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Mitochondrial DNA (mtDNA) control region sequences from hair samples of 213 individuals from Thailand were analyzed using Sanger sequencing. A total of 170 different haplotypes were identified, of which 146 occurred only once (unique haplotypes). The dataset showed a random match probability of 0.87% and a haplotype diversity of 0.9960. The samples were assigned to 85 different haplogroups with B5a, F1a1a, and M being the most frequent ones. Pairwise FST-values between this and other Southeast and East Asian populations revealed significant but relatively low differences, indicating a close relation. Heteroplasmic positions were observed in 12.2% of hair samples confirming the frequent appearance of heteroplasmic positions in hairs. This dataset will complement existing data as an mtDNA reference for forensic investigations. Electronic supplementary material The online version of this article (10.1007/s00414-020-02303-2) contains supplementary material, which is available to authorized users.

CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation ofQPCTexpression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereasQPCTLwas not regulated. In thyroid carcinoma cell lines,QPCTgene expression correlates with the mRNA levels of its substrateCCL2. BothQPCTandCCL2are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi ofp50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, andQPCTRNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

The goal of the study was to develop a STR multiplex ("Paterniplex") that is - as supplement to commercially available multiplex kits like the Identifiler® kit (Applied Biosystems, Foster City, CA) - suitable for solving complex paternity cases such as deficiency cases or cases with mutations. The Paterniplex comprises the nine highly polymorphic STRs D8S1132, D7S1517, D10S2325, D12S391, Se33, D17S976, Penta E, Penta D and FGA in addition to Amelogenin as sex determination marker. The loci were selected because of their high degree of polymorphism (higher than that of the widely used TH01 marker). Only one locus, FGA, is shared with the Identifiler kit to avoid sample mix up. The study further gives details on the population genetics of the loci in a German Caucasian population (allelic distribution, Hardy-Weinberg Equilibrium and forensic efficiency markers such as the Discriminating Power) and three examples for cases that could not be solved using commercially available kits alone, but using the Paterniplex in addition to a commercial kit.

The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood. We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR. Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine. Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions. Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.

Background Periodontitis is a chronic inflammatory disease triggered by the host immune response. Epigenetic modifications also affect the immune response. We assessed CpG methylation in 22 inflammatory candidate genes (ATF2, CCL25, CXCL14, CXCL3, CXCL5, CXCL6, FADD, GATA3, IL10RA, IL12A, IL12B, IL13, IL13RA1, IL15, IL17C, IL17RA, IL4R, IL6R, IL6ST, IL7, INHA, and TYK2) with respect to the occurrence of aggressive periodontitis (AgP). Patients and methods In this study 15 AgP patients (53.3% males, 41.4 ± 10.5 years) and 10 controls (40.0% males, 36.9 ± 17.5 years) were included. The methylation patterns of gingival biopsies were quantified using EpiTect® Methyl Signature PCR Array Human Inflammatory Response. Results In gingival biopsies taken from patients with AgP, CpG methylation of CCL25 (1.73% vs. 2.59%, p = 0.015) and IL17C (6.89% vs. 19.27%, p = 0.002) was significantly reduced as compared with periodontally healthy tissues. Discussion We showed for the first time a differential methylation pattern for CCL25 and IL17C in periodontitis. CCL25 plays an important role in T-cell development, whereas IL17C regulates innate epithelial immune responses. The decrease in CpG methylation is presumably accompanied by an increase in gene expression. This could lead to a greater availability of CCL25 and interleukin 17C and support periodontal loss of attachment.

In human populations, the correct historical interpretation of a genetic structure is often hampered by an almost inherent inability to differentiate between ancient and more recent influences upon extant gene pools. One method to trace recent population movements is the analysis of surnames, which, at least in Central Europe, can be thought of as traits 'linked' to the Y chromosome. Illegitimacy, extramarital birth and changes of surnames may have substantially obscured this linkage. In order to assess the actual extent of correlation between surnames and Y-chromosomal haplotypes in Central Europe, we typed Y-chromosomal short tandem repeat markers in 419 German males from Halle. These individuals were subdivided into three groups according to the origin of their respective surname, namely German (G), Slavic (S) or 'Mixed' (M). The distribution of the haplotypes was compared by Analysis of Molecular Variance. While the M group was indistinguishable from group G (PhiST=-0.0008, P0.5), a highly significant difference (PhiST=0.0277, P0.001) was observed between the S group and the combined G+M group. This surprisingly strong differentiation is comparable to that of European populations of much larger geographic and linguistic difference. In view of the major migration from Slavic countries into Germany in the 19th century, it appears likely that the observed concurrence of Slavic surnames and Y chromosomes is of a recent rather than an early origin. Our results suggest that surnames may provide a simple means to stratify, and thereby to render more efficient, Y-chromosomal analyses of Central Europeans that target more ancient events.

