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Base specific variation rates at mtDNA positions 16093 and 16183 in human hairs
Forensic Science International: Genetics 43 (2019) 102142
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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
Research paper
Base specific variation rates at mtDNA positions 16093 and 16183 in human hairs
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Stijn Desmytera, , Sophie Dognauxa, Fabrice Noela, Lourdes Prietob,c a
NICC - Belgian Institute for Forensic Science and Criminology, Vilvoordsesteenweg 100, B-1120, Brussels, Belgium Instituto de Ciencias Forenses. Grupo de Medicina Xenómica. Universidade de Santiago de Compostela, Spain c Laboratorio ADN. Comisaría General de Policía Científica, Madrid, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: mtDNA Heteroplasmy Hair Base-specific Variation rate
Small variations between haplotypes detected in different tissues from the same individual have been previously described. These differences complicate the interpretation of mtDNA results in real forensic casework. mtDNA haplotypes detected in hair strands collected at the crime scene have to be frequently compared with haplotypes of reference samples (buccal swabs) from victims or suspects. Nucleotide position 16093 is a well-known hot spot where differences can accumulate between different tissues of the same individual. Intra individual variation was also detected at positions 16182 and 16183 in haplotypes showing an uninterrupted HV1 poly-C stretch (with 16189C). In order to better characterize the type of variation in these positions between buccal cells and hair strands from the same individual, we have performed Sanger sequencing in 25–28 hair strands (411 in total) from 15 individuals showing either an uninterrupted HV1 polyC-stretch (16189C) or 16093C/Y in their buccal cells. The results have been evaluated by also taking into account our previous results published in [19]. We have found that no variation among hair strands was detected in individuals showing T16093 in buccal cells, while variation in hair strands (T16093, 16093C and 16093Y) were detected in individuals showing 16093C or 16093Y in buccal cells. Regarding nucleotide positions 16182 and 16183 in combination with an uninterrupted polyCstretch, no variation was detected in hairs from individuals showing A16182 16183C in their buccal cells. In contrast, individuals A16182 A16183 showed hair strands with A16182 16183 M and A16182 16183C. And finally, individuals with 16182C 16183C showed some variation in a small amount of their hair strands (some hairs with 16182 M 16183C). These results can be relevant for forensic practitioners when comparing reference samples with hair strands, which is the type of sample most tested by using mtDNA analysis in forensic casework.
1. Introduction The interpretation of mtDNA results is not usually easy or straightforward. Guidelines and recommendations were published to advise experts that they should consider not only the number of differences between the profiles of the known and unknown samples, but also the tissue type of the samples, the position of the variation and the instability of the variation within haplogroups [1–8]. For the latter, studies are needed to provide tissue- and site-specific data on mtDNA variation. As long as not much data is available, some forensic experts interpret mtDNA results in the most conservative way, which is solely based on the number of differences. If the difference between the mtDNA profiles of the known and unknown samples is only at a single position, some laboratories report the outcome as inconclusive. This way of interpretation completely neglects the weight of the difference ⁎
between the profiles. In addition, heteroplasmy, being the co-existence of multiple mitotypes in an individual, complicates the interpretation of mtDNA results in forensic casework. Providing quantitative data on site specific variations in hairs, which is the most analysed tissue in forensic casework, could help in this perspective aspect. In this study we focused on two positions: 16093 and 16183, with reference to [9]. 1.1. 16093 nucleotide position This position can show 3 different variants in the human phylogenetic tree: T16093, 16093A and 16093C. T16093 seems to be the ancestral variant in the phylogentic tree of global human mtDNA variation [10]. 16093C polymorphism has not been previously described as an
Corresponding author. E-mail address: [email protected] (S. Desmyter).
https://doi.org/10.1016/j.fsigen.2019.102142 Received 23 April 2019; Received in revised form 25 July 2019; Accepted 11 August 2019 Available online 12 August 2019 1872-4973/ © 2019 Elsevier B.V. All rights reserved.
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forensic field [33]. Therefore, the absence of conventions in length variant regions in the past and the fact that these variations have been ignored in direct forensic comparisons and database searches make it difficult to obtain the real picture of the evolutionary history of these positions. Forster et al. [31] indicate that the mutation rate of 16182C and 16183C is probably too fast between generations to be of reliable use in the phylogenetic reconstruction of the human mtDNA tree. Length heteroplasmy of the polyC-stretch of HV1 affects the length of the preceding polyA-stretch (16180–16183) in a directional way [31]. Usually, this short stretch contains four A’s, but shorter or longer stretches were also observed. Foster et al. studied 1012 individuals belonging to 248 pedigrees of up to 4 generations, and evaluated 802 transmissions [31]. In none of the transmissions, of which 440 belonged to families showing 16189C, did the polyA-stretch lengthen. The shortening of the A-stretch was observed in one of the pedigrees that contained 16189C, which shows that it is possible to observe the cooccurrence of the polyA-stretch shortening with 16189C. It is well known that the distribution of LHP variants varies among the tissues of an individual [34]. The co-existence of A and C at position 16183 (A16183 M) was observed in studies of single hairs [17–20], but only in the study of [19] were buccal swabs of the hair donors also analysed. In the case of one individual, the buccal swab showed an uninterrupted polyC-stretch preceded by four A’s (A16183 16189C), while heteroplasmic variation (16183 M 16189C) was observed in four of the 25 analysed hairs and homoplasmic variation (16183C 16189C) in one hair [Supplementary data Fig. 1]. No variation was observed at 16183 position in the hairs of the 10 other persons tested who had an interrupted HV1 polyC-stretch (T16189) in their buccal cells. This observation can lead us to the idea of a certain pattern of segregation in the hairs of individuals showing 16189C in their buccal cells, that is to say, 16183C/M variants are possible in hairs of individuals showing 16189C in their blood/buccal cells, but are not possible in T16189 individuals. To determine the base-specific variation rate at positions 16183 and 16182 in hairs, 11 more donors that showed an uninterrupted HV1 polyC-stretch were selected and a variable number of hairs from them were analysed. In order to provide quantitative data on the variation rate in single hairs at positions 16093 and 16183, we have studied hair shaft samples from individuals showing 16093C/Y or an uninterrupted HV1 polyCstretch in their buccal cells. The variant status at the particular positions for each single hair was reported and compared with data from a previous hair study of persons who either showed T16093 in their buccal swabs profiles or contained an interrupted HV1 polyC-stretch [19].
