DNA degradation in human teeth exposed to thermal stress.

Human identification from burned remains poses a challenge to forensic laboratories, and DNA profiling is widely used for this purpose. Our aim was to evaluate the effect of temperature on DNA degradation in human teeth. Thirty teeth were exposed to temperatures of 100, 200, or 400 °C for 60 min. DNA was quantified by Real-Time qPCR (Quantifiler Human DNA Quantification Kit) and fluorescence spectroscopy (Qubit 3.0 Fluorometer). DNA degradation was evaluated by using STR markers (AmpFLSTR Identifiler Plus PCR Amplification Kit) to determine the allele and locus dropout, inter-locus balance, and degradation slope (observed (Oa) to expected (Ea) locus peak height ratio against the molecular weight). Most of the genomic DNA was degraded between 100 °C and 200 °C. At 100 °C, locus dropout ratios showed significant differences between the largest loci (FGA, D7S820, D18S51, D16S539, D2S1338 and CSF1PO) and amelogenin. Inter-locus balance values significantly differed between all dye channels except between NED and PET. The dropout ratio between D18S51 (NED) and amelogenin (PET) can be recommended for the evaluation of DNA degradation. The Oa/Ea regression model can predict locus peak heights in DNA degradation (R2 = 0.7881). These findings may be useful to assess the reliability of DNA typing for human identification in teeth subjected to prolonged incineration.

Dental color measurement to predict DNA concentration in incinerated teeth for human identification.

Teeth exposed to thermal stress can shed light on the identification of incinerated individuals and on the circumstances of the fire. Changes in the color of burned teeth can provide information on structural changes and the temperature of exposure. The objective of this study was to correlate color modifications with the concentration of human DNA in teeth burned at different temperatures. Spectrophotometry was used to measure the color of 40 teeth heated at temperatures of 100, 200, and 400°C for 60 min. DNA was extracted by phenol-chloroform extraction and quantified by real-time quantitative PCR using the Quantifier human DNA quantification kit. Preliminary results indicated an association of higher temperature with changes in colorimetric variables and a decrease in DNA concentrations. A significant positive correlation was found between luminosity values and DNA concentration (r = 0.4727, p = 0.0128) and between chromaticity a* values and DNA concentration (r = 0.4154, p = 0.0250). Spectrophotometry analysis of the color of burned teeth may predict the feasibility of extracting human DNA for identification purposes.

Spectrophotometric analysis of color changes in teeth incinerated at increasing temperatures.

Color changes produced by histological alterations in burned teeth can provide conclusive forensic information on the temperature of exposure. The objective was to correlate heat-induced color changes in incinerated teeth with increases in temperature (to 1200°C). Spectrophotometry was used to measure lightness, chromaticity (a* and b*), whiteness, and yellowness in 80 teeth heated at temperatures of 100, 200, 400, 600, 800, 1000, or 1200°C for 60 min. Chromaticity a* was reduced at 100°C and lightness at 200 and 400°C, while chromaticity b* and yellowness were reduced at 400 and 600°C. Higher temperatures (800, 1000, and 1200°C) produced progressive increases in lightness and whiteness but reductions in chromaticity b* and yellowness. The accuracy of color values to determine the temperature of exposure was determined by Receiver Operating Characteristic analysis. High accuracy was shown by lightness, chromaticity b* and yellowness values for temperatures between 800° and 1200°C, by whiteness for temperatures of 1000° and 1200°C, and by lightness for temperatures of 200° and 400°C, with sensitivity and specificity values ranging from 90% to 100%. According to these results, colorimetric analysis of incinerated teeth can be used to estimate the temperature of exposure with high accuracy, with lightness being the most useful variable.

Time-dependent changes in DNA stability in decomposing teeth over 18 months.

