Functional and genetic survey of all known single-nucleotide polymorphisms within the human deoxyribonuclease I gene in wide-ranging ethnic groups

The single-nucleotide polymorphisms (SNPs) in the human DNase I gene (DNASE1) might be involved in susceptibility to some common diseases; however, only limited population data are available. Further, the effects of these SNPs on in vivo DNase I activity remain unknown. The genotype and haplotype of all the SNPs in DNASE1 were determined in 3 ethnic groups including 14 populations using newly developed methods. Together with our previous data on the nonsynonymous SNPs, two major haplotypes based on the five exonic SNPs were identified; genetic diversity in the Asian population was low. Among 10 SNPs, other than exonic SNPs in the gene, only 3 were polymorphic among all the populations. Haplotype distribution, based on all the polymorphic SNPs, was clarified to be generally varied in an ethnic-dependent manner. Thus, the genetic aspects of DNASE1 with regard to all the SNPs in wide-ranging ethnic groups could be first demonstrated. Further, there was no correlation of all the polymorphic SNPs other than nonsynonymous ones with serum DNase I activity levels. Polymorphic SNPs other than the exonic SNPs might not be directly related to common diseases through alterations in in vivo levels of the activity.     

Geographical north-south decline in DNASE1*2 in Japanese populations

Allele frequencies for human deoxyribonuclease I (DNase I) phenotypes were determined using blood samples from about 2000 Japanese subjects living in nine prefectures, and compared with one another. DNase I phenotyping was performed principally using isoelectric focusing electrophoresis and activity staining. The DNase I system was shown to have enhanced potential for anthropologic, genetic, and clinical studies of Japanese populations. DNase I phenotypes were analyzed to evaluate the degree of genetic variation at the DNASE1 locus. Our examination of DNase I types revealed a decreasing north-to-south gradient in the DNASE1 allele.     

Analysis of Genetic Polymorphism of Deoxyribonuclease I in Ovambo and Turk Populations Using a Genotyping Method

Deoxyribonuclease I (DNase I) polymorphism has been used as a valuable marker in genetic and clinical investigations. Six codominant alleles are known for DNase I, DNASE1*1, *2, *3, *4, and the recently discovered alleles *5 and *6. To detect these two new alleles, we added a new DNase I genotyping method based on both an allele-specific amplification and mismatched polymerase chain reaction (PCR). These methods were used to examine the distribution of DNase I genotypes in unrelated individuals from bloodstains of Ovambo and Turkish populations. The DNASE1*1 allele was found to be most dominant in the Ovambos. In contrast, Turks showed the highest allele frequency for DNASE1*2. This study is the first to demonstrate that there is a certain genetic heterogeneity in the worldwide distribution of DNase I polymorphism using the genotyping method of human DNase I polymorphism with PCR.     

Deoxyribonuclease I gene polymorphism in Han Chinese population: frequency and effect on glucose and lipid parameters

DNASE1, the encoding gene of deoxyribonuclease I (DNase I), exhibits polymorphisms, including a single nucleotide polymorphism (SNP A2317G) in exon 8 and a 56 bp variable number of tandem repeat, designated as HumDN1 in intron 4. Several different ethnic population studies have revealed both A2317G and HumDN1 demonstrate genetic heterogeneity in the worldwide distribution. Recently, G2317 allele was proposed as an independent risk factor for myocardial infarction in Japanese population. In the present study, we identified A2317G and HumDN1 genotypes in 402 unrelated healthy Han Chinese individuals. At the same time, the impact of different genotypes and diplotypes of DNase I on plasma lipids levels and fasting blood glucose was also illuminated. Polymerase chain reaction and restriction fragment length polymorphism were used for the detection of HumDN1 and A2317G polymorphisms. Plasma glucose and lipids were measured in fasting state by biochemical methods. Three genotypes of A2317G and 9 genotypes of HumDN1 were detected in Han Chinese population. Among them, the most predominate alleles were A2317 (frequency = 53.6%) and HumDN1*3 (frequency = 47.4%) respectively. Linkage disequilibrium between A2317G and HumDN1 polymorphisms was also observed (D' = 0.717). Haplotype A-3, presented in frequency of 46.5%, was most common. Compared to other ethnic populations, Han Chinese had its own unique DNase I gene distribution characteristics. As for the influence of DNase I gene polymorphisms on lipids and glucose levels, no association was found between either genotype or diplotype and these parameters. (all P > 0.05). Results obtained in this study could be used for anthropological investigation, probing into relations between DNase I gene and diseases.     