haplogroup motif in the main macro-haplogroups [11], although it was later described to appear in many sub-haplogroups [Phylotree, Build 17] In other sub-haplogroups this polymorphism seems to be recurrent or unstable within the respective clade, although this apparent "instability" could also be due to the absence of enough data currently available (L1c2b, L2a1a3, M31b'c, C4a1, G1b, G4, H3h2, H4b1, H10e'g, B4a1a1a7, K1a, K1b1a'b). 16093A polymorphism does not seem to define any haplogroup [Phylotree, Build 17], but can be found in EMPOP v4, R12 [12] in 2 samples from Asia (Uzbekistan and India) belonging to MRCA haplogroup U2a1. Searches of "16093Y" in the same version of EMPOP, using the match type "literal" and the edition range "16093", result in 262 matches in the whole database (42839 haplotypes). These matches involve haplotypes belonging to many different haplogroups. This number confirms the earlier observation in a large scale study on the blood/ buccal cells of 5015 individuals where 0.84% showed 16093Y [13]. The first reported variation between hair and the reference sample from a single donor was at position 16093 [14] and, since then, it has been denoted as a heteroplasmic hotspot in many large-scale hair studies [15–20]. In the studies where, besides sequence data of hair samples also reference data from buccal cells or blood were available, the observation of heteroplasmy at position 16093 in hairs seemed to be correlated with individuals showing 16093C in blood/buccal cells [17–19,21,22], although not exclusively [16,23]. In addition, variation in the C:T content throughout 55 single hair shafts was also described [22]. These sequencing data reveal a preference for C in about 90% of the hair shaft fragments. In several large-scale studies on blood and/or buccal cells samples, position 16093 was also classified as a heteroplasmic hotspot [13,24]. Besides, remarkable ratios between both variants C and T at this position were reported [13,24]. Although T was observed as the most common variant in population databases (about 95% in EMPOP v.4 R12), in heteroplasmic individuals a high C:T ratio was observed in the majority of the samples, that is to say, the C variant is usually the predominant one. Multiple-tissue studies with more sensitive sequencing technologies revealed a tissue- and base-specific variation rate at nucleotide 16093 [25–29]. A large-scale Sanger-sequencing based study on 9 tissues from 100 individuals included buccal cells and hair strands [30]. For all eight individuals who showed heteroplasmy at position 16093 in at least one sample, the ratio C:T was studied in all 9 tissues. These data confirm the high C-content in blood and the high T-content in skeletal muscle for all eight persons. The ratio in buccal cells was similar to the one in blood, while there was no preference for either nucleotide in hair.
2. Material and methods 1.2. 16183–16182 nucleotide positions 2.1. Sample collection and DNA extraction Length heteroplasmy (LHP) is observed when at least two mtDNA molecules that differ in their number of nucleotides are present in a detectable amount in an individual. In the Control Region, length heteroplasmy was seen to appear in the polyC-stretches of HV1 (16184–16193), HV2 (303–309) and HV3 (456–463 and 568–573), and in the AC tandem repeat (514–524) [13,31,32]. In the polyC-stretch of HV1, length heteroplasmy is mostly associated with the polymorphism 16189C if this mutation results in an uninterrupted polyC-stretch of more than eight C’s [13,31]. Positions 16182 and 16183 have usually been excluded from the mtDNA tree and are not considered for phylogenetic reconstruction [10]. 16182 M and 16183 M denote the (heteroplasmic) mixture of C and A at these positions; 16182C and 16183C denote the transversion A–C at these positions; and finally, 16182c and 16183c denote the (heteroplasmic) mixture of a transversion and a deletion at these nucleotide positions. Lower case nomenclature was introduced in 2014, after the ISFG recommendations about mtDNA nomenclature in the
Head hairs were collected from 15 staff members of the National Institute of Criminalistics and Criminology, Brussels, Belgium (NICC) and the Spanish Forensic Police, after informed consent. Their buccal cells mtDNA profiles were established over the years by Sanger sequencing as part of the quality control contamination databases. All donors belonged to the European population and were selected based on their mtDNA profiles containing either an uninterrupted HV1 polyCstretch (16189C) or the polymorphism 16093C/Y [Supplementary data Table 1]. Their haplotypes were assigned to haplogroups by using PhyloTree [10], build 17, aided by the EMMA software package [8]. Between 25 and 28 hairs per person were plucked. After collection, the roots and 0.5–1 cm proximal fragments of all hairs were removed. The first proximal 2 cm-shaft fragment of each of the 411 hairs were decontaminated and the DNA was extracted following the (a) protocol from [19].
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2.2. MtDNA amplification and sequencing
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Amplification, postPCR purification and Sanger-type cycle sequencing were performed as described in the (a) protocol of [19], while amplification was limited to only one fragment with the primer pair M13L15997-M13H16239. The sequencing was performed with primer M13 L [19]. 2.3. MtDNA sequence data generation and interpretation The sequences were assembled using SeqScape v.2.5 (AB, Oyster Point, CA, USA) and aligned to the revised Cambridge Reference Sequence (rCRS) [9]. Base calling was ensured by double independent data analysis and the sequencing results were checked on complementarity to the buccal cells reference profiles. The base calling results were only reported at the positions of interest (16093 or 16182 and 16183) in this work [Supplementary data Table 1].
