Knockout of M-LP/Mpv17L, a newly identified atypical PDE, induces physiological afferent cardiac hypertrophy in mice.

M-LP/Mpv17L (Mpv17-like protein) is an atypical cyclic nucleotide phosphodiesterase (PDE) without the molecular structure characteristic of the PDE family. Deficiency of M-LP/Mpv17L in mice has been found to result in development of ß-cell hyperplasia and improved glucose tolerance. Here, we report another phenotype observed in M-LP/Mpv17L-knockout (KO) mice: afferent cardiac hypertrophy. Although the hearts of M-LP/Mpv17L-KO mice did not differ in size from those of wild-type mice, there was marked narrowing of the left ventricular lumen and thickening of the ventricular wall. The diameter and cross-sectional area of cardiomyocytes in 8-month-old M-LP/Mpv17L-KO mice were increased 1.16-fold and 1.35-fold, respectively, relative to control mice, but showed no obvious abnormalities of cell structure, fibrosis or impaired cardiac function. In 80-day-old KO mice, the expression of hypertrophic marker genes, brain natriuretic peptide (BNF), actin alpha cardiac muscle 1 (ACTC1) and actin alpha 1 skeletal muscle (ACTA1), as well as the Wnt/ß-catenin pathway target genes, lymphoid enhancer-binding factor-1 (LEF1), axis inhibition protein 2 (AXIN2) and transcription factor 7 (TCF7), was significantly up-regulated relative to control mice, whereas fibrosis-related genes such as fibronectin 1 (FN1) and connective tissue growth factor (CTGF) were down-regulated. Western blot analysis revealed increased phosphorylation of molecules downstream of the cAMP/PKA signaling pathway, such as ß-catenin, ryanodine receptor 2 (RyR2), phospholamban (PLN) and troponin I (cTnI), as well as members of the MEK1-ERK1/2 signaling pathway, which is strongly involved in afferent cardiac hypertrophy. Taken together, these findings indicate that M-LP/Mpv17L is one of the PDEs actively functioning in the heart and that deficiency of M-LP/Mpv17L in mice promotes physiological cardiac hypertrophy.

Deficiency of M-LP/Mpv17L leads to development of ß-cell hyperplasia and improved glucose tolerance via activation of the Wnt and TGF-ß pathways.

M-LP/Mpv17L is a protein that was initially identified during screening of age-dependently expressed genes in mice. We have recently demonstrated that M-LP/Mpv17L-knockout (M-LP/Mpv17L-KO) in human hepatoma cells leads to a reduction of cellular cyclic nucleotide phosphodiesterase (PDE) activity, and that in vitro-synthesized M-LP/Mpv17L possesses PDE activity. These findings suggest that M-LP/Mpv17L functions as an atypical PDE, even though it has none of the well-conserved catalytic region or other structural motifs characteristic of the PDE family. In this study, we found that M-LP/Mpv17L-KO mice developed ß-cell hyperplasia and improved glucose tolerance. Deficiency of M-LP/Mpv17L in islets from KO mice at early postnatal stages or siRNA-mediated suppression of M-LP/Mpv17L in rat insulinoma cells led to marked upregulation of lymphoid enhancer binding factor 1 (Lef1) and transcription factor 7 (Tcf7), key nuclear effectors in the Wnt signaling pathway, and some of the factors essential for the development and maintenance of ß-cells. Moreover, at the protein level, increases in the levels of phosphorylated ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) were observed, indicating activation of the Wnt and TGF-ß signaling pathways. Taken together, these findings suggest that protein kinase A (PKA)-dependent phosphorylations of ß-catenin and GSK-3ß, the key mediators of the Wnt and/or TGF-ß signaling pathways, are the most upstream events triggering ß-cell hyperplasia and improved glucose tolerance caused by M-LP/Mpv17L deficiency.

Human Mpv17-like protein with a mitigating effect on mtDNA damage is involved in cAMP/PKA signaling in the mitochondrial matrix.