Extremely high prevalence of DNASE1*1 allele in African populations

The single nucleotide polymorphism (SNP) at deoxyribonuclease I (DNase I), designated as DNASE1 (NCBI SNP number; 1053874), in exon 8 (A2317G) has been shown to be associated with liver disease, colorectal carcinoma, and gastric carcinoma in Japanese patients. In this study, we investigated the frequency of the DNASE1 polymorphism in Ghanaian (n = 96) and Xhosa (n = 78) populations and compared the results with those of other studies. The single nucleotide polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The frequencies of DNASE1*1 in the Ghanaian and Xhosa populations were 0.90 and 0.88, respectively. These two African populations had an extremely high frequency of DNASE1*1, similar to that of the Ovambos living in Namibia. Caucasians and Asians had a lower frequency of DNASE1*1 than the African groups. This study is the first to reveal an extremely high frequency of DNASE1*1 among African populations.     

Deoxyribonuclease 1-like 3 Inhibits Hepatocellular Carcinoma Progression by Inducing Apoptosis and Reprogramming Glucose Metabolism

HCC has remained one of the challenging cancers to treat, owing to the paucity of drugs targeting the critical survival pathways. Considering the cancer cells are deficient in DNase activity, the increase of an autonomous apoptisis endonuclease should be a reasonable choice for cancer treatment. In this study, we investigated whether DNASE1L3, an endonuclease implicated in apoptosis, could inhibit the progress of HCC. We found DNASE1L3 was down-regulated in HCC tissues, whereas its high expression was positively associated with the favorable prognosis of patients with HCC. Besides, serum DNASE1L3 levels were lower in HCC patients than in healthy individuals. Functionally, we found that DNASE1L3 inhibited the proliferation of tumor cells by inducing G0/G1 cell cycle arrest and cell apoptosis in vitro. Additionally, DNASE1L3 overexpression suppressed tumor growth in vivo. Furthermore, we found that DNASE1L3 overexpression weakened glycolysis in HCC cells and tissues via inactivating the rate-limiting enzymes involved in PTPN2-HK2 and CEBPβ-p53-PFK1 pathways. Finally, we identified the HBx to inhibit DNASE1L3 expression by up-regulating the expression of ZNF384. Collectively, our findings demonstrated that DNASE1L3 could inhibit the HCC progression through inducing cell apoptosis and weakening glycolysis. We believe DNASE1L3 could be considered as a promising prognostic biomarker and therapeutic target for HCC.     

Distribution of Two Asian-Related Coding SNPs in the MC1R and OCA2 Genes

Very little is known about the genes and mechanisms affecting skin lightening in Asian populations. In this study, two coding SNPs, c.G1129A (R163Q) at the MC1R (melanocortin 1 receptor) gene and c.A1962G (H615R) at the OCA2 (oculocutaneous albinism type II) gene, were investigated in a total of 1,809 individuals in 16 populations from various areas. The Q163 and R615 alleles prevailed almost exclusively in East and Southeast Asian populations. Wright’s F _ST was 0.445 for R163Q and 0.385 for H615R among the 16 populations. The frequency of the Q163 allele was higher in Northeast Asians than in Southeast Asians. The frequency of the R615 allele was highest in South China and unlikely to be associated with levels of ultraviolet radiation. This allele may be a good marker to study the genetic affinity among East Asians because of its restricted distribution and marked difference in allele frequency.     

Insecticide-Driven Patterns of Genetic Variation in the Dengue Vector Aedes aegypti in Martinique Island

Effective vector control is currently challenged worldwide by the evolution of resistance to all classes of chemical insecticides in mosquitoes. In Martinique, populations of the dengue vector Aedes aegypti have been intensively treated with temephos and deltamethrin insecticides over the last fifty years, resulting in heterogeneous levels of resistance across the island. Resistance spreading depends on standing genetic variation, selection intensity and gene flow among populations. To determine gene flow intensity, we first investigated neutral patterns of genetic variability in sixteen populations representative of the many environments found in Martinique and experiencing various levels of insecticide pressure, using 6 microsatellites. Allelic richness was lower in populations resistant to deltamethrin, and consanguinity was higher in populations resistant to temephos, consistent with a negative effect of insecticide pressure on neutral genetic diversity. The global genetic differentiation was low, suggesting high gene flow among populations, but significant structure was found, with a pattern of isolation-by-distance at the global scale. Then, we investigated adaptive patterns of divergence in six out of the 16 populations using 319 single nucleotide polymorphisms (SNPs). Five SNP outliers displaying levels of genetic differentiation out of neutral expectations were detected, including the kdr-V1016I mutation in the voltage-gated sodium channel gene. Association tests revealed a total of seven SNPs associated with deltamethrin resistance. Six other SNPs were associated with temephos resistance, including two non-synonymous substitutions in an alkaline phosphatase and in a sulfotransferase respectively. Altogether, both neutral and adaptive patterns of genetic variation in mosquito populations appear to be largely driven by insecticide pressure in Martinique.     