In general, at 16093 the highest C-content was observed in blood, while the lowest C-content was observed in skeletal muscle [29]. The large-scale study of [29] identified in total seven nucleotides at which the heteroplasmy was position-, base- and tissue-dependent. This phenomenon was the most prominent at position 16093, which explains why it was observed in the past in smaller and Sanger-sequencing based studies. Even though many different tissues were studied by using MPS, none of them analysed buccal cells or hairs, in spite of the fact that they are the most common tissues in forensic casework. In another study, Naue et al. [30] demonstrated a high variation in C-content at position 16093 between 9 different tissues of 8 individuals who were predominantly 16093C in their blood and buccal cells. For these persons, the C-content at position 16093 was more variable in hairs (40–80%) compared to buccal cells (85–100%). Those tissuespecific variation rates could explain why in our study heteroplasmy was not observed in the buccal cell samples but in the hair samples. Nevertheless, in this large-scale study, each hair sample actually contained multiple hairs. This way of sampling is quite different from the one usually performed in real casework (each sample contains only one single hair strand) and therefore does not allow us to evaluate the homoplasmic and heteroplasmic variation rate between single hairs. Contrary to the observation that 16093C individuals are highly variable in hairs at this particular position, the base status was invariable for all the 310 hairs of the 10 individuals with T16093 in their reference profile. This observed base-specific variation at position 16093 made us believe that the high ranking of 16093 as a heteroplasmic hotspot in many hair studies [15–20] is due to the high variation rate in 16093C and 16093Y individuals, and not because of a high variation rate in all persons (16093C, 16093Y and T16093). Inter-individual variation was observed [Supplementary data Table 1], but not enough donors were represented in this study to statistically evaluate these differences in variation rates.
3. Results and discussion In order to study the mtDNA variation at positions 16093, 16182 and 16183 with Sanger sequencing, we collected hairs from 3 individuals with variant status 16093C, from 1 individual with an obvious status 16093Y and from 11 individuals with an uninterrupted polyCstretch in their buccal mtDNA profiles. These reference profiles were previously obtained by Sanger sequencing. Heteroplasmy detection is known to be influenced by different factors like amplification strategy, DNA polymerase, primers and sequencing chemistry [15,35]. Since the protocol we used in this study was identical to the one of our previous study on the latitudinal variation of CR mtDNA amongst hair shafts [19], we were able to combine the results of both studies. 3.1. Variation at position 16093 We have combined the results of hairs of the three 16093C (X1-X3) and the one 16093Y (X4) individual in this study with data from Desmyter et al. in which we had analysed 25 hairs from individual P11 who showed 16093C in her buccal cells. In total, we ended up with 109 analysed hairs from four individuals with 16093C in the reference profile (X1-X3 and P11) and 26 analysed hairs from one individual with 16093Y (X4). In addition, the variation at position 16093 of these hairs was also compared to the base status of the 310 analysed hairs of the 10 individuals P1-P10 who were all T16093 in their buccal cells profile [19]. Table 1 shows the results of both studies, but to briefly summarize:
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observed in other Sanger sequencing studies [21,22] where some hair shaft fragments of 16093C persons were analysed. Individual showing 16093Y in buccal swab - variation in hairs: As expected, a similar variation rate was also observed in the hairs of the person X4 whose reference sample was shown to be heteroplasmic at position 16093 (35% T16093, 27% 16093Y and 38% 16093C). In former studies, similar segregation patterns at other positions, different from 16093, were observed in hairs from individuals being heteroplasmic at this particular position in their buccal cells or blood. [19,22,36,37].
3.2. Variation at positions 16182 and 16183 Individuals showing 16093C in buccal swabs - variation in hairs: Variation rate at position 16093 in the three 16093C individuals from this study (X1-X3) confirmed the previous observations made in [19] for person P11, who also showed 16093C in her buccal cells and T16093, 16093Y and 16093C variants in her hair shafts. On average, half of the hairs of the four 16093C persons showed heteroplasmic variation (16093Y) and about 15% of the hairs showed homoplasmic variation (T16093). All observed variations were transitions. Variation at position 16093 was also previously
To study the variation at positions 16182 and 16183, in relation to an uninterrupted HV1 polyC-stretch, we have combined the present results with data from [19] (individual P5, 25 hair fragments). In total we have obtained sequencing data from 12 individuals: (i) six showing A16182 A16183 in their buccal cells reference profile, (ii) three showing A16182 16183C and (iii) three showing 16182C 16183C (Table 2). The haplotype obtained from buccal cells of all twelve persons contained an uninterrupted HV1 polyC-stretch (16189C). In the case of the six A16182 A16183 individuals, 164 hair shafts
Table 1 The average percentage (with standard deviation) of hairs with a particular base status at position 16093 compared to their respective reference profile from buccal cells. Base status at position 16093 in buccal cells T Y C a
Number of persons a
10 1 4 (3 + 1a)
Number of hairs a
310 26 109 (84 + 25a)
% of hairs T16093
% of hairs 16093Y
% of hairs 16093C
100 34.6 14.7 ± 11.3
0 26.9 49.5 ± 19.2
0 38.5 35.8 ± 10.5
310 hairs from ten T16093 individuals and 25 hairs from one 16093C individual were analysed in study [19]. 3
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0 0 94.0 ± 10.3 5.5 ± 5.1 100 0 15.2 ± 8.8 0 0
0 0 6.0 ± 10.3
25 hairs of one A16182 A16183 person were analysed in the study [19].