Human Mpv17-like protein (M-LPH/Mpv17L) is thought to play a role in minimizing mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. We have recently demonstrated that, in addition to an increase of mtDNA damage, M-LPH-knockout (M-LPH-KO) in HepG2 cells causes a significant reduction of mitochondrial transcription factor A (TFAM) protein, an essential factor for mtDNA maintenance, along with an increase in its phosphorylation. These intracellular changes suggested an association of M-LPH with the cAMP/PKA signaling pathway, as selective degradation of TFAM by mitochondrial protease is driven by protein kinase A (PKA)-dependent phosphorylation. In the present study, we observed that M-LPH-KO in HepG2 cells caused an increase in the level of mitochondrial cAMP and a reduction of total cellular cyclic nucleotide phosphodiesterase (PDE) activity. In vitro-synthesized M-LPH showed PDE activity, which was inhibited by IBMX, a non-selective inhibitor of PDE. Furthermore, M-LPH-KO promoted PKA-dependent phosphorylation of some mitochondrial proteins. Taken together, the present findings suggest that M-LPH, which has structural features atypical of PDE family members, might be a novel human PDE involved in cAMP/PKA signaling in the mitochondrial matrix.

Evaluation of the functional effects of genetic variants‒missense and nonsense SNPs, indels and copy number variations‒in the gene encoding human deoxyribonuclease I potentially implicated in autoimmunity.

Genetic variants, such as single nucleotide polymorphisms (SNPs), in the deoxyribonuclease I (DNase I) gene which remarkably reduce or abolish the activity are assumed to be substantially responsible for the genetic backgrounds determining susceptibility to autoimmune dysfunction. Here, we evaluated many genetic variants, including missense and nonsense SNPs, and indel (inframe) variants in the gene, potentially implicated in autoimmune diseases as functional variants resulting in altered activity levels. Eighteen missense and 7 nonsense SNPs, and 9 indel (inframe) variants were found to result in loss of function and disappearance of DNase I activity. Furthermore, considering the positions in the DNase I protein corresponding to the various nonsense SNPs, all of the other nonsense SNPs and frameshift variants registered in the Ensembl database ( https://asia.ensembl.org ) appear likely to exert a pathogenetic effect through loss of the activity. Accordingly, a total of 60 genetic variants in the DNase 1 gene (DNASE1) inducing abolishment or marked reduction of the DNase I activity could be identified as genetic risk factors for autoimmunity, irrespective of how sparsely they were distributed in the population. It was noteworthy that SNP p.Gln244Arg, reportedly associated with autoimmunity and reducing the activity to about half of that of the wild type, and SNP p.Arg107Gly, abolishing the activity completely, were distributed worldwide and in African populations at the polymorphic level, respectively. On the other hand, with regard to copy number variations in DNASE1 where loss of copy leads to a reduction of the in vivo enzyme activity, only 2 diploid copy numbers were distributed in Japanese and German populations, demonstrating no loss of copy. These exhaustive data for genetic variants in DNASE1 resulting in loss or marked reduction of the DNase I activity are highly informative when considering genetic predisposition leading to autoimmune dysfunction.

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

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.

Analysis of copy number variation in the NEDD4L gene potentially implicated in body height in the Japanese population.

Recently it has been recognized that a considerable number of copy number variations (CNVs) are associated with diseases and other complex human traits. In our previous study, we developed a simple quantitative real-time PCR (Q-PCR) method for analysis of CNV copy number, which had the advantage of obviating the need for reference DNA with a known copy number. Using DNA samples obtained from 231 Japanese individuals, we applied this method for analyzing the copy number of a candidate CNV associated with body height, located in the neural precursor cell expressed, developmentally down-regulated 4-like, E3 ubiquitin protein ligase (NEDD4L) gene. In addition, the appropriateness of the results was evaluated and confirmed by quantification of amplicons with an Agilent 2100 Bioanalyzer. The NEDD4L gene encodes a member of the Nedd4 family of HECT domain E3 ubiquitin ligases. The target CNV located in the intron has been found to be significantly associated with height variation in Chinese. However, it remains unknown whether such an association exists in other populations, including Japanese. Analysis of the correlations between copy number and body height using ANOVA revealed no statistically significant correlations in Japanese.

Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.