Aging is thought to occur through the accumulation of molecular and cellular damage. A key regulator of the cell's stress response is p53. In mice, the activity of p53 associates with lifespan. We were therefore interested whether SNPs in members of the p53-pathway are associated with longevity in humans. We genotyped the following SNPs: p53 - rs1042522 (Arg72Pro), MDM2 - rs2279744 (SNP309), MDM4 - rs4245739 (SNP34091), rs1563828 (SNP31826), PPP2R2B (rs319217) in 155 long-lived individuals (LLIs) who died at the age of 91 and over and in 171 ethnically-matched control subjects. Kaplan-Meier survival curves and log-Rank-test were used to determine the mean and median survival times. In female LLIs, the Pro-allele of rs1042522 (Arg72Pro) and the G-allele of rs2279744 (SNP309) were significantly associated with an increased survival time (P=0.026, P

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients.

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

Highly sensitive short tandem repeat-based stain analysis of weak biological stains has been improved during the last years. The risk of transfer of cellular material from handled items contaminated with biological stains such as blood and saliva is of forensic relevance. Although the policemen working with crime scene items are very careful, there exists a potential risk of transfer contamination. To obtain estimates for the risk of stain transfer by handling, we have carried out an experimental study on 288 dried blood and saliva stains in two laboratories. DNA quantification showed only small amounts of DNA that could be transferred, especially from stains on paper and cotton. The saliva and especially the blood stains from plastic surface resulted in higher amounts of transferred DNA, depending on the relation of blood volume to included area: Of 192 direct transfers, 17% gave extracts above 10 pg DNA/μl, and only 3% of 96 secondary transfers resulted in amounts above 10 pg DNA/μl.

In this study, orangutan (Pongo pygmaeus ssp.) DNA was extracted from excrements, obtained from individual zoo populations. To examine whether human short tandem repeat loci (STR) are suitable for the reconstruction of kinship in orangutans, nine STRs, commonly used in forensic studies in man and the amelogenin system, were amplified in a multiplex polymerase chain reaction (PCR) approach. Five of the nine human autosomal STRs in question amplified successfully in orangutans. Furthermore, the experiments revealed that orangutans possess allele distributions comparable to humans in these systems. Fragment length determination allowed kinship structures to be reconstructed.

Objective: Fetal microchimerism (MCH) has been implicated in the etiology of autoimmune diseases such as autoimmune thyroiditis. The goal of the study was to reliably estimate the number of fetal engrafted cells and to further investigate factors influencing the development of MCH. Methods: Quantitative real-time PCR amplification using Y-chromosome specific (DYS14) and autosomal (β-globin) loci was performed on thyroid gland specimens. Furthermore, we compared the distribution of ABO and rhesus systems in mothers with and without blood MCH in relation to the blood groups of the children. Results: MCH was detected in eight of 21 Hashimoto patients in a frequency range of 15 to 4900 male cells per 100 000 total cells (median 97 cells), but in none of 17 healthy thyroid glands. In a third group, consisting of 18 nodular goiters, only one sample was positive (182 male cells/100 000 total cells). No woman who had not had a prior pregnancy with a male fetus showed MCH. Mothers both with and without MCH showed the same rate of mother/child incompatibilities for the ABO and rhesus systems. Conclusions: The percentage of microchimeric cells varies to a great extent in Hashimoto’s thyroiditis, and this phenomenon can occur in nodular goiter in rare instances, but it appears to be absent from normal thyroid glands. Nevertheless, the biological significance of MCH remains unclear. Moreover, we have concluded that the tested blood group systems (as opposed to their role in graft vs host disease after transplantations) have no effect on fetal MCH.

TH01 is a tetrameric short tandem repeat locus located in intron 01 of the tyrosine hydroxylase gene. The tyrosine hydroxylase catalyzes the hydroxylation of L-tyrosine to L-DOPA and is the rate limiting enzyme in the synthesis of catecholamines like noradrenaline or adrenaline, which are pivotal in the regulation of blood pressure. In a clinical study a strong correlation between alleles *9.3 and *10 and essential hypertension was observed ([2] Hypertension 32: 676–682). To further investigate this association, we typed TH01 in 296 autopsy cases and correlated the genotypes to the heart weight as parameter for myocardial hypertrophy. No significant correlation was observed. Moreover, dividing the studied cases into 2 groups, one including 172 casualties from hypertension-associated diseases (myocardial infarction, left heart failure, aortic aneurysm, spontaneous intracerebral bleeding and cerebral infarction) and one consisting of 124 cases of death unrelated to hypertension, revealed similar allelic frequencies for both groups. Our data thus suggest that TH01 long alleles appear not to lead to a significant increase in the incidence of myocardial hypertrophy or other hypertension associated diseases. This could be explained by a relatively small impact of the TH01 genotype on the blood pressure or by counteraction of another mechanism related to catecholamines and their effect on the human body.

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.

We have performed a population genetic study on a population from South Saxony-Anhalt, Germany. The allele distributions of the systems DYS19, DYS385, DYS389I/II DYS390, DYS391, DYS392 and DYS393 were investigated in a sample of 234 unrelated males. PCR products were detected using capillary electrophoresis on the ABI Prism 310 DNA sequencer. Two hundred and six different haplotypes were obtained. The haplotype diversity was 0.8915. Using AMOVA significant differences were observed to populations from Poland and Croatia.