164 (139 + 25a) 80 82 6 (5 + 1a) 3 3 AA AC CC
79.3 ± 10.5 0 0
Heteroplasmy at position 16093 is known to be tissue- and base specific. Our data confirmed the lack of preference for a certain variant (C or T) at position 16093 in hairs of individuals that show 16093C in their buccal cells haplotypes. Indeed, of a total number of 109 hairs originating from 16093C individuals, an average of 15% showed homoplasmic variation (T16093) and 50% heteroplasmic variation (16093Y), while no variation was observed in all 310 analysed hairs from T16093 individuals. The shortening of the polyA-stretch, preceding the HV1 polyCstretch, is known to be associated with 16189C in germline transmissions. Our data on single hairs confirmed this co-occurrence of an uninterrupted HV1 polyC-stretch with the shortening of the preceding polyA-stretch. Variation at position 16183 was observed in hairs from individuals with an uninterrupted polyC-stretch, while no variation was previously observed in 310 analysed hairs with T16189 or 16186 T 16189C. The observed variation rate at position 16183 was variantspecific in hairs, favouring shortening over lengthening of the polyAstretch. On average, 6% of the 164 analysed hairs from individuals with A16182 A16183 in their reference profiles showed homoplasmic variation (16183C) and 15% heteroplasmic variation (16183 M). The 162 analysed hairs from individuals with A16182 16183C or 16182C 16183C showed no variation at position 16183. Variation at position 16182 was not observed in this study, probably due to both the lower variation rate when compared to position 16183 and not a sufficient amount of analysed hairs needed to detect any variation. In this study, we provide quantitative data for the base-specific variation rates in single human hairs at positions 16093 and 16183 of the mtDNA. These data could be useful in the interpretation of mtDNA results in forensic casework in a quantitative manner, in which not only the number of differences are considered but also the weight of the difference (see [38] as an example). For instance, let’s consider the following two possible scenarios:
a
% of hairs 16182C 16183C % of hairs 16182 M 16183C % of hairs A16182 16183C % of hairs A16182 16183 M Number of hairs Number of persons
% of hairs A16182 A16183
were studied. Variation at position 16183 was found in these hairs. On average about 15% of the hairs showed heteroplasmic variation (16183 M) and about 6% of the hairs showed homoplasmic variation (16183C). In the case of the three individuals showing A16182 16183C, 80 hair shafts were studied, and finally, in the case of the three individuals showing 16182C 16183C, 82 hair shafts were studied. No or hardly any variation was found in these cases. These data suggest that variation at position 16183 between hairs is characterized by directionality. The shortening of the succession of four A’s preceding the uninterrupted polyC stretch seems to be favoured over the lengthening. This base-specific variation rate at position 16183 in association with an uninterrupted polyC-stretch was also observed in a germline transmission study involving 248 pedigrees with up to four generations [31]. While no lengthening was observed in a total of 802 transmissions, of which 440 were within families with 16189C, the shortening of the polyA-stretch occurred in a pedigree with 16189Y. In the present study, variation was only observed at position 16183 and not at position 16182. The absence of variation was most probably related to a lower variation rate at position 16182 compared to 16183. Apparently more hairs should be studied in order to obtain a variation rate at position 16182. While variation was observed at position 16183 in some hairs of individuals with an uninterrupted HV1 polyC-stretch, no variation was observed in previous data from 310 hairs of 10 individuals P1-P10 [19]. All of them had an interrupted polyC-stretch in HV1 either by T16189 (Persons P1-P3, P6-P11) or by 16186 T (Person P4) and all of them showed A16182 A16183. These findings confirm the association between variation at position 16183 and the presence of an uninterrupted HV1 polyC-stretch.
4. Conclusions
Base status at positions 16182 and 16183 in buccal cells
Table 2 The average percentage (with standard deviation) of hairs with a particular base status at positions 16182 and 16183 compared to their respective buccal cells reference profile. All profiles contained an uninterrupted polyC-stretch in HV1.
S. Desmyter, et al.
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a) a suspect of a crime shows haplotype 16519C 263G 315.1C in his buccal cells, and a hair strand collected from the hand of the victim shows haplotype 16093C 16519C 263G 315.1C b) a suspect of a crime shows haplotype 16093C 16519C 263G 315.1C in his buccal cells, and a hair strand collected from the hand of the victim shows haplotype 16519C 263G 315.1C
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In both cases, the suspect cannot be excluded as the donor of the hair strand. But taking into account what we have described in this study, scenario b) is more probable than scenario a) if the hair strand really belongs to that suspect. Nevertheless, the results presented here are only based on Sanger sequencing, but other more sensitive techniques, such as massive parallel sequencing, could shed more light on the real base-content at these positions. Sanger sequencing has a limited capacity to detect heteroplasmies and therefore, some samples described here as homoplasmic, could contain some level of heteroplasmy. For both positions 16093 and 16183, the variation rates obtained show inter-individual differences (e.g. T16093 varies from 0% in individual P11 to 25% in individual X2). The current data set is too small to evaluate if the observed differences between individuals are statistically significant. Therefore, more research would be needed in order to use inter-individual variation in casework interpretation. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2019.102142. References [1] M.M. Holland, T.J. Parsons, Mitochondrial DNA sequence analysis - validation and use for forensic casework, Forensic Sci. Rev. 11 (1999) 21–50. [2] A. Carracedo, W. Bär, P. Lincoln, W. Mayr, N. Morling, B. Olaisen, P. Schneider, B. Budowle, B. Brinkmann, P. Gill, M. Holland, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [3] W. Bär, B. Brinkmann, B. Budowle, A. Carracedo, P. Gill, M. Holland, P.J. Lincoln, W. Mayr, N. Morling, B. Olaisen, P.M. Schneider, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Int. J. Legal Med. 113 (2000) 193–196. [4] G. Tully, W. Bär, B. Brinkmann, A. Carracedo, P. Gill, N. Morling, W. Parson, P. Schneider, Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles, Forensic Sci. Int. 124 (2001) 83–91. [5] B. Budowle, M.W. Allard, M.R. Wilson, R. Chakraborty, Forensics and mitochondrial DNA: applications, debates, and foundations, Annu. Rev. Genomics Hum. Genet. 4 (2003) 119–141, https://doi.org/10.1146/annurev.genom.4.070802. 110352. [6] A. Salas, H.-J. Bandelt, V. Macaulay, M.B. Richards, Phylogeographic investigations: the role of trees in forensic genetics, Forensic Sci. Int. 168 (2007) 1–13, https://doi.org/10.1016/j.forsciint.2006.05.037. [7] G. Tully, J. Wetton, Mitochondrial DNA: interpretation, in: A. Moenssens (Ed.), Encycl. Forensic Sci. Wiley, Chichester, UK, 2009, pp. 1823–1832. [8] A.W. Röck, A. Dür, M. van Oven, W. Parson, Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA), Forensic Sci. Int. Genet. 7 (2013) 601–609, https://doi.org/10.1016/j.fsigen.2013.07.005. [9] R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 147, https://doi.org/10.1038/13779. [10] M. van Oven, M. Kayser, Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation, Hum. Mutat. 30 (2009) E386–394, https://doi.org/ 10.1002/humu.20921. [11] M. Richards, V. Macaulay, E. Hickey, E. Vega, B. Sykes, V. Guida, C. Rengo, D. Sellitto, F. Cruciani, T. Kivisild, R. Villems, M. Thomas, S. Rychkov, O. Rychkov, Y. Rychkov, M. Gölge, D. Dimitrov, E. Hill, D. Bradley, V. Romano, F. Calì, G. Vona, A. Demaine, S. Papiha, C. Triantaphyllidis, G. Stefanescu, J. Hatina, M. Belledi, A. Di Rienzo, A. Novelletto, A. Oppenheim, S. Nørby, N. Al-Zaheri, S. SantachiaraBenerecetti, R. Scozari, A. Torroni, H.J. Bandelt, Tracing European founder lineages in the Near Eastern mtDNA pool, Am. J. Hum. Genet. 67 (2000) 1251–1276. [12] W. Parson, A. Dür, EMPOP–a forensic mtDNA database, Forensic Sci. Int. Genet. 1 (2007) 88–92, https://doi.org/10.1016/j.fsigen.2007.01.018. [13] J.A. Irwin, J.L. Saunier, H. Niederstätter, K.M. Strouss, K.A. Sturk, T.M. Diegoli, A. Brandstätter, W. Parson, T.J. Parsons, Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples, J. Mol. Evol. 68 (2009) 516–527, https://doi.
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[37] M.R. Wilson, D. Polanskey, J. Replogle, J.A. DiZinno, B. Budowle, A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes, Hum. Genet. 100 (1997) 167–171. [38] R.S. Just, O.M. Loreille, J.E. Molto, D.A. Merriwether, S.R. Woodward, C. Matheson, J. Creed, S.E. McGrath, K. Sturk-Andreaggi, M.D. Coble, J.A. Irwin, A. Ruffman, R.L. Parr, Titanic’s unknown child: the critical role of the mitochondrial DNA coding region in a re-identification effort, Forensic Sci. Int. Genet. 5 (2011) 231–235, https://doi.org/10.1016/j.fsigen.2010.01.012.
[34] A. Salas, M.V. Lareu, A. Carracedo, Heteroplasmy in mtDNA and the weight of evidence in forensic mtDNA analysis: a case report, Int J Leg. Med. 114 (2001) 186–190. [35] J. Naue, T. Sänger, U. Schmidt, R. Klein, S. Lutz-Bonengel, Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing, Int. J. Legal Med. 125 (2011) 427–436, https://doi.org/10.1007/s00414-011-0549-6. [36] K.E. Bendall, V.A. Macaulay, B.C. Sykes, Variable levels of a heteroplasmic point mutation in individual hair roots, Am. J. Hum. Genet. 61 (1997) 1303–1308, https://doi.org/10.1086/301636.
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Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
Research paper
Base specific variation rates at mtDNA positions 16093 and 16183 in human hairs
T
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Stijn Desmytera, , Sophie Dognauxa, Fabrice Noela, Lourdes Prietob,c a
NICC - Belgian Institute for Forensic Science and Criminology, Vilvoordsesteenweg 100, B-1120, Brussels, Belgium Instituto de Ciencias Forenses. Grupo de Medicina Xenómica. Universidade de Santiago de Compostela, Spain c Laboratorio ADN. Comisaría General de Policía Científica, Madrid, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: mtDNA Heteroplasmy Hair Base-specific Variation rate
Small variations between haplotypes detected in different tissues from the same individual have been previously described. These differences complicate the interpretation of mtDNA results in real forensic casework. mtDNA haplotypes detected in hair strands collected at the crime scene have to be frequently compared with haplotypes of reference samples (buccal swabs) from victims or suspects. Nucleotide position 16093 is a well-known hot spot where differences can accumulate between different tissues of the same individual. Intra individual variation was also detected at positions 16182 and 16183 in haplotypes showing an uninterrupted HV1 poly-C stretch (with 16189C). In order to better characterize the type of variation in these positions between buccal cells and hair strands from the same individual, we have performed Sanger sequencing in 25–28 hair strands (411 in total) from 15 individuals showing either an uninterrupted HV1 polyC-stretch (16189C) or 16093C/Y in their buccal cells. The results have been evaluated by also taking into account our previous results published in [19]. We have found that no variation among hair strands was detected in individuals showing T16093 in buccal cells, while variation in hair strands (T16093, 16093C and 16093Y) were detected in individuals showing 16093C or 16093Y in buccal cells. Regarding nucleotide positions 16182 and 16183 in combination with an uninterrupted polyCstretch, no variation was detected in hairs from individuals showing A16182 16183C in their buccal cells. In contrast, individuals A16182 A16183 showed hair strands with A16182 16183 M and A16182 16183C. And finally, individuals with 16182C 16183C showed some variation in a small amount of their hair strands (some hairs with 16182 M 16183C). These results can be relevant for forensic practitioners when comparing reference samples with hair strands, which is the type of sample most tested by using mtDNA analysis in forensic casework.