Analysis of phenylalanine hydroxylase gene mutations in phenylketonuria patients from Kemerovo oblast and the Sakha Republic

This paper presents the results of a molecular genetic study on the phenylalanine hydroxylase (PAH) gene among phenylketonuria (PKU) patients and their family members residing in Kemerovo oblast and the Sakha Republic. To reveal the PAH gene mutations, the researchers applied exon amplification and a direct determination of their nucleotide sequences. The study has revealed both well-known mutations (R158Q, R252W, R261Q, P281L, IVS10 ? 11G > A, R408W, and IVS12 + 1G > A) and some rarely encountered ones (IVS2 + 5G > A, R155H, Y168H, W187R, E221-D222 > Efs, A342T, Y386C, and IVS11 + 1G > C). Some of the studied populations with a mixed ethnic ancestry have been shown to demonstrate a wider spectrum of their PKU-associated alleles.     

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACT

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.     

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.     

Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrP^sc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrP^sc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.     

Human loci involved in drug biotransformation: worldwide genetic variation, population structure, and pharmacogenetic implications

Understanding the role of inheritance in individual variation in drug response is the focus of pharmacogenetics (PGx). A key part of this understanding is quantifying the role of genetic ancestry in this phenotypic outcome. To provide insight into the relationship between ethnicity and drug response, this study first infers the global distribution of PGx variation and defines its structure. Second, the study evaluates if geographic population structure stems from all PGx loci in general, or if structure is caused by specific genes. Lastly, we identify the genetic variants contributing the greatest proportion of such structure. Our study describes the global genetic structure of PGx loci across the 52 populations of the Human Genome Diversity Cell-Line Panel, the most inclusive set of human populations freely available for studies on human genetic variation. By analysing genetic variation at 1,001 single nucleotide polymorphisms (SNPs) involved in biotransformation of exogenous substances, we describe the between-populations PGx variation, as well geographical groupings of diversity. In addition, with discriminant analysis of principal component (DAPC), we infer how many and which groups of populations are supported by PGx variation, and identify which SNPs actually contribute to the PGx structure between such groups. Our results show that intergenic, synonymous and non-synonymous SNPs show similar levels of genetic variation across the globe. Conversely, loci coding for Cytochrome P450s (mainly metabolizing exogenous substances) show significantly higher levels of genetic diversity between populations than the other gene categories. Overall, genetic variation at PGx loci correlates with geographic distances between populations, and the apportionment of genetic variation is similar to that observed for the rest of the genome. In other words, the pattern of PGx variation has been mainly shaped by the demographic history of our species, as in the case of most of our genes. The population structure defined by PGx loci supports the presence of six genetic clusters reflecting geographic location of samples. In particular, the results of the DAPC analyses show that 27 SNPs substantially contribute to the first three discriminant functions. Among these SNPs, some, such as the intronic rs1403527 of NR1I2 and the non-synonymous rs699 of AGT, are known to be associated with specific drug responses. Their substantial variation between different groups of populations may have important implications for PGx practical applications.     

The role of common and rare MC4R variants and FTO polymorphisms in extreme form of obesity

Melanocortin 4 receptor (MC4R) is an important regulator of food intake and number of studies report genetic variations influencing the risk of obesity. Here we explored the role of common genetic variation from MC4R locus comparing with SNPs from gene FTO locus, as well as the frequency and functionality of rare MC4R mutations in cohort of 380 severely obese individuals (BMI > 39 kg/m²) and 380 lean subjects from the Genome Database of Latvian Population (LGDB). We found correlation for two SNPs—rs11642015 and rs62048402 in the fat mass and obesity-associated protein (FTO) with obesity but no association was detected for rs17782313 located in the MC4R locus in these severely obese individuals. We sequenced the whole gene MC4R coding region in all study subjects and found five previously known heterozygous non-synonymous substitutions V103I, I121T, S127L, V166I and I251L. Expression in mammalian cells showed that the S127L, V166I and double V103I/S127L mutant receptors had significantly decreased quantity at the cell surface compared to the wild type MC4R. We carried out detailed functional analysis of V166I that demonstrated that, despite low abundance in plasma membrane, the V166I variant has lower EC₅₀ value upon αMSH activation than the wild type receptor, while the level of AGRP inhibition was decreased, implying that V166I cause hyperactive satiety signalling. Overall, this study suggest that S127L may be the most frequent functional MC4R mutation leading to the severe obesity in general population and provides new insight into the functionality of population based variants of the MC4R.     

