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mtDNA role in mixtures deconvolution
Accepted Manuscript Title: mtDNA ROLE IN MIXTURES DECONVOLUTION Author: M. Omedei G.S. Amayeh S. Gino PII: DOI: Reference:
S1875-1768(15)00003-7 http://dx.doi.org/doi:10.1016/j.fsigss.2015.09.226 FSIGSS 1223
To appear in: Please cite this article as: M.Omedei, G.S.Amayeh, S.Gino, mtDNA ROLE IN MIXTURES DECONVOLUTION, Forensic Science International: Genetics Supplement Series http://dx.doi.org/10.1016/j.fsigss.2015.09.226 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Page 1 of 5
mtDNA ROLE IN MIXTURES DECONVOLUTION
M. Omedei2 [email protected], G.S. Amayeh1, S. Gino1 1 Department of Public Health Sciences and Pediatrics, University of Turin, Turin, Italy 2 University of Rome ‘‘Tor Vergata’’, Department of Public Health, Rome, Italy *Corresponding author.
Page 2 of 5
Abstract Mitochondrial DNA (mtDNA) usually has a negligible role in forensic casework and is only taken into account by forensic scientists if familiar testing is necessary or when genomic DNA (gDNA) is degraded. The forensic workflow normally does not include validated protocols to calculate the amount of mtDNA in a trace or the ratio between gDNA and mtDNA Because of the great inter-individual and tissue-specific variability of the number of mitochondria, the absence of this information may create misunderstandings in results interpretation. The aim of this work was to study mtDNA behaviour in mixtures composed of blood-saliva, blood-skin and saliva-skin; the GEP-ISFG group made similar analyses in the past, but only mixtures with semen were involved (representative of rape samples after differential lysis) and an almost complete absence of mtDNA was observed for the sperm fractions. In this project mixtures were prepared using two donor subjects with formerly known sequence information, having at least three different variants on the HV1 sequence, and DNA extraction was carry out on all samples. Autosomal STR markers were also analyzed in order to compare sequence results with gDNA electropherograms. Our experiment is the first time where skin tissue is included as a DNA source in mixtures where the role of mitochondria may be pivotal. These results could represent the first step of a more wide analysis involving real-time detection of mtDNA, in order to understand real gDNA/mtDNA ratios in different contexts Keywords: skin; mtDNA; mixture interpretation
Page 3 of 5 1. Introduction The analysis of body fluid mixtures is a common practice in forensic casework, and is of primary importance in sexual assault cases where semen traces are to be identified. In these instances, the routine workflow generally includes a preferential lysis, followed by STR typing and, where necessary, mtDNA sequencing [1]. However, quantitation results are related to the sole genomic DNA fraction, and information regarding the mtDNA amount and its ratio to gDNA is never taken into account. The GEP-ISFG working group organized some collaborative exercises to shed a light on this discrepancy, revealing the existence of different relative amounts of nuclear and mtDNA in saliva and semen and hypothesizing that similar condition may be found in other tissues [2, 3]. The purpose of this study is to analyze the mtDNA amounts in body fluid mixtures in which semen is not present, focusing on blood, saliva and skin tissues. This is intended to be suitable in case autosomal STRs results may be in apparent contradiction with mtDNA results. Skin tissue is here considered for the first time, giving the increasing relevance of new touch DNA techniques in forensic routine casework, and secondly, because it is the sole way of recreating the de-epithelialization of a rotting corpse. Different SNPs were selected on the mitochondrial HV1 region, and a quantitative SNaPshot analysis was conducted to compare the amount of mtDNA with that of gDNA. 2. Materials and methods In this preliminary work, a first screening was conducted to identify two appropriate donors for body fluids mixtures, having different nucleotides at mtDNA positions 16126 (T/C) and 16344 (A/G). A hundred microliters of blood and saliva were collected from selected volunteers, and subjected to DNA extraction using separate kits: fluids were processed with the Nucleomag 96 Blood kit (Macherey-Nagel), whereas epithelial debris were extracted with the QIAamp DNA Investigator kit (Qiagen) following manufacturer’s instructions. RT PCR was performed using the Plexor® HY System (Promega) on a BioRad CFX 96 Real-time PCR System (Bio-Rad). In order to create balanced DNA mixtures blood-skin and saliva-skin were mixed with 1:1, 1:10, 1:20 ratios, with a fixed skin amount and an increasing addition of the other body fluid. mtDNA typing was performed with a SNaPshot minisequencing reaction. In order to compare genomic STR profiles with mtDNA ratios, STR analysis was conducted with the Identifiler Plus Amplification kit (Life Technologies). DNA fragments were separated and detected by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Life Technologies) and analyzed with the GeneMapper® v.3.1 software. 