1. Introduction The interpretation of mtDNA results is not usually easy or straightforward. Guidelines and recommendations were published to advise experts that they should consider not only the number of differences between the profiles of the known and unknown samples, but also the tissue type of the samples, the position of the variation and the instability of the variation within haplogroups [1–8]. For the latter, studies are needed to provide tissue- and site-specific data on mtDNA variation. As long as not much data is available, some forensic experts interpret mtDNA results in the most conservative way, which is solely based on the number of differences. If the difference between the mtDNA profiles of the known and unknown samples is only at a single position, some laboratories report the outcome as inconclusive. This way of interpretation completely neglects the weight of the difference ⁎
between the profiles. In addition, heteroplasmy, being the co-existence of multiple mitotypes in an individual, complicates the interpretation of mtDNA results in forensic casework. Providing quantitative data on site specific variations in hairs, which is the most analysed tissue in forensic casework, could help in this perspective aspect. In this study we focused on two positions: 16093 and 16183, with reference to [9]. 1.1. 16093 nucleotide position This position can show 3 different variants in the human phylogenetic tree: T16093, 16093A and 16093C. T16093 seems to be the ancestral variant in the phylogentic tree of global human mtDNA variation [10]. 16093C polymorphism has not been previously described as an
Corresponding author. E-mail address: [email protected] (S. Desmyter).
https://doi.org/10.1016/j.fsigen.2019.102142 Received 23 April 2019; Received in revised form 25 July 2019; Accepted 11 August 2019 Available online 12 August 2019 1872-4973/ © 2019 Elsevier B.V. All rights reserved.
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forensic field [33]. Therefore, the absence of conventions in length variant regions in the past and the fact that these variations have been ignored in direct forensic comparisons and database searches make it difficult to obtain the real picture of the evolutionary history of these positions. Forster et al. [31] indicate that the mutation rate of 16182C and 16183C is probably too fast between generations to be of reliable use in the phylogenetic reconstruction of the human mtDNA tree. Length heteroplasmy of the polyC-stretch of HV1 affects the length of the preceding polyA-stretch (16180–16183) in a directional way [31]. Usually, this short stretch contains four A’s, but shorter or longer stretches were also observed. Foster et al. studied 1012 individuals belonging to 248 pedigrees of up to 4 generations, and evaluated 802 transmissions [31]. In none of the transmissions, of which 440 belonged to families showing 16189C, did the polyA-stretch lengthen. The shortening of the A-stretch was observed in one of the pedigrees that contained 16189C, which shows that it is possible to observe the cooccurrence of the polyA-stretch shortening with 16189C. It is well known that the distribution of LHP variants varies among the tissues of an individual [34]. The co-existence of A and C at position 16183 (A16183 M) was observed in studies of single hairs [17–20], but only in the study of [19] were buccal swabs of the hair donors also analysed. In the case of one individual, the buccal swab showed an uninterrupted polyC-stretch preceded by four A’s (A16183 16189C), while heteroplasmic variation (16183 M 16189C) was observed in four of the 25 analysed hairs and homoplasmic variation (16183C 16189C) in one hair [Supplementary data Fig. 1]. No variation was observed at 16183 position in the hairs of the 10 other persons tested who had an interrupted HV1 polyC-stretch (T16189) in their buccal cells. This observation can lead us to the idea of a certain pattern of segregation in the hairs of individuals showing 16189C in their buccal cells, that is to say, 16183C/M variants are possible in hairs of individuals showing 16189C in their blood/buccal cells, but are not possible in T16189 individuals. To determine the base-specific variation rate at positions 16183 and 16182 in hairs, 11 more donors that showed an uninterrupted HV1 polyC-stretch were selected and a variable number of hairs from them were analysed. In order to provide quantitative data on the variation rate in single hairs at positions 16093 and 16183, we have studied hair shaft samples from individuals showing 16093C/Y or an uninterrupted HV1 polyCstretch in their buccal cells. The variant status at the particular positions for each single hair was reported and compared with data from a previous hair study of persons who either showed T16093 in their buccal swabs profiles or contained an interrupted HV1 polyC-stretch [19].
haplogroup motif in the main macro-haplogroups [11], although it was later described to appear in many sub-haplogroups [Phylotree, Build 17] In other sub-haplogroups this polymorphism seems to be recurrent or unstable within the respective clade, although this apparent "instability" could also be due to the absence of enough data currently available (L1c2b, L2a1a3, M31b'c, C4a1, G1b, G4, H3h2, H4b1, H10e'g, B4a1a1a7, K1a, K1b1a'b). 16093A polymorphism does not seem to define any haplogroup [Phylotree, Build 17], but can be found in EMPOP v4, R12 [12] in 2 samples from Asia (Uzbekistan and India) belonging to MRCA haplogroup U2a1. Searches of "16093Y" in the same version of EMPOP, using the match type "literal" and the edition range "16093", result in 262 matches in the whole database (42839 haplotypes). These matches involve haplotypes belonging to many different haplogroups. This number confirms the earlier observation in a large scale study on the blood/ buccal cells of 5015 individuals where 0.84% showed 16093Y [13]. The first reported variation between hair and the reference sample from a single donor was at position 16093 [14] and, since then, it has been denoted as a heteroplasmic hotspot in many large-scale hair studies [15–20]. In the studies where, besides sequence data of hair samples also reference data from buccal cells or blood were available, the observation of heteroplasmy at position 16093 in hairs seemed to be correlated with individuals showing 16093C in blood/buccal cells [17–19,21,22], although not exclusively [16,23]. In addition, variation in the C:T content throughout 55 single hair shafts was also described [22]. These sequencing data reveal a preference for C in about 90% of the hair shaft fragments. In several large-scale studies on blood and/or buccal cells samples, position 16093 was also classified as a heteroplasmic hotspot [13,24]. Besides, remarkable ratios between both variants C and T at this position were reported [13,24]. Although T was observed as the most common variant in population databases (about 95% in EMPOP v.4 R12), in heteroplasmic individuals a high C:T ratio was observed in the majority of the samples, that is to say, the C variant is usually the predominant one. Multiple-tissue studies with more sensitive sequencing technologies revealed a tissue- and base-specific variation rate at nucleotide 16093 [25–29]. A large-scale Sanger-sequencing based study on 9 tissues from 100 individuals included buccal cells and hair strands [30]. For all eight individuals who showed heteroplasmy at position 16093 in at least one sample, the ratio C:T was studied in all 9 tissues. These data confirm the high C-content in blood and the high T-content in skeletal muscle for all eight persons. The ratio in buccal cells was similar to the one in blood, while there was no preference for either nucleotide in hair.
