASE1 and DNASE1L3 by the PCR-RFLP method

Genotyping assays for each of 32 non-synonymous SNPs in DNASE1 and 20 non-synonymous SNPs in DNASE1L3 were separately performed using PCR followed by RFLP analysis [22], in the same manner as the other SNPs [11, 18]. Primers for the specific amplification of the DNA fragments encompassing a substitution site corresponding to each SNP were designed on the basis of the nucleotide sequences of the human DNASE1 and DNASE1L3 (GenBank accession no. D83195 and EMBL accession no. AC137936, respectively) (Tables S1 and S2). Nucleotide and amino acid residues of each DNase were numbered from the 5′-terminus of the translation initiation codon and the N-terminal amino acid residue of the precursor protein, respectively. SNP nomenclature is based on the recommendations for description of sequence variants (http://www.hgvs.org/mutnomen/examplesDNA.htlm); the sequence of each DNase (GenBank accession no. AB188151 and NCBI Reference Sequence NM_004944.3) has been used as the coding DNA reference sequence.

PCR amplification was performed in a 25 μL reaction mixture using approximately 5 ng of DNA. The reaction mixture contained 1× buffer (15 mm Tris/HCl, pH 8.0, 50 mm KCl), 1.5 mm MgCl2, 0.5 μm of each primer, 200 μm dNTPs and 1.25 U of Taq polymerase (AmpliTaq Gold; Applied Biosystems, Foster City, CA, USA). PCR was performed with a protocol consisting of initial denaturation at 94 °C for 7 min, followed by 30 cycles with denaturation at 94 °C for 30 s, annealing at 55–65 °C (Tables S1 and S2) for 30 s and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min.

Two microliters of the PCR product obtained using each pair of primers was digested with 5 U of each enzyme listed in Tables S1 and S2 (New England Biolabs, Ipswich, MA, USA) at 37 °C for 3 h in a final reaction mixture volume of 15 μL to determine the genotype of each SNP. The digested products (5 μL) were separated in an 8% polyacrylamide gel, and the patterns on the gels were visualized by silver or ethidium bromide staining, as described previously [11, 13].

Direct sequencing of the genomic DNA region including the substitution site of each SNP in DNASE1 and DNASE1L3 for the representative subjects was performed by the dideoxy chain-terminating method with the BigDye®Terminator Cycle Sequencing Kit (Applied Biosystems). The sequencing run was performed on a Genetic Analyzer 310 (Applied Biosystems).

Construction of expression vectors encoding human DNases I and 1L3 and the amino acid substituted forms corresponding to each SNP

Expression pcDNA3.1(+) vectors (Invitrogen, San Diego, CA, USA) inserted with the entire coding sequence of human DNase I or 1L3 cDNA were separately prepared [11, 13] and were used as a wild-type construct; inserted cDNAs were derived from the predominant haplotype in Japanese. The 44 and 25 amino acid substituted constructs, corresponding to each non-synonymous SNP in DNASE1 and DNASE1L3, respectively, including those previously prepared [11, 18], were constructed using the KOD-Plus Mutagenesis Kit (Toyobo Co. Ltd, Osaka, Japan) with the wild-type construct as a template. In these constructs, the amino acid residue was replaced by the counterpart derived from a minor allele in each substitution site; e.g. the Q31E construct of DNase I, in which the Gln residue at position 31 in the protein is replaced by Glu derived from the minor allele, corresponds to SNP p.Gln31Glu. Nucleotide sequences of all the constructs were confirmed by DNA sequence analysis. Purification of each construct used for transfection was performed using the Plasmid Midi Kit (Qiagen).

Transient expression of the expression vectors and assay for each DNase activity

COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 1 mm l-glutamine, 50 U·mL−1 penicillin, 50 μg·mL−1 and 10% (v/v) fetal bovine serum at 37 °C under 5% CO2 in air. The cells were transiently transfected four times with 2 μg of each DNase I related expression vector, together with 600 ng of pSV-β-galactosidase vector (Promega, Madison, WI, USA; for estimation of transfection efficiency) using Lipofectamine 2000 reagent (Invitrogen) according to the method described previously [43]. At 48 h after transfection, the cells were harvested. On the other hand, at 24 h after transfection of 2 μg of each DNase 1L3-related expression vector and pSV-β-galactosidase vector in the same manner as above, the cells were washed with phosphate-buffered saline and transferred to Dulbecco's modified Eagle's medium containing 100-fold-diluted insulin–transferrin–selenium-X (Invitrogen). The transfected cells were harvested after 72 h. Then the cells were subjected to sonication using Bioruptor UCD-250 (Cosmo Bio Co. Ltd, Tokyo, Japan) to prepare lysates for the subsequent assay. DNase I and 1L3 activities in the transfected cells were assayed by the SRED method using a LAS-3000 imaging analyzer (Fuji Film, Tokyo, Japan) according to our previous reports [11, 13, 44]. The activity of wild-type DNase was defined as 1.0, and that of the amino acid substituted DNase was expressed relative to the wild type. The relative activity is expressed as mean ± standard deviation derived from four transfections.