Human Mpv17-like protein (M-LPH) has been suggested to participate in prevention of mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. To clarify the molecular mechanism of M-LPH function, we knocked out M-LPH in human hepatoma HepG2 using CRISPR-Cas9 technology. An increase in mtDNA damage in M-LPH-KO HepG2 cells was demonstrated by PCR-based quantitation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) measurement. Furthermore, confocal immunofluorescence analysis and Western blot analysis of mitochondrial extracts demonstrated that M-LPH-KO caused reductions in the protein levels of mitochondrial transcription factor A (TFAM), an essential factor for transcription and maintenance of mtDNA, and two DNA repair enzymes, 8-oxoguanine DNA glycosylase (OGG1) and DNA ligase 3 (LIG3), both involved in mitochondrial base excision repair (BER). Accordingly, it was suggested that the increase in mtDNA damage was due to a cumulative effect of mtDNA instability resulting from deficiencies of TFAM and diminished ability for BER arising from deficiencies in BER-related enzymes. These findings suggest that M-LPH could be involved in the maintenance of mtDNA, and therefore mitochondrial function, by protecting proteins essential for mtDNA stability and maintenance, in an integrated manner.

Simple screening method for copy number variations associated with physical features.

Recent studies of copy number variations (CNVs) associated with physical features, such as body mass index, body height or bone length, have suggested that such CNVs could serve as markers in forensic cases involving unidentified individuals. However, the process of cataloging CNVs has been slow because of the cumbersome nature and low reliability of the procedures involved. Here we describe a simple quantitative real-time PCR (Q-PCR) method for screening of medicolegally useful CNVs, which does not require reference DNA with known copy number. The first step is to prepare a chimeric plasmid vector including one copy each of the single-copy gene-specific sequence as the internal standard, and the target CNV-specific sequence. To assess the validity of this new method, we analyzed CNVs in the LTBP1 and ETV6 gene regions, both of which are candidate CNVs associated with body height. The PCR efficiencies for the single-copy (reference) gene and the target CNV were similar, indicating that quantitation was reliable. Furthermore, simulated analysis of the LTBP1 CNV using mock samples prepared by mixing vectors in varying proportions showed that this analytical method allowed correct determination of the LTBP1 copy number. These results demonstrated that our simple method has considerable potential for screening of trait-related CNVs that would be useful for forensic casework.

Survey of single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 2 producing loss of function potentially implicated in the pathogenesis of parakeratosis.

Dysfunction of DNase I-like 2 (DNase 1L2) has been assumed to play a role in the etiology of parakeratosis through incomplete degradation of DNA in the epidermis. However, the pathogenetic background factor for such pathophysiologic conditions remains unknown. In this context, non-synonymous single-nucleotide polymorphisms (SNPs) in DNASE1L2 that would potentially result in loss of in vivo DNase 1L2 activity might serve as a genetic risk factor for such pathophysiologic conditions. Our aim was to effectively survey the non-synonymous SNPs of DNASE1L2 that would produce a loss-of-function variant of the enzyme together with a genetic distribution in the various populations. Here, the effects of all of the SNPs predicted by PolyPhen-2 analysis to be "probably damaging" (score = 1.000), and derived from frameshift/nonsense mutations, on the activity of DNase 1L2 were examined using the corresponding DNase 1L2 variants expressed in COS-7 cells. Genotyping of these SNPs was also performed in three ethnic groups including 14 different populations. Among the 28 SNPs examined, the minor allele of 23 SNPs was defined as a loss-of-function variant resulting in loss of DNase 1L2 function, indicating that Polyphen-2 analysis could be effective for surveys of at least non-synonymous SNPs resulting in loss of function. On the other hand, these minor alleles were not distributed worldwide, thereby avoiding any marked reduction of the enzyme activity in human populations. Furthermore, all of the 19 SNPs originating from frameshift/ nonsense mutations found in DNASE1L2 resulted in loss of function of the enzyme. Thus, the present findings suggest that each of the minor alleles for these SNPs may serve as one of genetic risk factors for parakeratotic skin diseases such as psoriasis, even though they lack a worldwide genetic distribution.

Functional Single Nucleotide Polymorphisms (SNPs) in the Genes Encoding the Human Deoxyribonuclease (DNase) Family Potentially Relevant to Autoimmunity.