2. Material and methods 1.2. 16183–16182 nucleotide positions 2.1. Sample collection and DNA extraction Length heteroplasmy (LHP) is observed when at least two mtDNA molecules that differ in their number of nucleotides are present in a detectable amount in an individual. In the Control Region, length heteroplasmy was seen to appear in the polyC-stretches of HV1 (16184–16193), HV2 (303–309) and HV3 (456–463 and 568–573), and in the AC tandem repeat (514–524) [13,31,32]. In the polyC-stretch of HV1, length heteroplasmy is mostly associated with the polymorphism 16189C if this mutation results in an uninterrupted polyC-stretch of more than eight C’s [13,31]. Positions 16182 and 16183 have usually been excluded from the mtDNA tree and are not considered for phylogenetic reconstruction [10]. 16182 M and 16183 M denote the (heteroplasmic) mixture of C and A at these positions; 16182C and 16183C denote the transversion A–C at these positions; and finally, 16182c and 16183c denote the (heteroplasmic) mixture of a transversion and a deletion at these nucleotide positions. Lower case nomenclature was introduced in 2014, after the ISFG recommendations about mtDNA nomenclature in the
Head hairs were collected from 15 staff members of the National Institute of Criminalistics and Criminology, Brussels, Belgium (NICC) and the Spanish Forensic Police, after informed consent. Their buccal cells mtDNA profiles were established over the years by Sanger sequencing as part of the quality control contamination databases. All donors belonged to the European population and were selected based on their mtDNA profiles containing either an uninterrupted HV1 polyCstretch (16189C) or the polymorphism 16093C/Y [Supplementary data Table 1]. Their haplotypes were assigned to haplogroups by using PhyloTree [10], build 17, aided by the EMMA software package [8]. Between 25 and 28 hairs per person were plucked. After collection, the roots and 0.5–1 cm proximal fragments of all hairs were removed. The first proximal 2 cm-shaft fragment of each of the 411 hairs were decontaminated and the DNA was extracted following the (a) protocol from [19].
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2.2. MtDNA amplification and sequencing
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Amplification, postPCR purification and Sanger-type cycle sequencing were performed as described in the (a) protocol of [19], while amplification was limited to only one fragment with the primer pair M13L15997-M13H16239. The sequencing was performed with primer M13 L [19]. 2.3. MtDNA sequence data generation and interpretation The sequences were assembled using SeqScape v.2.5 (AB, Oyster Point, CA, USA) and aligned to the revised Cambridge Reference Sequence (rCRS) [9]. Base calling was ensured by double independent data analysis and the sequencing results were checked on complementarity to the buccal cells reference profiles. The base calling results were only reported at the positions of interest (16093 or 16182 and 16183) in this work [Supplementary data Table 1].
In general, at 16093 the highest C-content was observed in blood, while the lowest C-content was observed in skeletal muscle [29]. The large-scale study of [29] identified in total seven nucleotides at which the heteroplasmy was position-, base- and tissue-dependent. This phenomenon was the most prominent at position 16093, which explains why it was observed in the past in smaller and Sanger-sequencing based studies. Even though many different tissues were studied by using MPS, none of them analysed buccal cells or hairs, in spite of the fact that they are the most common tissues in forensic casework. In another study, Naue et al. [30] demonstrated a high variation in C-content at position 16093 between 9 different tissues of 8 individuals who were predominantly 16093C in their blood and buccal cells. For these persons, the C-content at position 16093 was more variable in hairs (40–80%) compared to buccal cells (85–100%). Those tissuespecific variation rates could explain why in our study heteroplasmy was not observed in the buccal cell samples but in the hair samples. Nevertheless, in this large-scale study, each hair sample actually contained multiple hairs. This way of sampling is quite different from the one usually performed in real casework (each sample contains only one single hair strand) and therefore does not allow us to evaluate the homoplasmic and heteroplasmic variation rate between single hairs. Contrary to the observation that 16093C individuals are highly variable in hairs at this particular position, the base status was invariable for all the 310 hairs of the 10 individuals with T16093 in their reference profile. This observed base-specific variation at position 16093 made us believe that the high ranking of 16093 as a heteroplasmic hotspot in many hair studies [15–20] is due to the high variation rate in 16093C and 16093Y individuals, and not because of a high variation rate in all persons (16093C, 16093Y and T16093). Inter-individual variation was observed [Supplementary data Table 1], but not enough donors were represented in this study to statistically evaluate these differences in variation rates.