Analytical methods

Orthologs of human DNases I and 1L3 in other available animal species were surveyed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/); the amino acid sequences of human DNases I or 1L3 were used as query sequences for blast searches of each genome database. Multiple alignment analyses of the amino acid sequences of animal DNases I and 1L3 were performed using dnasis pro V3.0 (Hitachi Solutions Ltd, Tokyo, Japan).

Allele frequency and heterozygosity of each SNP in DNASE1 and DNASE1L3 were calculated based upon the total subjects (= 1752) enrolled in this study regardless of ethnic group; the heterozygosity of each SNP = 1 − (f1)2 − (f2)2, where f1 and f2 are the allele frequency of the major and minor alleles, respectively, in the SNP.

DNase I and 1L3 activities were compared between wild-type and substituted enzymes by means of the unpaired Student's t-test. Differences at < 0.05 were considered to be statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

DNA samples of bloodstain samples and blood samples were kindly provided by Dr B. Brinkmann and Dr K. Shiwaku, respectively. This study was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (22249023 to T.Y.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 1
    Counis MF & Torriglia A (2000) DNases and apoptosis. Biochem Cell Biol 78, 405414.
  • 2
    Napirei M, Gültekin A, Kloeckl T, Möröy T, Frostegård J & Mannherz HG (2006) Systemic lupus-erythematosus: deoxyribonuclease I in necrotic chromatin disposal. Int J Biochem Cell Biol 38, 297306.
  • 3
    Mizuta R, Mizuta M, Araki S, Shiokawa D, Tanuma S & Kitamura D (2006) Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates. Biochem Biophys Res Commun 345, 560567.
  • 4
    Rodriguez AM, Rodin D, Nomura H, Morton CC, Weremowicz S & Schneider MC (1997) Identification, localization, and expression of two novel human genes similar to deoxyribonuclease I. Genomics 42, 507513.
  • 5
    Shiokawa D & Tanuma S (2001) Characterization of human DNase I family endonuclease and activation of DNase gamma during apoptosis. Biochemistry 40, 143152.
  • 6
    Tsukumo S & Yasutomo K (2004) DNase I in pathogenesis of systemic lupus erythematosus. Clin Immunol 113, 1418.
  • 7
    Valle FM, Balada E, Ordi-Ros J & Vilardell-Tarres M (2008) DNase I and systemic lupus erythematosus. Autoimmun Rev 7, 359363.
  • 8
    Hedberg A, Mortensen ES & Rekvig OP (2011) Chromatin as a target antigen in human and murine lupus nephritis. Arthritis Res Therapy 13, 214222.
  • 9
    Yasutomo K, Horiuchi T, Kagami H, Tsukamoto H, Hashimura C, Urushihara M & Kuroda Y (2001) Mutation of DNASE1 in people with systemic lupus erythematosus. Nat Genet 28, 313314.
  • 10
    Dittmar M, Bischofs Matheis N, Poppe R & Kahaly GJ (2009) A novel mutation in the DNASE1 gene is related with protein instability and decreased activity in thyroid autoimmunity. J Antoimmun 32, 713.
  • 11
    Yasuda T, Ueki M, Takeshita H, Fujihara J, Kimura K, Iida R, Tsubota E, Soejima M, Koda Y, Kato H et al. (2010) A biochemical and genetic study on all non-synonymous single nucleotide polymorphisms of the gene encoding human deoxyribonuclease I potentially relevant to autoimmunity. Int J Biochem Cell Biol 42, 12161225.
  • 12
    Al-Mayouf SM, Sunker A, Adbwani R, Al Abrawi S, Almurshedi F, Alhashmi N, Al Sonbul A, Sewairi W, Qari A, Adballah E et al. (2011) Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus. Nat Genet 43, 11861188.
  • 13
    Ueki M, Takeshita H, Fujihara J, Iida R, Yuasa I, Kato H, Panduro A, Nakajima T, Kominato Y & Yasuda T (2009) Caucasian-specific allele in non-synonymous single nucleotide polymorphisms of the gene encoding deoxyribonuclease I-like 3, potentially relevant to autoimmunity, produces an inactive enzyme. Clin Chim Acta 407, 2024.