For several years Y-chromosomal microsatellites (short tandem repeats, STRs) have been well established in forensic practice. In this context, the genetic characteristics of the Y chromosome (i.e. its paternal inheritance and lack of recombination) render STRs particularly powerful. However, genetic differences between male populations appear to be larger for Y-STRs than for autosomal STRs, a fact that is most likely due to the higher sensitivity of Y-chromosomal lineages to genetic drift (Forensic Sci Int 118 (2001) 153). The assessment of probabilities for matches between haplotyped male persons or traces/persons requires the typing of a large number of haplotypes in the appropriate reference populations. The haplotype data of a large number of European as well as South and North American populations have been collected and are continuously published online (Y-STR Haplotype Reference Database--YHRD; http://www.ystr.org). The most recent multicentric effort has led to the establishment of an Asian YHRD (http://www.ystr.org/asia) which has been available since January 2002. All databases are maintained and curated at the Institute of Legal Medicine, Humboldt-University, Berlin and will soon be fused to a global repository including populations from all continents.

Allele frequencies for the short tandem repeat loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, THO1 and FGA were determined in 231 German and 100 Austrian unrelated Caucasoids using the AmpFlSTR SGM plus kit (Applied Biosystems). All loci met Hardy-Weinberg expectations. In 212 meioses per locus, one mutation event for D19S433 was observed.

Genetic analysis is a useful tool for assigning biological relationships. Thus, it will improve genetic management of wild animal populations and breeding colonies. Kinship analysis will give new insights into the behavior, sociobiology and genetic management of orangutans. In this study, chromosomal DNA from orangutan (Pongo pygmaeus ssp.) was extracted from excrements. Feces samples were screened for up to nine microsatellite markers from related zoo populations of orangutans (Pongo pygmaeus ssp.) kept at the Zoological Garden Berlin and the Zoological Garden Heidelberg, Germany. Family structures are documented in the "International Studybook of the Orangutan" (Perkins 1995) and the "Europaisches Erhaltungszucht Programm 1998" (Becker 1998). To examine whether human short tandem repeat loci (STR) are suitable for the reconstruction of kinship in orangutans, nine STRs, commonly used in forensic studies and the amelogenin system, were amplified in a multiplex-PCR approach (AmpFlSTR Profiler Plus). We were able to show that five of the nine human autosomal STRs in question amplified successfully in orangutans. Thus, we could reconstruct kinship structures of the Berlin and Heidelberg populations.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent. Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marino, Miguel Eduardo. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Analisis de ADN; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Purps, Josephine. Charité-Universitätsmedizin; Alemania Fil: Siegert, Sabine. University of Cologne; Alemania Fil: Willuweit, Sascha. Charité-Universitätsmedizin; Alemania Fil: Nagy, Marion. Charité-Universitätsmedizin; Alemania Fil: Alves, Cíntia. Universidad de Porto; Portugal Fil: Salazar, Renato. Universidad de Porto; Portugal Fil: Angustia, Sheila M. T.. Philippine National Police Crime Laboratory; Filipinas Fil: Santos, Lorna H.. Philippine National Police Crime Laboratory; Filipinas Fil: Anslinger, Katja. Universitat Genzentrum Der Ludwing-maximilians; Alemania Fil: Bayer, Birgit. Universitat Genzentrum Der Ludwing-maximilians; Alemania Fil: Ayub, Qasim. The Wellcome Trust Sanger Institute; Reino Unido Fil: Wei, Wei. The Wellcome Trust Sanger Institute; Reino Unido Fil: Xue, Yali. The Wellcome Trust Sanger Institute; Reino Unido Fil: Tyler Smith, Chris. The Wellcome Trust Sanger Institute; Reino Unido Fil: Baeta Bafalluy, Miriam. Universidad de Zaragoza; España Fil: Martínez Jarreta, Begoña. Universidad de Zaragoza; España Fil: Egyed, Balazs. Eotvos University, Budapest; Argentina Fil: Balitzki, Beate. Universidad de Basilea; Suiza Fil: Tschumi, Sibylle. Universidad de Basilea; Suiza Fil: Ballard, David. King; Reino Unido Fil: Syndercombe Court, Denise. King; Reino Unido Fil: Barrantes, Xinia. Poder Judicial, Forensic Sciences Department; Costa Rica Fil: Bäßler, Gerhard. Landeskriminalamt Baden-Württemberg; Alemania Fil: Berger, Burkhard. Universidad de Innsbruck; Austria Fil: Niederstätter, Haral. Universidad de Innsbruck; Austria Fil: Parson, Walther. Universidad de Innsbruck; Austria. University Park; Estados Unidos Fil: Davis, Carey. Department of Molecular and Medical Genetics; Estados Unidos. Institute of Applied Genetics; Estados Unidos Fil: Furfuro, Sandra. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentina Fil: Locarno, Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentina