Tomaž Zupanc

The association between brain-derived neurotrophic factor polymorphism (BDNF Val66Met) and suicide

BACKGROUND Brain-derived neurotrophic factor (BDNF) mediates neural plasticity, mood, different behaviours, and stress response. A functional BDNF polymorphism (BDNF Val66Met) was reported to influence the effects of stressful life events or childhood adversity on depression and suicidal behaviour in various psychopathologies. The study evaluated the association between BDNF Val66Met variants and suicide, committed with violent or non-violent methods, in victims with or without stressful childhood experience. METHODS BDNF Val66Met polymorphism was genotyped on 560DNA samples from 359 suicide victims and 201 control subjects collected on autopsy from unrelated Caucasian subjects and subdivided according to gender, method of suicide, and influence of childhood adversity. RESULTS A similar frequency of BDNF Val66Met variants was found between all included suicide victims and the control groups, and also between the male groups. The frequency of the combined Met/Met and Met/Val genotypes and the homozygous Val/Val genotype was significantly different between the female suicide victims and female controls, between the female suicide victims who used violent suicide methods and female controls, and between all included suicide victims with or without stressful life events. The combined Met/Met and Met/Val genotypes contributed to this significance. LIMITATION A small group of suicide victims with available data on childhood adversity was studied. CONCLUSIONS The combined Met/Met and Met/Val genotypes of the BDNF Val66Met variant could be the risk factor for violent suicide in female subjects and for suicide in victims exposed to childhood trauma. These results confirm a major role of BDNF in increased vulnerability to suicide.

Promoter and functional polymorphisms of HTR2C and suicide victims

In Europe, the countries with the highest suicide rates form a so‐called J‐curve, which starts in Finland and extends down to Slovenia—a country with one of the worlds highest suicide rates. So far, the strongest association between suicide and genes has been shown for the serotonergic system. A functional polymorphism 68G>C (Cys23Ser) and a promoter polymorphism–995G>A of serotonin receptor 2C (HTR2C) have already been investigated, but no associations with suicide were determined. In the present study 334 suicide victims and 211 controls of Slovenian origin were genotyped for the above‐mentioned polymorphisms using standard methods. In the case of the polymorphism–995G>A no association with suicide was found. However, a significant association was observed between female suicide victims and polymorphism 68G>C. The significance remained when we combined alleles of female and male populations. An excess of GG genotype and allele G was observed. However, no statistically important differences were present when only males were analyzed. Haplotype analysis on female population showed marginal association of haplotype G‐C with suicide. The present study speaks for the plausible implication of the HTR2C in suicide susceptibility.

TPH2 polymorphisms and alcohol-related suicide

Substantial evidence from family, twin, and adoption studies corroborates implication of genetic and environmental factors, as well as their interactions, on suicidal behavior and alcoholism risk. Serotonergic disfunction seems to be involved in the pathophysiology of substance abuse, and has also an important role in suicidal behavior. Recent studies of the tryptophan hydroxylase 2 showed mild or no association with suicide and alcohol-related suicide. We performed SNP and alcohol analysis on 388 suicide victims and 227 controls. The results showed association between suicide (Pχ²=0.043) and alcohol-related suicide (Pχ²=0.021) for SNP Rs1843809. A tendency for association was determined also for polymorphism Rs1386493 (Pχ²=0.055) and alcohol-related suicide. Data acquired from psychological autopsies in a subsample of suicide victims (n=79) determined more impulsive behavior (Pχ²=0.016) and verbal aggressive behavior (Pχ²=0.025) in the subgroup with alcohol misuse or dependency. In conclusion, our results suggest implication of polymorphisms in suicide and alcohol-related suicide, but further studies are needed to clarify the interplay among serotonergic system disfunction, suicide, alcohol dependence, impulsivity and the role of TPH2 enzyme.

Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system

Retrieving information about externally visible characteristics from DNA can provide investigative leads to find unknown perpetrators, and can also help in disaster victim and other missing person identification cases. Aiming for the application to both types of forensic casework, we previously developed and forensically validated the HIrisPlex test system enabling parallel DNA prediction of eye and hair colour. Although a recent proof-of-principle study demonstrated the general suitability of the HIrisPlex system for successfully analysing DNA from bones and teeth of various storage times and conditions, practical case applications to human remains are scarce. In this study, we applied the HIrisPlex system to 49 DNA samples obtained from bones or teeth of World War II victims excavated at six sites, mostly mass graves, in Slovenia. PCR-based DNA quantification ranged from 4pg/μl to 313pg/μl and on an average was 41pg/μl across all samples. All 49 samples generated complete HIrisPlex profiles with the exception of one MC1R DNA marker (N29insA) missing in 83.7% of the samples. In 44 of the 49 samples (89.8%) complete 15-loci autosomal STR (plus amelogenin) profiles were obtained. Of 5 pairs of skeletal remains for which STR profiling suggested an origin in the same individuals, respectively, 4 showed the same HIrisPlex profiles and predicted eye and hair colours, respectively, while discrepancies in one pair (sample 26 and 43) are likely to be explained by DNA quantity and quality issues observed in sample 43. Sample 43 had the lowest DNA concentration of only 4pg/μl, producing least reliable STR results and could be misleading in concluding that samples 43 and 26 originate from the same individual. The HIrisPlex-predicted eye and hair colours from two skeletal samples, suggested to derive from two brothers via STR profiling together with a living sister, were confirmed by the living sisters report. Overall, we demonstrate that after more than 70 years, HIrisPlex-based eye and hair colour prediction from skeletal remains is feasible with high success rate. Our results further encourage the use of the HIrisPlex system in missing person/disaster victim identification to aid the identification process in cases where ante-mortem samples or putative relatives are not directly available, and DNA predicted eye and hair colour information provides leads for locating them, allowing STRbased individual identification.

Prediction of autosomal STR typing success in ancient and Second World War bone samples

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.

Reduced Blood Alcohol Concentration in Suicide Victims in Response to a New National Alcohol Policy in Slovenia

Background: Addiction is a major social and health problem. Studies on suicide and alcohol at the individual and aggregated level have confirmed a link between alcohol and suicide. Aim: To assess the impact of the new national alcohol policy in Slovenia on the blood alcohol concentration (BAC) in BAC-positive suicide victims before, during and after the implementation of the new national alcohol policy in 2003. Method: Blood samples were collected by forensic pathologists during medicolegal autopsies of suicide victims in order to establish their BAC levels at the time of death. BAC was measured using two routine independent headspace gas chromatography methods (HSS-GC-FID) and expressed in grams per kilogram. Results: During the period before the implementation of the act which limited the availability of alcohol in Slovenia, the BACs of BAC-positive suicide victims were higher than those tested in the period after the implementation of the act. Conclusion: Despite certain limitations, this study demonstrates that legislation measures restricting alcohol availability may be an effective measure of BAC reduction in BAC-positive suicide victims.

Rapidly mutating Y-STR analyses of compromised forensic samples

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were identified to improve differentiation of unrelated males and also to enable separating closely and distantly related males in human identity testing in forensic and other applications. RM-Yplex assay was developed as a single multiplex that is capable of simultaneously amplifying all currently known RM Y-STRs, and reproducibility and sensitivity testing were performed on reference samples. Additional analyses are necessary to test its suitability for analysing compromised forensic samples. For this purpose, we applied the RM-Yplex assay to approximately 70-year-old skeletons that were used as a model for poorly preserved, challenging forensic samples. We analysed 57 male skeletal remains (bones and teeth) from 55 skeletons excavated from the Second World War (WWII) mass graves in Slovenia. The RM-Yplex typing was successful in all 57 samples; there were 56% full profiles obtained, and in partial profiles, up to 7 locus drop-outs were observed and they appeared correlated with low DNA quantities and degradation of DNA obtained from WWII bone and tooth samples. The longest loci, DYS403S1b, DYS547, DYS627 and DYS526b, were the most often dropped-out RM Y-STRs. In spite of high frequency of drop-out events, the RM-Yplex typing was successful in all WWII samples, showing the possibility of successful amplification of at least half of the RM Y-STRs even from the most compromised samples analysed.