3. Results and Discussion As shown in electropherograms above, both blood-skin and saliva-skin mixture ratios of 1:1, 1:10 and 1:20, created based on RT-PCR data, resulted to be in line with STR typing results, but not with those of mtDNA: in fact, these last seem to be consistently higher than expected by about three times (only one SNP variant is here represented for a more comprehensive image). Skin seems to contain a greater mtDNA amount if compared with blood: in fact, 1:1 STR typing corresponds to a 1:2 in mtDNA (blood variant 1974 RFU vs. skin variant 4899 RFU). This condition is preserved in each dilution. Saliva-skin mixtures preserve the 1:1 ratios both in STR and in mtDNA (saliva 4921 RFU vs. skin 3921 RFU), while in 1:10 and 1:20 dilutions shows the same trend of blood-skin samples. On the contrary, blood-saliva mixtures revealed DNA proportions to be, in both genomic and mitochondrial DNA fractions, in accordance with the expectations (data not shown). Within skin cells, mtDNA is better preserved than nuclear DNA, and our data indicate that in the presence of a more unbalanced ratio than 1:20 with STR results indicating only a single-source contribution, mtDNA analysis may reveal contradictory outcomes, i.e. a DNA mixture. These preliminary results suggest that mtDNA analysis may indicate the presence of a mixture even when autosomal STR typing excluded the presence of multiple contributors to a trace not only in semen samples, but also in other body fluids or tissues, such as skin. 4. Figures 5. Conflict of interest: none
Page 4 of 5 6. References 1. K.D. Anslinger, B.R. Bayer, B. Rolf, W. Eisenmenger, mtDNA investigations after differential lysis, Journal of Forensic Sciences 50 (2005) 579-581. 2. M. Crespillo, et al., Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain. Forensic Science International 160 (2000) 157-167. 3. M. Montesino, et al., Analysis of body fluid mixtures by mtDNA sequencing: an inter-laboratory study of the GEP-ISFG working group, Forensic Science International. 168 (2007) 42-56.
Page 5 of 5 Figure Captions
Fig. 1. On the left, STR locus of skin-blood mixtures in which both donors are heterozygote. Peaks are balanced and 1:1, 1:10, 1:20 ratios are in accordance with the expectation.[Blood (28, 31); Skin (30, 33.2)] On the right mtDNA variants in blood-skin and saliva-skin samples. mtDNA ratios are different from expected results. Where the STR profile appears as a 1:1 ratio, skin mtDNA amount is about three times higher than the expected, and this condition is maintained in each dilution (for more detailed information look at peaks height display under each SNP). The only exception is saliva-skin 1:1, where SNP peaks height is more or less balanced between two donors (orange circle).
S1875-1768(15)00003-7 http://dx.doi.org/doi:10.1016/j.fsigss.2015.09.226 FSIGSS 1223
To appear in: Please cite this article as: M.Omedei, G.S.Amayeh, S.Gino, mtDNA ROLE IN MIXTURES DECONVOLUTION, Forensic Science International: Genetics Supplement Series http://dx.doi.org/10.1016/j.fsigss.2015.09.226 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Page 1 of 5
mtDNA ROLE IN MIXTURES DECONVOLUTION
M. Omedei2 [email protected], G.S. Amayeh1, S. Gino1 1 Department of Public Health Sciences and Pediatrics, University of Turin, Turin, Italy 2 University of Rome ‘‘Tor Vergata’’, Department of Public Health, Rome, Italy *Corresponding author.
Page 2 of 5
Abstract Mitochondrial DNA (mtDNA) usually has a negligible role in forensic casework and is only taken into account by forensic scientists if familiar testing is necessary or when genomic DNA (gDNA) is degraded. The forensic workflow normally does not include validated protocols to calculate the amount of mtDNA in a trace or the ratio between gDNA and mtDNA Because of the great inter-individual and tissue-specific variability of the number of mitochondria, the absence of this information may create misunderstandings in results interpretation. The aim of this work was to study mtDNA behaviour in mixtures composed of blood-saliva, blood-skin and saliva-skin; the GEP-ISFG group made similar analyses in the past, but only mixtures with semen were involved (representative of rape samples after differential lysis) and an almost complete absence of mtDNA was observed for the sperm fractions. In this project mixtures were prepared using two donor subjects with formerly known sequence information, having at least three different variants on the HV1 sequence, and DNA extraction was carry out on all samples. Autosomal STR markers were also analyzed in order to compare sequence results with gDNA electropherograms. Our experiment is the first time where skin tissue is included as a DNA source in mixtures where the role of mitochondria may be pivotal. These results could represent the first step of a more wide analysis involving real-time detection of mtDNA, in order to understand real gDNA/mtDNA ratios in different contexts Keywords: skin; mtDNA; mixture interpretation