OBJECTIVE: To continue our previous investigations, we have extensively investigated the function of the 61, 41, and 35 non-synonymous single nucleotide polymorphisms (SNPs) in the human genes encoding DNASE1, DNASE1L3, and DNASE2, respectively, potentially relevant to autoimmune diseases. METHODS: The site-directed mutagenesis was employed to amino acid-substituted constructs corresponding to each SNP. The COS-7 cells were transfected with each vector and DNase activity was assayed by the single radial enzyme diffusion method. By using PolyPhen-2, changes in the DNase function of each non-synonymous SNP were predicted. Genotyping of all the non-synonymous SNPs was performed in 14 different populations including 3 ethnic groups using the polymerase chain reaction followed by the restriction fragment length polymorphism method. RESULTS: Expression analysis demonstrated these SNPs to be classified into four categories with regard to the effect on DNase activity: SNPs not affecting the activity level, ones reducing it, ones abolishing it, and ones elevating it. In particular, 9, 5, and 4 SNPs producing a loss-of-function variant of the enzymes in DNASE1, DNASE1L3, and DNASE2, respectively, were confirmed. SNPs producing DNase loss of function can be estimated by PolyPhen-2 to be "probably damaging" with a high accuracy of prediction. Almost all of these functional SNPs producing a loss of function or substantially low activity-harboring forms exhibited a mono-allelic distribution in all of the populations. CONCLUSION: A minor allele of functional SNPs, despite the remarkably low genetic heterogeneity of the SNPs, might be a genetic risk factor for autoimmune diseases.

Identification of interacting partners of Human Mpv17-like protein with a mitigating effect of mitochondrial dysfunction through mtDNA damage.

Human Mpv17-like protein (M-LPH) has been suggested to participate in mitochondrial function. In this study, we investigated the proteins that interact with M-LPH, and identified four: H2A histone family, member X (H2AX), ribosomal protein S14 (RPS14), ribosomal protein S3 (RPS3) and B-cell receptor-associated protein 31 (Bap31). Immunofluorescence and subcellular fractionation studies revealed that M-LPH is localized predominantly in the nucleus, to some extent in a subset of mitochondria, and marginally in the cytosol. Mitochondrial M-LPH appeared as punctate foci, and these were co-localized with a subset of mitochondrial transcription factor A (TFAM) and mtDNA, indicating that M-LPH is localized in or in close proximity to mitochondrial nucleoids. RNAi-mediated knockdown of M-LPH resulted in an increase of mtDNA damage and reduced the expression of mtDNA-encoded genes. A ROS inducer, antimycin A, caused an increase in both the number and size of the mitochondrial M-LPH foci, and these foci were co-localized with two enzymes, DNA polymerase γ (POLG) and DNA ligase III (LIG3), both involved in mtDNA repair. Furthermore, knockdown of M-LPH hampered mitochondrial localization of these enzymes. Taken together, these observations suggest that M-LPH is involved in the maintenance of mtDNA and protects cells from mitochondrial dysfunction.

Global analysis of genetic variations in a 56-bp variable number of tandem repeat polymorphisms within the human deoxyribonuclease I gene.

A 56-bp variable number of tandem repeat polymorphism is confirmed in intron 4 of the human deoxyribonuclease I (DNase I) gene (HumDN1). The purpose of the present study was to document global ethnic variations of allelic frequencies in HumDN1 VNTR polymorphisms. In this study, HumDN1 VNTR polymorphisms in 11 worldwide populations were examined by polymerase chain reaction and compared with those reported previously. Fifteen genotypes were identified in these 11 populations. Novel genotypes were found: 1/2 was observed in Ghanaians and mestizos, 3/6 was in Tamangs, 4/6 was in Tibetans and Nahuas, 6/6 was in Sinhalese. The African population showed the highest frequency for the HumDN1(∗)3 allele. Among Asian populations, the different genotype distribution was observed. The predominant allele in Mongolian, Korean, Japanese, and Chinese populations was HumDN1(∗)3, followed by HumDN1(∗)4, and then HumDN1(∗)5. In Chinese from South China, Tamangs, and Sinhalese, HumDN1(∗)4 and HumDN1(∗)5 were predominant. The allele frequency for HumDN1(∗)4 was high in three Mexican populations, but a significant difference was observed between Nahuas and Huicoles. Germans and Turks showed a similar distribution. This study is the first to show the existence of a certain genetic heterogeneity in the worldwide distribution of HumDN1 VNTR polymorphism.

Identification of functional SNPs potentially served as a genetic risk factor for the pathogenesis of parakeratosis in the gene encoding human deoxyribonuclease I-like 2 (DNase 1L2) implicated in terminal differentiation of keratinocytes.