Single nucleotide polymorphisms in the BDNF gene and suicide in the Slovenian sample.

In recent years, brain-derived neurotrophic factor (BDNF) and sequence variations within and near the BDNF gene have been studied for associations with various psychiatric disorders, including suicidal behavior. Since our previous work on completed suicide in Slavic population showed an association of the functional single nucleotide polymorphism (SNP) rs6265 in the BDNF gene, we decided to extend the investigation and test additional SNPs in the BDNF gene, rs7124442, rs10767664, rs962369, rs12273363, rs908867, rs1491850, and rs1491851, for association with completed suicide. Our study subjects were Caucasians, and included 486 suicide completers and 289 controls. The case/control comparisons of allele, genotype and haplotype frequency distributions were performed by means of Pearsons X(2) tests. Analyses of allele and genotype frequency distributions of the sudied SNPs did not reveal any significant differences between the controls and suicide completers. Haplotype analysis (rs7124442-rs10767664-rs962369-rs12273363-rs908867) showed an association of the haplotype C-A-T-C-C (p(corr)=0.038) with completed suicide, indicating that these SNPs on a haplotype level may play a role in completed suicide phenotype in our study sample.

Secular trends in body height in Balkan populations from 1945 to 1995

The aim of this study was to look for any secular trend in the stature of Balkan populations from the time of World War II (1939-1945) to the Balkans War (1991-1995). The research was based on the examination of exhumed skeletons of 202 men killed in World War II in the area of the Republic of Slovenia, and 243 men killed in the Bosnian War in Bosnia and Herzegovina. The length measurements of the right and left humerus, femur, tibia and fibula were taken. Since the results revealed no significant differences and the left-sided bones were more complete and recurrent in the sample, the bones of the left side were used in the analysis. Since the increase in height depends mostly on the increase in length of the long bones, with an average absolute change of about 0.28 cm for humerus, 0.55 cm for femur, 0.49 cm for tibia and 0.20 cm for fibula per decade in our case, these results suggest a significant increase of the height of the Balkans population. The difference of the sum of the average femur and tibia length for the study period was 4.13 cm. Recalculated average length increase of the sum length of femur and tibia per decade was 0.88 cm for the left side. Our study revealed that there was a trend towards increased long bone lengths, at least in the male population analyzed.

Analysis of promoter polymorphism in monoamine oxidase A (MAOA) gene in completed suicide on Slovenian population

Suicide is a well-defined public health problem and is a complex phenomenon influenced by a number of different risk factors, including genetic ones. Numerous studies have examined serotonin system genes. Monoamine oxidase A (MAO-A) is an outer mitochondrial membrane enzyme which is involved in the metabolic pathway of serotonin degradation. Upstream variable number of tandem repeats (uVNTR) in the promoter region of MAOA gene affects the activity of transcription. In the present study we genotyped MAOA-uVNTR polymorphism in 266 suicide victims and 191 control subjects of Slovenian population, which ranks among the European and world populations with the highest suicide rate. Genotyping was performed with polymerase chain reaction and agarose gel electrophoresis. Using a separate statistical analysis for female and male subjects we determined the differences in genotype distributions of MAOA-uVNTR polymorphism between the studied groups. Statistical analysis showed a trend towards 3R allele and suicide, and associated 3R allele with non-violent suicide method on stratified data (20 suicide victims). This is the first study associating highly suicidal Slovenian population with MAOA-uVNTR polymorphism.