Page 3 of 5 1. Introduction The analysis of body fluid mixtures is a common practice in forensic casework, and is of primary importance in sexual assault cases where semen traces are to be identified. In these instances, the routine workflow generally includes a preferential lysis, followed by STR typing and, where necessary, mtDNA sequencing [1]. However, quantitation results are related to the sole genomic DNA fraction, and information regarding the mtDNA amount and its ratio to gDNA is never taken into account. The GEP-ISFG working group organized some collaborative exercises to shed a light on this discrepancy, revealing the existence of different relative amounts of nuclear and mtDNA in saliva and semen and hypothesizing that similar condition may be found in other tissues [2, 3]. The purpose of this study is to analyze the mtDNA amounts in body fluid mixtures in which semen is not present, focusing on blood, saliva and skin tissues. This is intended to be suitable in case autosomal STRs results may be in apparent contradiction with mtDNA results. Skin tissue is here considered for the first time, giving the increasing relevance of new touch DNA techniques in forensic routine casework, and secondly, because it is the sole way of recreating the de-epithelialization of a rotting corpse. Different SNPs were selected on the mitochondrial HV1 region, and a quantitative SNaPshot analysis was conducted to compare the amount of mtDNA with that of gDNA. 2. Materials and methods In this preliminary work, a first screening was conducted to identify two appropriate donors for body fluids mixtures, having different nucleotides at mtDNA positions 16126 (T/C) and 16344 (A/G). A hundred microliters of blood and saliva were collected from selected volunteers, and subjected to DNA extraction using separate kits: fluids were processed with the Nucleomag 96 Blood kit (Macherey-Nagel), whereas epithelial debris were extracted with the QIAamp DNA Investigator kit (Qiagen) following manufacturer’s instructions. RT PCR was performed using the Plexor® HY System (Promega) on a BioRad CFX 96 Real-time PCR System (Bio-Rad). In order to create balanced DNA mixtures blood-skin and saliva-skin were mixed with 1:1, 1:10, 1:20 ratios, with a fixed skin amount and an increasing addition of the other body fluid. mtDNA typing was performed with a SNaPshot minisequencing reaction. In order to compare genomic STR profiles with mtDNA ratios, STR analysis was conducted with the Identifiler Plus Amplification kit (Life Technologies). DNA fragments were separated and detected by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Life Technologies) and analyzed with the GeneMapper® v.3.1 software. 3. Results and Discussion As shown in electropherograms above, both blood-skin and saliva-skin mixture ratios of 1:1, 1:10 and 1:20, created based on RT-PCR data, resulted to be in line with STR typing results, but not with those of mtDNA: in fact, these last seem to be consistently higher than expected by about three times (only one SNP variant is here represented for a more comprehensive image). Skin seems to contain a greater mtDNA amount if compared with blood: in fact, 1:1 STR typing corresponds to a 1:2 in mtDNA (blood variant 1974 RFU vs. skin variant 4899 RFU). This condition is preserved in each dilution. Saliva-skin mixtures preserve the 1:1 ratios both in STR and in mtDNA (saliva 4921 RFU vs. skin 3921 RFU), while in 1:10 and 1:20 dilutions shows the same trend of blood-skin samples. On the contrary, blood-saliva mixtures revealed DNA proportions to be, in both genomic and mitochondrial DNA fractions, in accordance with the expectations (data not shown). Within skin cells, mtDNA is better preserved than nuclear DNA, and our data indicate that in the presence of a more unbalanced ratio than 1:20 with STR results indicating only a single-source contribution, mtDNA analysis may reveal contradictory outcomes, i.e. a DNA mixture. These preliminary results suggest that mtDNA analysis may indicate the presence of a mixture even when autosomal STR typing excluded the presence of multiple contributors to a trace not only in semen samples, but also in other body fluids or tissues, such as skin. 4. Figures 5. Conflict of interest: none
Page 4 of 5 6. References 1. K.D. Anslinger, B.R. Bayer, B. Rolf, W. Eisenmenger, mtDNA investigations after differential lysis, Journal of Forensic Sciences 50 (2005) 579-581. 2. M. Crespillo, et al., Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain. Forensic Science International 160 (2000) 157-167. 3. M. Montesino, et al., Analysis of body fluid mixtures by mtDNA sequencing: an inter-laboratory study of the GEP-ISFG working group, Forensic Science International. 168 (2007) 42-56.
Page 5 of 5 Figure Captions
Fig. 1. On the left, STR locus of skin-blood mixtures in which both donors are heterozygote. Peaks are balanced and 1:1, 1:10, 1:20 ratios are in accordance with the expectation.[Blood (28, 31); Skin (30, 33.2)] On the right mtDNA variants in blood-skin and saliva-skin samples. mtDNA ratios are different from expected results. Where the STR profile appears as a 1:1 ratio, skin mtDNA amount is about three times higher than the expected, and this condition is maintained in each dilution (for more detailed information look at peaks height display under each SNP). The only exception is saliva-skin 1:1, where SNP peaks height is more or less balanced between two donors (orange circle).