OBJECTIVE: The objective was to use a dual quantitative and qualitative approach to analyze the dental DNA degradation produced by the passage of time since tooth death under controlled environmental conditions. MATERIALS AND METHODS: Sixty human teeth were stored at room temperature for 0, 1, 3, 6, 12 or 18 months post-extraction. DNA quantification was determined by real-time quantitative PCR using a Quantifiler(TM) kit. DNA quality was assessed by the allelic dropout ratio between the smallest and largest loci obtained after STR genotyping and using an AmpFlSTR® Identifiler™ PCR kit. We also evaluated differences of DNA concentration related to gender and tooth position. RESULTS: DNA concentration significantly reduced in 1 month post-extraction, stabilized between 1-12 months post-extraction, but decreased again at 18 months post-extraction. Interestingly, a significant reduction of the allelic dropout ratio (DNA quality) was only detected at 18 months post-extraction. CONCLUSIONS: Stability of dental DNA decreased over time, differently affecting the amount and quality of the DNA in a time-dependent process over the first 18 months post-extraction. These results have a potential use in post-mortem intervals in human teeth in controlled environmental conditions.

Human genetic selection on the MTHFR 677C>T polymorphism.

BACKGROUND: The prevalence of genotypes of the 677C>T polymorphism for the MTHFR gene varies among humans. In previous studies, we found changes in the genotypic frequencies of this polymorphism in populations of different ages, suggesting that this could be caused by an increase in the intake of folate and multivitamins by women during the periconceptional period. The aim was to analyze changes in the allelic frequencies of this polymorphism in a Spanish population, including samples from spontaneous abortions (SA). METHODS: A total of 1305 subjects born in the 20th century were genotyped for the 677C>T polymorphism using allele specific real-time PCR with Taqman probes. A section of our population (n = 276) born in 1980-1989 was compared with fetal samples (n = 344) from SA of unknown etiology from the same period. RESULTS: An increase in the frequency of the T allele (0.38 vs 0.47; p < 0.001) and of the TT genotype (0.14 vs 0.24; p < 0.001) in subjects born in the last quarter of the century was observed. In the 1980-1989 period, the results show that the frequency of the wild type genotype (CC) is about tenfold lower in the SA samples than in the controls (0.03 vs 0.33; p < 0.001) and that the frequency of the TT genotype increases in the controls (0.19 to 0.27) and in the SA samples (0.20 to 0.33 (p < 0.01)); r = 0.98. CONCLUSION: Selection in favor of the T allele has been detected. This selection could be due to the increased fetal viability in early stages of embryonic development, as is deduced by the increase of mutants in both living and SA populations.

Influence of gender and genetic variability on plasma angiotensin peptides.

INTRODUCTION: We analysed the influence of three polymorphisms of the renin-angiotensin system (RAS) (I/D from angiotensin-converting enzyme [ACE], M235T from angiotensinogen gene [ATG] and A1166C from AT1 receptors) on plasma levels of angiotensin I (Ang I), angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)]. MATERIALS AND METHODS: The study population consisted of a homogeneous group of 93 healthy subjects (43 men and 50 women, mean age: 20.67+/-2.75 years). The mean blood pressure (BP) was 126+/-7/76+/-5 (SD) mmHg and the mean body mass index (BMI) was 22.4+/-2.5 kg/m2. Angiotensin peptides were separated by high performance liquid chromatography (HPLC) and quantified by radio immuno assay (RIA). Genotypes were determined by polymerase chain reaction (PCR) and restriction enzyme analysis. RESULTS: Mean peptide levels were 92.48+/-102.12 pg/ml for Ang I, 22.35+/-10 pg/ml for Ang II, and 31.65+/-27.46 pg/ml for Ang-(1-7). Men had significantly higher levels of Ang-(1-7) (37.76+/-36.47 pg/ml) than women (26.04+/-13.98 pg/ml) (p<0.05). Among genotypes of each polymorphism, men with the T allele showed higher Ang- (1-7) levels compared with those with the MM genotype (p<0.05). Genotype analysis in women showed that higher Ang I levels were related with the DD genotype. When both genders were compared according to genotype, higher values of Ang-(1-7) levels and its molar ratios were found in men, and there was significantly greater Ang I levels in DD genotypes in women than men (136.72+/-112.43 vs . 65.36+/-46.83 pg/mL). CONCLUSIONS: Significant correlations were found between Ang I and Ang II as well as between Ang II and Ang-(1-7) in the different study group distributions. No correlation was found between levels of Ang I and Ang-(1-7). Certain genotypes exert an influence on angiotensin peptide plasma levels which can only be seen when the population is divided according to gender.