Nonsynonymous single-nucleotide polymorphisms of the human apoptosis-related endonuclease--DNA fragmentation factor beta polypeptide, endonuclease G, and Flap endonuclease-1--genes show a low degree of genetic heterogeneity

DNA fragmentation factor beta (DFFB) polypeptide, endonuclease G (EndoG), and Flap endonuclease-1 (FEN-1) are responsible for DNA fragmentation, a hallmark of apoptosis. Although the human homologs of these genes show three, four, and six nonsynonymous single-nucleotide polymorphisms (SNPs), respectively, data on their genotype distributions in populations worldwide are limited. In this context, the objectives of this study were to elucidate the genetic heterogeneity of all these SNPs in wide-ranging populations, and thereby to clarify the genetic background of these apoptosis-related endonucleases in human populations. We investigated the genotype distribution of their SNPs in 13 different populations of healthy Asians, Africans, and Caucasians using novel genotyping methods. Among the 13 SNPs in the 3 genes, only 3 were found to be polymorphic: R196K and K277R in the DFFB gene, and S12L in the EndoG gene. All 6 SNPs in the FEN-1 gene were entirely monoallelic. Although it remains unclear whether each SNP would exert any effect on endonuclease functions, these genes appear to exhibit low degree of genetic heterogeneity with regard to nonsynonymous SNPs. These findings allow us to conclude that human apoptosis-related endonucleases, similarly to other human DNase genes, revealed previously, are well conserved at the protein level during the course of human evolution.     

Prospects for applying breeding for resistance to control scrapie in goats: The current situation in Italy

Genetic selection has been used to control transmissible spongiform encephalopathies (TSEs) in sheep populations based on the association between PRNP polymorphisms and resistance to scrapie. In goats even if a protective role has been suggested for several polymorphisms (I142M, H143R, N146D/S, R154H, R211Q, Q222K) by different European studies, a similar, univocal association has not been proved so far.The aim of this paper was to identify target polymorphisms in goats and their potential applicability in a selection plan in Italy. For this purpose, the existing genetic data on modulating resistance to classical and atypical scrapie in goats in this country will be used as starting point.Two Italian case–control studies concluded that the polymorphism at codon 222 (from glutamine to lysine Q/K) is important in influencing the susceptibility of goats to classical scrapie. Moreover, goats are susceptible to an unusual form of scrapie, named Nor98, and the H154 mutation was shown to be statistically associated with this disease in goats in an Italian case–control study. Currently, a strategy based on killing goats carrying the H154 mutation is being applied to manage atypical scrapie outbreaks in Italy.The current situation in Italy bodes well for the applicability of breeding plans based on the K222 mutation; data from independent studies on the role of K222 as a protective or even a resistance factor and its frequency in several Italian breeds are available. In the near future, as new data on K222 will be reported, testing the application of selective culling in classical scrapie goat outbreaks may become feasible.     

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy

Aims:  To determine the variability of the prion protein gene ( PRNP) in goats from Northern and Southern Italy.
Methods and results:  Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis.
Conclusions:  Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters.
Significance and impact of the study:  In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.     

Site-directed Mutagenesis of the Catalytic Residues of Bovine Pancreatic Deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (DNase I) is a well characterised endonuclease which cleaves double-stranded DNA to yield 5′ phosphory lated polynucleotides. Co-crystal structures of DNase I with two different oligonucleotides have revealed the presence of several residues (R9, E78, H134, D168, D212 and H252) close to the scissile phosphate. The roles that these amino acids play in the catalytic mechanism have been investigated using site-directed mutagenesis. The following variants were used: R9A, E78T, H134Q, D168S, D212S and H252Q. The kinetics of all six mutants with both DNA and a small chromophoric substrate, thymidine-3′,5′-di(p- nitrophenyl)-phosphate, were studied. Only R9A and E78T showed any significant turnover of the two substrates. D168S, H134Q, D212S and H252Q showed vanishingly low activities towards DNA and no detectable activity with thymidine-3′,5′-di-n(p-nitrophenyl)-phosphate. These results demonstrate that H134, D168, D212 and H252 play a critical role in the catalytic mechanism. It is suggested that H134 and H252 (which are hydrogen-bonded to E78 and D212, respectively) provided general acid and general base catalysis. DNase I also requires Mg^{2 +}and E39 has been identified as a ligand for this metal ion. We propose that D168 serves as a ligand for a second Mg^{2 +}, and thus DNase I, uses a two metal-ion hydrolytic mechanism. Both magnesium ions are used to supply electrophilic catalysis. Role assignment is based on the mutagenesis results, structural information, homologies between DNase I from different species and a comparison with exonuclease III. However, it is still not feasible to unequivocally assign a particular catalytic role to each amino acid/metal ion.