Multiplex-Genotypisierung forensisch relevanter SNPs unter Verwendung von Microarrays auf chemisch aktivierten Glasflächen

Die Analyse tandem-repetitiver DNA-Sequenzen hat einen festen Platz als genetisches Typisierungsverfahren in den Breichen der stammesgeschichtlichen Untersuchung, der Verwandtschaftsanalyse und vor allem in der forensischen Spurenkunde, bei der es durch den Einsatz der Multiplex-PCR-Analyse von Short Tandem Repeat-Systemen (STR) zu einem Durchbruch bei der Aufklärung und sicheren Zuordnung von biologischen Tatortspuren kam. Bei der Sequenzierung des humanen Genoms liegt ein besonderes Augenmerk auf den genetisch polymorphen Sequenzvariationen im Genom, den SNPs (single nucleotide polymorphisms). Zwei ihrer Eigenschaften – das häufige Vorkommen innerhalb des humanen Genoms und ihre vergleichbar geringe Mutationsrate – machen sie zu besonders gut geeigneten Werkzeugen sowohl für die Forensik als auch für die Populationsgenetik.rnZum Ziel des EU-Projekts „SNPforID“, aus welchem die vorliegende Arbeit entstanden ist, wurde die Etablierung neuer Methoden zur validen Typisierung von SNPs in Multiplexverfahren erklärt. Die Berücksichtigung der Sensitivität bei der Untersuchung von Spuren sowie die statistische Aussagekraft in der forensischen Analyse standen dabei im Vordergrund. Hierfür wurden 52 autosomale SNPs ausgewählt und auf ihre maximale Individualisierungsstärke hin untersucht. Die Untersuchungen der ersten 23 selektierten Marker stellen den ersten Teil der vorliegenden Arbeit dar. Sie umfassen die Etablierung des Multiplexverfahrens und der SNaPshot™-Typisierungsmethode sowie ihre statistische Auswertung. Die Ergebnisse dieser Untersuchung sind ein Teil der darauf folgenden, in enger Zusammenarbeit der Partnerlaboratorien durchgeführten Studie der 52-SNP-Multiplexmethode. rnEbenfalls im Rahmen des Projekts und als Hauptziel der Dissertation erfolgten Etablierung und Evaluierung des auf der Microarray-Technologie basierenden Verfahrens der Einzelbasenverlängerung auf Glasobjektträgern. Ausgehend von einer begrenzten DNA-Menge wurde hierbei die Möglichkeit der simultanen Hybridisierung einer möglichst hohen Anzahl von SNP-Systemen untersucht. Die Auswahl der hierbei eingesetzten SNP-Marker erfolgte auf der Basis der Vorarbeiten, die für die Etablierung des 52-SNP-Multiplexes erfolgreich durchgeführt worden waren. rnAus einer Vielzahl von Methoden zur Genotypisierung von biallelischen Markern hebt sich das Assay in seiner Parallelität und der Einfachheit des experimentellen Ansatzes durch eine erhebliche Zeit- und Kostenersparnis ab. In der vorliegenden Arbeit wurde das „array of arrays“-Prinzip eingesetzt, um zur gleichen Zeit unter einheitlichen Versuchsbedingungen zwölf DNA-Proben auf einem Glasobjektträger zu typisieren. Auf der Basis von insgesamt 1419 typisierten Allelen von 33 Markern konnte die Validierung mit einem Typisierungserfolg von 86,75% abgeschlossen werden. Dabei wurden zusätzlich eine Reihe von Randbedingungen in Bezug auf das Sonden- und Primerdesign, die Hybridisierungsbedingungen sowie physikalische Parameter der laserinduzierten Fluoreszenzmessung der Signale ausgetestet und optimiert. rn

A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

Background: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. Methods: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in antimalarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e. g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. Conclusion: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.

Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha-238 and-308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study

Background The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. Methods Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher`s test. Results More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06-9.71; P = 0.04) in the group with severe disease. Conclusions Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

Development and Validation of Single Nucleotide Polymorphisms (SNPs) Markers from Two Transcriptome 454-Runs of Turbot (Scophthalmus maximus) Using High-Throughput Genotyping

The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genesrelated to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot

Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus

Most of the cultivated species of citrus have narrow genetic basis. Relationships among species and cultivars are obscured by sexual compatibility, polyembryony, apomixis and a high incidence of somatic mutations. DNA analysis is crucial in genetic studies not only for citrus breeding programs but also for characterization of hybrids and species. In this paper, single nucleotide polymorphisms ( SNPs) were investigated in 58 accessions of Citrus, hybrids and related genera. Genomic sequences of 'Pera IAC' sweet orange ( Citrus sinensis L. Osbeck) were used for primer design and selection of sequence tagged sites (STSs) for identification of SNPs. Analysis of 36 STSs showed identical sequences among 40 of the 41 sweet orange accessions studied. However, these accessions were heterozygous for many SNPs. Ten selected STSs were analyzed in 17 additional accessions from 13 species and hybrids. Comparing to the 'Pera IAC' sweet orange accession, a total of 150 polymorphic nucleotides were identified and most of the alterations were transitions ( 52.7%). The greatest number of SNPs was observed in Poncirus trifoliata ( L.) Raf. and the smallest in 'Ponkan' mandarin ( Citrus reticulata Blanco). At the intra-specific level, 'Bafa Gigante' ( Citrus sinensis L. Osbeck) was the only sweet orange accession with a divergent SNPs genotype, which corroborates the hypothesis of a hybrid origin for this accession. Although the STSs analyzed represent randomly sampled genomic sequences, they provided consistent information about the level of polymorphism and showed the potential of SNPs markers for characterization and phylogenetic studies.

Identificação de SNPs (Single Nucleiotide Polymorphisms) no gene colina monooxigenase relacionado ao metabolismo da glicina betaína em Eucalyptus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

SNPs (Single Nucleotide Polymorphisms) em genes relacionados a absorção e translocação de Boro em Eucalyptus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de marcadores SNPs (Single Nucleotide Polymorphisms) para estudos populacionais do mosquito Aedes aegypti no Brasil

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds

Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.

Single nucleotide polymorphisms (SNPs) associated with TGF-beta pathway and their significance in systemic sclerosis - A multilevel analysis

Systemic sclerosis (SSc) or Scleroderma is a complex disease and its etiopathogenesis remains unelucidated. Fibrosis in multiple organs is a key feature of SSc and studies have shown that transforming growth factor-β (TGF-β) pathway has a crucial role in fibrotic responses. For a complex disease such as SSc, expression quantitative trait loci (eQTL) analysis is a powerful tool for identifying genetic variations that affect expression of genes involved in this disease. In this study, a multilevel model is described to perform a multivariate eQTL for identifying genetic variation (SNPs) specifically associated with the expression of three members of TGF-β pathway, CTGF, SPARC and COL3A1. The uniqueness of this model is that all three genes were included in one model, rather than one gene being examined at a time. A protein might contribute to multiple pathways and this approach allows the identification of important genetic variations linked to multiple genes belonging to the same pathway. In this study, 29 SNPs were identified and 16 of them located in known genes. Exploring the roles of these genes in TGF-β regulation will help elucidate the etiology of SSc, which will in turn help to better manage this complex disease. ^

Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

Analysis of Single-Nucleotide Polymorphisms in the crt-o and mdr1 Genes of Plasmodium vivax among Chloroquine-Resistant Isolates from the Brazilian Amazon Region

Plasmodium vivax parasites with chloroquine resistance (CQR) are already circulating in the Brazilian Amazon. Complete single-nucleotide polymorphism (SNP) analyses of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes revealed no associations with CQR, even if some mutations had not been randomly selected. In addition, striking differences in the topologies and numbers of SNPs in these transporter genes between P. vivax and P. falciparum reinforce the idea that mechanisms other than mutations may explain this virulent phenotype in P. vivax.

Influence of Single-Nucleotide Polymorphisms on C-Reactive Protein Levels in Chronic Kidney Disease Before and After Kidney Transplantation

Introduction. We sought to evaluate 2 sing] e-nucleotide polymorphisms (SNPs) in the C-reactive protein (CRP) gene promoter region for their effects on CRP levels in chronic kidney disease (CKD) patients before and after a successful kidney transplantation. Methods. Fifty CKD patients were evaluated before and at the first and second years after the graft. Two SNPs were studied, a bi-allelic (G -> A) at the -409 and a tri-allelic (C -> T -> A) variation at the -390 position in the CRP gene. Results. All patients presented the -409GG genotype. At the -390 position, the ""A"" allele was not found; there were 15 ""CC"" patients, 11 ""TT"" patients, and 24 ""CT"" patients. CRP levels were different among patients with various genotypes (P < .019). Also the presence of the allele ""T"" was sufficient to determine differences in CRP levels both in pretransplantation (P = .045) and at 1 year posttransplantation (P = .011), but not at the second year (P = .448). Conclusion. SNPs at the -390 position of the CRP gene promoter region influence CRP basal levels in such a way that the ""C"" allele correlated with the lowest and the ""T"" with the highest. We did not observe this influence in our patients at the second year posttransplantation.