Dagmar Schmid

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Correlation of inter-individual variations of amitriptyline metabolism examined in hairs with CYP2C19 and CYP2D6 polymorphisms

The metabolite ratio of amitriptyline (AT), nortriptyline (NT) and its 10-hydroxy metabolites (E10-OHAT, Z10-OHAT, E10-OHNT and Z10-OHNT) was examined by liquid chromatography-mass spectrometry in hair samples of 23 white infants after long-term administration of AT. High inter-individual variation of the metabolite ratios were observed (e.g. NT/AT = 0.8–8.1, E10-OHNT/Z10-OHNT = 1.6–10.3). The significance of these variations was proven by confirmation of the temporary stability of these ratios within a hair fibre. Moreover, an association of the metabolic phenotype with genetic disposition was observed. The genotypes of CYP2C19 (alleles *2, *3 and *4) and of CYP2D6 (*3, *4, and *6) were examined by conventional polymerase chain reaction genotyping experiments. The relative amount of demethylation (NT/AT) is clearly affected by the number of functional alleles of CYP2C19. The demethylation capacity of CYP2C19 poor metabolizers (3 individuals, compared to 15 extensive metabolizers) was 4.3 times depleted. Moreover, the selectivity of hydroxylation (e.g. E10-OHNT/Z10-OHNT) is significantly correlated with CYP2C19.

Hair Analysis for Determination of Isoniazid Concentrations and Acetylator Phenotype during Antituberculous Treatment

Background. Analysis of isoniazid (INH) uptake has been based on measurement of plasma concentrations providing a short-term and potentially biased view. Objectives. To establish hair analysis as a tool to measure long-term uptake of INH and to assess whether acetylator phenotype in hair reflects N-acetyltransferase-2 (NAT2) genotype. Design and Methods. INH and acetyl-INH concentrations in hair were determined in patients on INH treatment for M. tuberculosis infection using high pressure liquid chromatography/mass spectrometry. Acetyl-INH/INH ratios were correlated with NAT-2 genotype. Results. Hair concentrations of INH, determined in 40 patients, were not dependent on ethnic group or body mass index and were significantly higher in male compared to female patients (median (range) 2.37 ng/mg (0.76–4.9) versus 1.11 ng/mg (0.02–7.20) (P = 0.02). Acetyl-INH/INH ratios were a median of 15.2% (14.5 to 31.7) in homozygous rapid acetylator NAT-2 genotype and 37.3% (1.73 to 51.2) in the heterozygous rapid acetylator NAT-2 genotype and both significantly higher than in the slow acetylator NAT-2 genotype with 5.8% (0.53 to 14.4) (P < 0.05). Conclusions. Results of hair analysis for INH showed lower concentrations in females. Acetyl-INH/INH ratios were significantly lower in patients with slow acetylator versus rapid acetylator genotypes.

DNA adducts of ortho-toluidine in human bladder

Background: 4-Aminobiphenyl (4-ABP) and o-toluidine are known human bladder carcinogens, but only 4-ABP-releasing DNA adducts are known. Methods: Determination of 4-ABP and o-toluidine-releasing DNA adducts in epithelial and submucosal bladder tissues of sudden death victims (SDV: n = 46), and bladder tumours (n = 12) by gas chromatography/mass spectrometry. Results: Above background, 4 and 11 of 12 tumour samples contained adducts of 4-ABP (0.057 ± 0.125 fmol/µg DNA) and o-toluidine (8.72 ± 4.49 fmol/µg DNA), respectively. Lower adduct levels were present in both epithelial and submucosal bladder tissues of SDV (4-ABP: 0.011 ± 0.022 and 0.019 ± 0.047 fmol/µg DNA; o-toluidine: 0.24 ± 0.63 and 0.27 ± 0.70 fmol/µg DNA). Conclusion: Detection of o-toluidine-releasing DNA adducts support the carcinogenicity of o-toluidine in the human bladder.

