Mitochondrial DNA control region variation in a population sample from Thailand.

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.

Normoxic accumulation of HIF1α is associated with glutaminolysis.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. CLINICAL RELEVANCE: 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.

Epigenetic characteristics in inflammatory candidate genes in aggressive periodontitis.

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.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Germline genetics of the p53 pathway affect longevity in a gender specific manner.

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<0.001, respectively, log-Rank-test). In contrast, there was no difference regarding the survival time in male LLIs (rs1042522: P=0.58, rs2279744: P=0.503, log-Rank-test). There was no difference regarding the average age of death for the genotypes of the respective SNPs in the MDM4 gene (rs1563828: P=0.99; rs4245739: P=0.179, respectively). Here we show for the first time that the G-allele of rs2279744 (SNP309) is associated with increased lifespan. Importantly, this effect is gender-specific. Our data support the hypothesis that genetic variants that are associated with lower activity of p53--and therefore increased tumor risk--are associated with prolonged lifespan in a gender-specific manner.

High coexpression of CCL2 and CX3CL1 is gender-specifically associated with good prognosis in soft tissue sarcoma patients.

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.

Role of glutaminyl cyclases in thyroid carcinomas.

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 of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are 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 of p50. 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, and QPCT RNAi 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.

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome.

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.

PopAffiliator: online calculator for individual affiliation to a major population group based on 17 autosomal short tandem repeat genotype profile.

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15-17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator ( http://cracs.fc.up.pt/popaffiliator ) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification.

"Paterniplex", a highly discriminative decaplex STR multiplex tailored for investigating special problems in paternity testing.

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.

Fetal microchimerism in Hashimoto's thyroiditis: a quantitative approach.

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 (beta-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.

Y-chromosomal STR haplotype analysis reveals surname-associated strata in the East-German population.

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, P>0.5), a highly significant difference (PhiST=0.0277, P<0.001) 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.

Y chromosome polymorphisms and haplotypes in South Saxony-Anhalt (Germany).

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.

TH01, a tetrameric short tandem repeat locus in the tyrosine hydroxylase gene: association with myocardial hypertrophy and death from myocardial infarction?

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.

Haplotype studies support slippage as the mechanism of germline mutations in short tandem repeats.

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.