In the present study, we evaluated all of the 35 non-synonymous SNPs in the gene encoding DNase I-like 2 (DNase 1L2), implicated in terminal differentiation of keratinocytes, to seek a functional SNP that would potentially affect the levels of in vivo DNase 1L2 activity. Based on a compiled expression analysis of the amino acid-substituted DNase 1L2 corresponding to each of the 35 non-synonymous SNPs in the gene, these 35 SNPs were grouped into 4 classes according to the alteration of catalytic activity caused by the corresponding amino acid substitution in the DNase 1L2 protein; we were able to identify 12 non-synonymous SNPs as functional SNPs abolishing or substantially reducing the activity. Almost all of the amino acid residues corresponding to the SNPs abolishing the activity were completely or highly conserved in not only the DNase I family, but also animal DNase 1L2. Each of the minor alleles of these functional SNPs producing a loss-of-function or low activity-harboring variant was absent in 14 different populations derived from 3 ethnic groups, allowing us to assume that DNASE1L2 is generally well conserved with regard to these non-synonymous SNPs, thereby avoiding any marked reduction of the enzyme activity in human populations. However, it seems likely that each of the minor alleles for these SNPs may serve as a genetic risk factor for multiple skin diseases such as psoriasis, in which there is an aberrant retention of nuclear chromatin in cornified keratinocytes through incomplete DNA degradation.

Identification of the functional alleles of the nonsynonymous single-nucleotide polymorphisms potentially implicated in systemic lupus erythematosus in the human deoxyribonuclease I gene.

In the present study, we have extensively continued our previous investigations of the nonsynonymous single-nucleotide polymorphisms (SNPs) in the human DNase I (DNASE1) gene potentially relevant to systemic lupus erythematosus (SLE); therefore, all of the 58 nonsynonymous SNPs registered in the NCBI dbSNP database could be evaluated and it could be checked as to whether these SNPs might serve as a functional SNP. From a compiled expression analysis of the amino-acid-substituted DNase I corresponding to each of the SNPs, it was possible to sort them into 23 SNPs while not affecting the activity: 12 abolishing it, 14 reducing it, and 9 increasing it. Among a total of 58 nonsynonymous SNPs, only 4 SNPs exhibited genetic polymorphisms in some of the populations examined; a minor allele producing a loss-of-function variant of each SNP was not distributed in 14 different populations derived from three ethnic groups. It could be assumed that a minor allele of these functional SNPs, despite their remarkably low genetic heterogeneity, could directly serve as a genetic risk factor for SLE. Furthermore, among the human DNase family genes, it seems that DNASE1 is able to tolerate the generation of nonsynonymous SNPs, and that the amino-acid substitutions resulting from the SNPs in DNASE1 easily alter the activity.

Evaluation of all non-synonymous single nucleotide polymorphisms (SNPs) in the genes encoding human deoxyribonuclease I and I-like 3 as a functional SNP potentially implicated in autoimmunity.

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.

Evaluation of all nonsynonymous single-nucleotide polymorphisms in the gene encoding human deoxyribonuclease I-like 1, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer.

Many nonsynonymous single-nucleotide polymorphisms (SNPs) in the human deoxyribonuclease I-like 1 (DNase 1L1) gene, possibly implicated in the blocking of endocytosis-mediated foreign gene transfer, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all 21 nonsynonymous human DNase 1L1 SNPs was performed in 16 different populations representing three ethnic groups using the PCR-restriction fragment length polymorphism technique. All of the nonsynonymous SNPs, except for SNP p.Val122Ile in Caucasian populations, exhibited a monoallelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, two activity-abolishing and four activity-reducing SNPs were confirmed to be functional. Although all of the nonsynonymous SNPs that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of six SNPs producing a loss-of-function or extremely low-activity variant could serve directly as a genetic risk factor for diseases. Especially, the amino acid residues in activity-abolishing SNPs were conserved in animal DNases 1L1. Furthermore, results of phylogenetic analysis suggest that DNase 1L1 might have appeared latest among the DNase I family during the course of molecular evolution.

Seven nonsynonymous SNPs in the gene encoding human deoxyribonuclease II may serve as a functional SNP potentially implicated in autoimmune dysfunction.

