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Abstract Background Most cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer. Methods We investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls. Results Methylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity. Conclusions In this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes.

Background: Most cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer. Methods: We investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls. Results: Methylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity. Conclusions: In this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes. [ABSTRACT FROM AUTHOR]

The high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA material for further analysis. Furthermore, human hypervariable regions - HV1 and HV2, located on mtDNA were sequenced to define human haplogroups. For extraction, samples of buccal mucosa from ten human volunteers were taken and three different protocols were used (a method with ammonium acetate, a salting-out method and GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific)). Concentration and purity of DNA were measured on BioPhotometer. Sequencing was performed by Sanger method on 3130 Genetic Analyzer (Applied Biosystems) with commercial BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were processed using Chromas and MEGA software and compared with sequence databases with BLAST tool on NCBI. Haplogroups and mutations were determined in the MITOMAP online database. Results of extraction indicated that the Purification Kit gives the highest concentration of DNA (11.86±0.63) and low purity (1.17±0.02), while a method with ammonium acetate gave the best purity (1.67±0.17). Sequencing obtained on DNA extracted with a commercial kit, gave 5 different human haplogroups U2e, T1a, J1c, H2a, K. In conclusion, it is recommended to use the Purification Kit for obtaining high integrity DNA that can be used for effective sequencing. Determination of haplogroups and haplotypes can reveal our ancestry and have important implications in molecular systematics, phylogenetics and phylogeography.

Some studies have pointed to the relationship between mitochondrial DNA (mtDNA) mutations and age in different tissues, which are potentially interesting in aging research and in forensic identification because they could help to improve the estimation of age-at-death. The present study aims to evaluate the mutations in mtDNA from dentin and pulp and their relation with age. Healthy erupted third molars were extracted from individuals from two Spanish populations, aged 20-70. When analyzing the amplification of hypervariable region 2 of the mtDNA by real-time polymerase chain reaction, a negative strong linear correlation was found between the mtDNA amplification and age in dentin from both populations. In contrast, a significant correlation between mtDNA amplification and age in pulp was not discovered, probably due to the majority of the mitochondria are placed in dentin. A difference in mtDNA damage between these two populations was also detected, indicating the role of ancestry as a component. The findings from this research enrich the current studies related to aging and mitochondrial damage and provide a new quantitative tool for estimating the age-at-death that, in combination with traditional age markers, could improve identification accuracy in forensic cases. [ABSTRACT FROM AUTHOR]

Examines the utility of mitochondrial DNA typing for forensic purposes in Uruguay. Blood and saliva samples from unrelated individuals; DNA extraction method; Equipment and software used; Compliance with the Hardy-Weinberg equilibrium; Variation of mtDNA HV1 and HV2 regions confirmed in the 146 position.

Abstract: Analysis of mitochondrial DNA (mtDNA) has been widely applied in the field of human identification, and features a large number of mtDNA molecules per cell, the exclusively maternal inheritance, lack of recombination and high mutation rate found in the control region, and ensure the high level of polymorphism, mainly in hypervariable regions HV1, HV2 and HV3. However, despite highly polymorphic, one of the limitations of using these regions is that many polymorphisms are highly common, resulting in the presence of sequences shared by more than one individual, or maternal lineages, in different populations. The aim of this study was to evaluate the discrimination power of the analysis of hypervariable regions HV1 (16,024–16,365), HV2 (73–340) and HV3 (438–574) of mtDNA in a Brazilian sample containing 290 unrelated individuals. Sequencing was performed using BygDye Terminator v3.1 and capillary electrophoresis was performed on ABI3130. All samples were validated by EMPOP and were classified into 15 haplogroups. Eighty out of 290 individuals presented European haplotypes, 108 showed Amerindian haplotypes and 102 individuals presented African haplotypes. Sixty-nine individuals (23.8%) could not be discriminated by the analysis of hypervariable regions HV1, HV2 and HV3 of mtDNA, and they were distributed in 24 different groups of common sequences. The most common sequence belonged to European haplogroup R0 with 10 individuals showing the same sequence. Trying to increase the discrimination power of common haplotypes and, therefore, individuals, analysis of SNPs in the coding region has been done. [Copyright &y& Elsevier]