Mass spectrometric base composition profiling: Implications for forensic mtDNA databasing

In forensic genetics mitochondrial DNA (mtDNA) is usually analyzed by direct Sanger-type sequencing (STS). This method is known to be laborious and sometimes prone to human error. Alternative methods have been proposed that lead to faster results. Among these are methods that involve mass-spectrometry resulting in base composition profiles that are, by definition, less informative than the full nucleotide sequence. Here, we applied a highly automated electrospray ionization mass spectrometry (ESI-MS) system (PLEX-ID) to an mtDNA population study to compare its performance with respect to throughput and concordance to STS. We found that the loss of information power was relatively low compared to the gain in speed and analytical standardization. The detection of point and length heteroplasmy turned out to be roughly comparable between the technologies with some individual differences related to the processes. We confirm that ESI-MS provides a valuable platform for analyzing mtDNA variation that can also be applied in the forensic context.

Comparison of telogen hair analyses: genRES® MPX-2SP kit versus genRES® MPX-SP1 and genRES® MPX-SP2 kits

STR investigations of telogen hair are invariably difficult due to the small amounts of nuclear DNA and its degradation products. However, in recent years there has been a considerable improvement. This study examined the suitability of a new STR kit with shortened amplicons for the investigation of hair in routine casework. This kit allows the simultaneous amplification of the eight STR-loci D3S1358, VWA, FGA, TH01, SE33, D8S1179, D18S51, and D21S11, and the sex-determining amelogenin system. It was tested against the genRES MPX-SP1 and genRES MPX-SP2 kits. The sensitivity of the new genRES MPX-2SP kit was demonstrated to be inferior to that of the genRES MPX-SP1, but almost equal to that of the genRES MPX-SP2 kit.

Abstract 3461: DNA adducts of ortho-toluidine in human bladder

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Recently, ortho-toluidine (o-Tol) has been added to the list of aromatic amines in group 1 of human carcinogens by the International Agency for Research on Cancer. High levels of o-Tol hemoglobin adducts have been found in workers of a chemical factory with high excess of bladder cancer (Ward et al., JNCI 88:1046-52, 1996) as well as in patients treated with the local anesthetic prilocaine (Gaber et al., Toxicology 229:157-64, 2007). In contrast to 4-aminobiphenyl (4-ABP), the presence of DNA adducts of o-Tol has not yet been proven in humans. For the present study we obtained bladder tissue from 46 sudden death victims (SDV) and 12 samples from human bladder tumors. DNA was isolated from the epithelial and a submucosal layer of the SDV tissue samples and from tumor samples. For analysis of DNA adducts releasing o-Tol and 4-ABP, the aromatic amines were hydrolyzed from DNA by addition of 100 μL 4 N HCl. As internal standards d9-o-Tol and d5-4-ABP were added prior to hydrolysis. The acidic DNA solution was washed with dichloromethane and the amines were extracted with hexane after alkalinization. Determination of o-Tol and 4-ABP was achieved after derivatization with heptafluorobutyric anhydride by capillary gas chromatography/mass spectrometry with negative chemical ionization. Water samples were analyzed with each batch of samples to control for background contamination. Values less than 2-fold higher than background values were designated as not detectable and included as zero values in calculation of mean±standard error. Adducts of o-Tol well above background levels could be detected in 11 of 12 tumor samples (8.72±1.30 fmol/µg DNA). In contrast, only 4 of 12 tumor samples were positive for ABP (0.057±0.036 fmol/µg; p<0.0001 vs o-Tol). In 46 samples of bladder tissue from SDV, o-Tol DNA adduct levels were significantly lower compared to tumor samples but still detectable in 13 samples from the epithelial (0.24±0.09 fmol/µg DNA; p<0.0001) and 10 samples from the submucosal layer (0.27±0.10 fmol/µg DNA; p<0.0001), respectively. 4-ABP was detectable at significantly lower levels compared to tumor samples in 32 samples from the epithelial (0.011±0.003 fmol/µg DNA; p<0.0001) and 28 samples from the submucosal layer (0.019±0.007 fmol/µg DNA; p<0.005) of the 46 samples of SDV bladder tissue. Further characterization of the data concerning active smoking and polymorphism of N-acetyltransferase 2 genotype is underway. In conclusion, for the first time DNA adducts releasing o-Tol were detected in human urinary bladder. The presence of DNA adducts in the human target tissue of o-Tol strongly support a carcinogenic risk which should be taken into account in preventive hazard minimization in both occupational medicine and drug therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3461.