Many nonsynonymous SNPs in the human DNase II gene (DNASE2), potentially relevant to autoimmunity in conditions such as rheumatoid arthritis, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all the 15 nonsynonymous human DNase II SNPs was performed in three ethnic groups including 16 different populations using the PCR-restriction fragment length polymorphism technique. A series of constructs corresponding to each SNP was examined. Fifteen nonsynonymous SNPs in the gene, except for p.Val206Ile in a Korean population, exhibited a mono-allelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, four activity-abolishing and five activity-reducing SNPs were confirmed to be functional. The amino acid residues in activity-abolishing SNPs were conserved in animal DNase II. All the nonsynonymous SNPs that affected the catalytic activity of human DNase II showed extremely low genetic heterogeneity. However, a minor allele of seven SNPs producing a loss-of-function or extremely low activity-harboring variant could serve as a genetic risk factor for autoimmune dysfunction. These functional SNPs in DNASE2 may have clinical implications in relation to the prevalence of autoimmune diseases.

Distribution and haplotype analysis of all the non-synonymous and autoimmunity-related single nucleotide polymorphisms in the human deoxyribonuclease II gene using worldwide populations.

We have focused on the 14 SNPs including all the non-synonymous and autoimmunity-related ones in the DNase II gene (DNASE2). The distribution of each allele and haplotype in these SNPs was examined in eight Asian, three African, three Mexican and two Caucasian populations using the newly developed PCR-RFLP methods. Eight SNPs among nine non-synonymous ones were monomorphic, indicating that a specific allele generating the intact activity-harboring DNase II in these SNPs is well conserved in worldwide populations. On the other hand, five other SNPs (-1951G>A, -1066G>C, -390A>C, +2630T>C, and +6235G>C) related to autoimmunity exhibited polymorphism common in worldwide populations, and especially their distributions were ethnic-dependent in the same manner as those of haplotypes. Furthermore, a strong linkage between SNPs -1951G>A and -1066G>C was confirmed in most populations. This study was the first to report any worldwide population analysis regarding all the non-synonymous and autoimmunity-related SNPs in the DNASE2, providing genetic information on the DNASE2 as a genetic marker for personal identification and/or genetic factor for susceptibility to autoimmunity.

Five non-synonymous SNPs in the gene encoding human deoxyribonuclease I-like 2 implicated in terminal differentiation of keratinocytes reduce or abolish its activity.

Several non-synonymous SNPs in the human deoxyribonuclease I-like 2 (DNase 1L2) gene responsible for DNA degradation during terminal differentiation of epidermal keratinocytes have been identified. However, only limited population data are available, and furthermore the effect of these SNPs on the DNase 1L2 activity remains unknown. Genotyping of all of the 17 SNPs was performed using the PCR-RFLP method in three ethnic groups including 14 different populations. A series of amino acid-substituted DNase 1L2 corresponding to each SNP was expressed, and its activity was measured. All of the six non-synonymous SNPs exhibited a mono-allelic distribution, whereas the distribution of some SNPs other than exonic ones was ethnicity-dependent. Each of the minor alleles in SNPs, p.Ala20Asp, p.Val104Leu, p.Asp197Ala, p.Glu274Lys and p.Asp287Asn, among the non-synonymous SNPs produced low or no activity-harbouring DNase 1L2. DNase 1L2 is well conserved, retaining full levels of enzymatic activity, with regard to these exonic SNPs in human populations. It seems plausible to assume that these SNPs affecting the activity may be one of the factors responsible for a genetic pre-disposition for failure of differentiation-associated cell death in various keratinocyte lineages, thereby leading to the development of parakeratosis. Our results may have clinical implications in relation to the pathogenesis of parakeratosis.

Three Nonsynonymous Single Nucleotide Polymorphisms in the RhitH Gene Cause Reduction of the Repression Activity That Leads to Upregulation of M-LPH, a Participant in Mitochondrial Function.

Human Mpv17-like protein (M-LPH) has been suggested to play a role in mitochondrial function. In this study, we identified a RhitH (human regulator of heat-induced transcription) binding site in intron 1 of the M-LPH gene. Tissue distribution analysis showed that M-LPH was specifically distributed in tissues with high mitochondrial metabolism. Functional and genetic analyses of nonsynonymous single nucleotide polymorphisms (SNPs) in the RhitH gene revealed that p.Cys461Ser, p.Thr465Ala, and p.Leu495Gln, corresponding to substitutions in the zinc fingers, cause reductions in the repression activity that lead to upregulation of M-LPH expression. The analyses also showed that the minor allele frequencies of these SNPs are extremely low in worldwide populations.