Abstract: In biological samples with low copy DNA or DNA degraded samples, such as hair and pubic hair, there is a greater probability of obtaining a mitochondrial DNA profile. Hair and pubic hair are often found at crime scenes; in cases of sexual violence is common to find the suspect''s pubic hair in the victim''s body. This study analyzed, blood, head and pubic hair samples of 25 males to perform the analysis of mtDNA. Each three hair shafts were grouped and divided into 0.5cm sections: with the bulbs, and the other with 2cm, the proximal portion, without the bulbs. The DNA extraction of hair samples was performed using QIAamp DNA Micro Kit (QIAGEN) and the PCR amplification of HV1 and HV2 carried out. The amplified products were purified and sequenced using Big Dye™ Terminator and ABI 3130 (Applied Biosystems). To insure that hair samples were from the same individual, the AmpFLSTR Identifiler Kit® (Applied Biosystems) were used. The success rate of genotyping of hair samples with the bulb was: 56% for head hairs and in 64% for pubic hair samples, for a complete profile of the 15 STR (Identifiler). There was no statistically significant difference between the DNA concentration samples from hair with bulb and without bulb, as well as samples of pubic hair with bulb and without bulb. The overall success rate obtained with the extraction method used was approximately 60% and the success rate obtained in the sequencing was higher for pubic hair, both with and without bulb, than in head hair samples. Partial profiles of HV1 and HV2 were observed in most cases. Point heteroplasmy was detected in a sample of hair without bulb at hotspot position 16192 and another individual showed point heteroplasmy in pubic hair sample with bulb and without bulb at position 204, also observed in the blood sample of the same individual. In the preliminary results, 193 different haplotypes were detected; the most frequent haplotype was shared by 3 individuals. The most frequent lineage observed was African, showing the contribution of this haplotype group in São Paulo City population. This data were used to analyze and to compare the haplotypes of hair samples. [Copyright &y& Elsevier]

Abstract: Sequences from the two hypervariable regions (HV1 and HV2) of the control region of the mitochondrial DNA were obtained from a total of 201 Danes and five individuals who later were recognized to be of non-West European origin. Two fractions of each region were amplified separately and sequenced at least twice. The samples were sequenced using flanking sequencing primes and both terminator and primer chemistry. Sequence evaluation was performed by two independent scientists. The haplogroup distribution of the samples resembles that found in other European population. All the sequences have been made available in the EMPOP database. [Copyright &y& Elsevier]

Objectives: The aim of this study was to develop ribonucleic acid (RNA) profiles that could serve as novel biomarkers for the response to aspirin., Background: Aspirin reduces death and myocardial infarction (MI), suggesting that aspirin interacts with biological pathways that may underlie these events., Methods: Aspirin was administered, followed by whole-blood RNA microarray profiling, in a discovery cohort of healthy volunteers (HV1) (n = 50) and 2 validation cohorts of healthy volunteers (HV2) (n = 53) and outpatient cardiology patients (OPC) (n = 25). Platelet function was assessed using the platelet function score (PFS) in HV1 and HV2 and the VerifyNow Aspirin Test (Accumetrics, Inc., San Diego, California) in OPC. Bayesian sparse factor analysis identified sets of coexpressed transcripts, which were examined for associations with PFS in HV1 and validated in HV2 and OPC. Proteomic analysis confirmed the association of validated transcripts in platelet proteins. Validated gene sets were tested for association with death or MI in 2 patient cohorts (n = 587 total) from RNA samples collected at cardiac catheterization., Results: A set of 60 coexpressed genes named the "aspirin response signature" (ARS) was associated with PFS in HV1 (r = -0.31, p = 0.03), HV2 (r = -0.34, Bonferroni p = 0.03), and OPC (p = 0.046). Corresponding proteins for the 17 ARS genes were identified in the platelet proteome, of which 6 were associated with PFS. The ARS was associated with death or MI in both patient cohorts (odds ratio: 1.2 [p = 0.01]; hazard ratio: 1.5 [p = 0.001]), independent of cardiovascular risk factors. Compared with traditional risk factors, reclassification (net reclassification index = 31% to 37%, p ≤ 0.0002) was improved by including the ARS or 1 of its genes, ITGA2B., Conclusions: RNA profiles of platelet-specific genes are novel biomarkers for identifying patients who do not respond adequately to aspirin and who are at risk for death or MI., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)