  • 14
    Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG & Möröy T (2000) Feature of systemic lupus erythematosus in DNase 1-deficient mice. Nat Genet 25, 177181.
  • 15
    Wilber A, O'Connor TP, Lu ML, Karimi A & Schneider MC (2003) DNase1 l3 deficiency in lupus-prone MRL and NZB/W F1 mice. Clin Exp Immunol 134, 4652.
  • 16
    Napirei M, Ludwig S, Mezrhab J, Klöckl T & Mannherz HG (2009) Murine serum nucleases – contrasting effects of plasmin and heparin on the activities of DNase 1 and DNase 1-like 3 (DNase 1 l3). FEBS J 276, 10591073.
  • 17
    Fujihara J, Ueki M, Yasuda T, Iida R, Soejima M, Koda Y, Kimura K, Kato H, Panduro A, Tongu M et al. (2011) Functional and genetic survey of all known single-nucleotide polymorphisms within the human deoxyribonuclease I gene in wide-ranging ethnic groups. DNA Cell Biol 30, 205217.
  • 18
    Ueki M, Fujihara J, Takeshita H, Kimura K, Iida R, Yuasa I, Kato H & Yasuda T (2011) Global genetic analysis of all single nucleotide polymorphisms in exons of the human deozyribonuclease I-like 3 gene and their effect on its catalytic activity. Electrophoresis 32, 14651472.
  • 19
    Yasuda T, Nadano D, Tenjo E, Takeshita H, Sawazaki K, Nakanaga M & Kishi K (1995) Genotyping of human deoxyribonuclease I polymorphism by the polymerase chain reaction. Electrophoresis 16, 18891893.
  • 20
    MacLea KS, Krieser RJ & Eastman A (2003) A family history of deoxyribonuclease II: surprise from Trichinella spiralis and Burkholderia pseudomallei. Gene 305, 112.
  • 21
    Takeshita H, Yasuda T, Iida R, Nakajima T, Mori S, Mogi K, Kaneko Y & Kishi K (2001) Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of a serine residue into the Ca2+-binding site. Biochem J 357, 473480.
  • 22
    Takeshita H, Yasuda T, Nakajima T, Mogi K, Kaneko Y, Iida R & Kishi K (2003) A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability: a clue to the molecular evolutionary mechanism from cold-blooded to warm-blooded vertebrates. Eur J Biochem 70, 307314.
  • 23
    Yasuda T, Takeshita H, Iida R, Ueki M, Nakajima T, Kaneko Y, Mogi K, Kominato Y & Kishi K (2004) A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analyses of the piscine DNase family. Biochim Biophys Acta 1672, 174183.
  • 24
    Yasuda T, Iida R, Ueki M, Kominato Y, Nakajima T, Takeshita H, Kobayashi T & Kishi K (2004) Molecular evolution of shark and other vertebrate DNases I. Eur J Biochem 271, 44284435.
  • 25
    Jones SJ, Worrall AF & Connolly BA (1996) Site-directed mutagenesis of the catalytic residues of bovine pancreatic deoxyribonuclease I. J Mol Biol 264, 11541163.
  • 26
    Oefner C & Suck D (1986) Crystallographic refinement and structure of DNase I at 2Å resolution. J Mol Biol 192, 605632.
  • 27
    Wu J-R & Zeng R (2012) Molecular basis for population variation: from SNPs to SAPs. FEBS Lett 586, 28412845.
  • 28
    Fujihara J, Yasuda T, Iida R, Kimura K, Soejima M, Koda Y, Kato H, Panduro A, Yuasa I & Takeshita H (2010) Global analysis of single nucleotide polymorphisms in the exons of human deoxyribonuclease I-like 1 and 2 genes. Electrophoresis 31, 35523557.
  • 29
    Ueki M, Fujihara J, Kimura K, Takeshita H, Iida R & Yasuda T (2013) Five non-synonymous SNPs in the gene encoding human deoxyribonuclease I-like 2 implicated in terminal differentiation of keratinocytes reduce or abolish its activity. Electrophoresis 34, 456462.
  • 30
    Kimura K, Yasuda T, Fujihara J, Toga T, Ono R, Otsuka Y, Ueki M, Iida R, Sano R, Nakajima T et al. (2012) Genetic and expression analysis of SNPs in the human deoxyribonuclease II: SNPs in the promoter region reduce its in vivo activity through decreased promoter activity. Electrophoresis 33, 28522858.
  • 31
    Iida R, Yasuda T, Aoyama M, Tsubota E, Kobayashi M, Yuasa I, Matsuki T & Kishi K (1997) The fifth allele of the human deoxyribonuclease I (DNase I) polymorphism. Electrophoresis 18, 19361939.
  • 32
    Chen W-J, Lee I-S, Chen C-Y & Liao T-H (2004) Biological functions of the disulfides in bovine pancreatic deoxyribonuclease I. Protein Sci 13, 875883.
  • 33
    Baron WF, Pan CQ, Spence SA, Tyan AM, Lazarus RA & Baker KP (1998) Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages. Gene 215, 291301.
  • 34
    Yasuda T, Takeshita H, Iida R, Kogure S & Kishi K (1999) A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability. Biochem Biophys Res Commun 260, 280283.
  • 35
    Pan CQ, Ulmar JS, Herzka A & Lazarus RA (1998) Mutation analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence. Protein Sci 7, 628636.
  • 36
    Chitrabamrung S, Ribin RL & Tan EM (1981) Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus. Rheumatol Int 1, 5560.
  • 37
    Dittmar M, Poppe R, Bischofs C, Fredenhagen C, Kanitz M & Kahaly GJ (2007) Impaired deoxyribonuclease I activity in monoglandular and polyglandular autoimmunity. Exp Clin Endocrinol Diabetes 115, 387391.
  • 38
    Sallai K, Nagy E, Derfalvy B, Müzes G & Gergely A (2005) Antinucleosome antibodies and decreased deoxyribonuclease I activity in sera of patients with systemic lupus erythematosus. Clin Diagn Lab Immunol 12, 5659.
  • 39
    Martinea-Valle F, Balada E, Ordi-Ros J, Bujan-Rivas S, Sellas-Fernadez A & Vilardell-Tarres M (2009) DNase I activity in patients with systemic lupus erythematosus: relationship with epidemiological, clinical, immunological and therapeutical features. Lupus 18, 418423.
  • 40
    Zykova SN, Tveita AA & Rekvig OP (2010) Renal Dnase 1 enzyme activity and protein expression is selectively shut down in murine and human membranoproliferative lupus nephritis. PLoS ONE 5, e12096.
  • 41
    Napirei M, Wulf S, Eulitz D, Mannherz HG & Kloeckl T (2005) Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1 l3). Biochem J 389, 355364.
  • 42
    Shin HD, Park BL, Kim LH, Lee H-S, Kim T-Y & Bae A-C (2004) Common DNase I polymorphism associated autoantibody production among systemic lupus erythermatosus. Hum Mol Genet 13, 23432350.
  • 43
    Ueki M, Fujihara J, Takeshita H, Kimura K, Iida R, Nakajima T, Kominato Y, Yuasa I & Yasuda T (2010) Genetic and expression analysis of all non-synonymous single nucleotide polymorphisms in the human deoxyribonuclease I-like 1 and 2 genes. Electrophoresis 31, 20632069.
  • 44
    Nadano D, Yasuda T & Kishi K (1993) Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme diffusion method. Clin Chem 39, 448452.
  • 45
    Takeshita H, Yasuda T, Nakashima Y, Mogi K, Kishi K, Shiono K, Sagisaka K, Yuasa I, Nishimukai H & Kimura H (2001) Geographical north–south decline in DNase I allele 2 in Japanese population. Hum Biol 73, 129134.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12608-sup-0001-TableS1-S4-FigS1-S2.zipapplication/ZIP586K

Fig. S1. Multiple alignment analysis on DNase I.

Fig. S2. Multiple alignment analysis on DNase 1L3.

Table S1. Primer sequence, annealing temperatures and restriction enzymes for PCR-based genotyping for DNASE1.

Table S2. Primer sequence, annealing temperatures and restriction enzymes for PCR-based genotyping for DNASE1L3.

Table S3. Genotype distribution and allele frequencies of non-synonymous SNPs in DNASE1.

Table S4. Genotype distribution and allele frequencies of non-synonymous SNPs in DNASE1L3.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.