3. Results and discussion In order to study the mtDNA variation at positions 16093, 16182 and 16183 with Sanger sequencing, we collected hairs from 3 individuals with variant status 16093C, from 1 individual with an obvious status 16093Y and from 11 individuals with an uninterrupted polyCstretch in their buccal mtDNA profiles. These reference profiles were previously obtained by Sanger sequencing. Heteroplasmy detection is known to be influenced by different factors like amplification strategy, DNA polymerase, primers and sequencing chemistry [15,35]. Since the protocol we used in this study was identical to the one of our previous study on the latitudinal variation of CR mtDNA amongst hair shafts [19], we were able to combine the results of both studies. 3.1. Variation at position 16093 We have combined the results of hairs of the three 16093C (X1-X3) and the one 16093Y (X4) individual in this study with data from Desmyter et al. in which we had analysed 25 hairs from individual P11 who showed 16093C in her buccal cells. In total, we ended up with 109 analysed hairs from four individuals with 16093C in the reference profile (X1-X3 and P11) and 26 analysed hairs from one individual with 16093Y (X4). In addition, the variation at position 16093 of these hairs was also compared to the base status of the 310 analysed hairs of the 10 individuals P1-P10 who were all T16093 in their buccal cells profile [19]. Table 1 shows the results of both studies, but to briefly summarize:
•
observed in other Sanger sequencing studies [21,22] where some hair shaft fragments of 16093C persons were analysed. Individual showing 16093Y in buccal swab - variation in hairs: As expected, a similar variation rate was also observed in the hairs of the person X4 whose reference sample was shown to be heteroplasmic at position 16093 (35% T16093, 27% 16093Y and 38% 16093C). In former studies, similar segregation patterns at other positions, different from 16093, were observed in hairs from individuals being heteroplasmic at this particular position in their buccal cells or blood. [19,22,36,37].
3.2. Variation at positions 16182 and 16183 Individuals showing 16093C in buccal swabs - variation in hairs: Variation rate at position 16093 in the three 16093C individuals from this study (X1-X3) confirmed the previous observations made in [19] for person P11, who also showed 16093C in her buccal cells and T16093, 16093Y and 16093C variants in her hair shafts. On average, half of the hairs of the four 16093C persons showed heteroplasmic variation (16093Y) and about 15% of the hairs showed homoplasmic variation (T16093). All observed variations were transitions. Variation at position 16093 was also previously
To study the variation at positions 16182 and 16183, in relation to an uninterrupted HV1 polyC-stretch, we have combined the present results with data from [19] (individual P5, 25 hair fragments). In total we have obtained sequencing data from 12 individuals: (i) six showing A16182 A16183 in their buccal cells reference profile, (ii) three showing A16182 16183C and (iii) three showing 16182C 16183C (Table 2). The haplotype obtained from buccal cells of all twelve persons contained an uninterrupted HV1 polyC-stretch (16189C). In the case of the six A16182 A16183 individuals, 164 hair shafts
Table 1 The average percentage (with standard deviation) of hairs with a particular base status at position 16093 compared to their respective reference profile from buccal cells. Base status at position 16093 in buccal cells T Y C a
Number of persons a
10 1 4 (3 + 1a)
Number of hairs a
310 26 109 (84 + 25a)
% of hairs T16093
% of hairs 16093Y
% of hairs 16093C
100 34.6 14.7 ± 11.3
0 26.9 49.5 ± 19.2
0 38.5 35.8 ± 10.5
310 hairs from ten T16093 individuals and 25 hairs from one 16093C individual were analysed in study [19]. 3
Forensic Science International: Genetics 43 (2019) 102142
0 0 94.0 ± 10.3 5.5 ± 5.1 100 0 15.2 ± 8.8 0 0
0 0 6.0 ± 10.3
25 hairs of one A16182 A16183 person were analysed in the study [19].
164 (139 + 25a) 80 82 6 (5 + 1a) 3 3 AA AC CC
79.3 ± 10.5 0 0
Heteroplasmy at position 16093 is known to be tissue- and base specific. Our data confirmed the lack of preference for a certain variant (C or T) at position 16093 in hairs of individuals that show 16093C in their buccal cells haplotypes. Indeed, of a total number of 109 hairs originating from 16093C individuals, an average of 15% showed homoplasmic variation (T16093) and 50% heteroplasmic variation (16093Y), while no variation was observed in all 310 analysed hairs from T16093 individuals. The shortening of the polyA-stretch, preceding the HV1 polyCstretch, is known to be associated with 16189C in germline transmissions. Our data on single hairs confirmed this co-occurrence of an uninterrupted HV1 polyC-stretch with the shortening of the preceding polyA-stretch. Variation at position 16183 was observed in hairs from individuals with an uninterrupted polyC-stretch, while no variation was previously observed in 310 analysed hairs with T16189 or 16186 T 16189C. The observed variation rate at position 16183 was variantspecific in hairs, favouring shortening over lengthening of the polyAstretch. On average, 6% of the 164 analysed hairs from individuals with A16182 A16183 in their reference profiles showed homoplasmic variation (16183C) and 15% heteroplasmic variation (16183 M). The 162 analysed hairs from individuals with A16182 16183C or 16182C 16183C showed no variation at position 16183. Variation at position 16182 was not observed in this study, probably due to both the lower variation rate when compared to position 16183 and not a sufficient amount of analysed hairs needed to detect any variation. In this study, we provide quantitative data for the base-specific variation rates in single human hairs at positions 16093 and 16183 of the mtDNA. These data could be useful in the interpretation of mtDNA results in forensic casework in a quantitative manner, in which not only the number of differences are considered but also the weight of the difference (see [38] as an example). For instance, let’s consider the following two possible scenarios:
a
% of hairs 16182C 16183C % of hairs 16182 M 16183C % of hairs A16182 16183C % of hairs A16182 16183 M Number of hairs Number of persons
% of hairs A16182 A16183
were studied. Variation at position 16183 was found in these hairs. On average about 15% of the hairs showed heteroplasmic variation (16183 M) and about 6% of the hairs showed homoplasmic variation (16183C). In the case of the three individuals showing A16182 16183C, 80 hair shafts were studied, and finally, in the case of the three individuals showing 16182C 16183C, 82 hair shafts were studied. No or hardly any variation was found in these cases. These data suggest that variation at position 16183 between hairs is characterized by directionality. The shortening of the succession of four A’s preceding the uninterrupted polyC stretch seems to be favoured over the lengthening. This base-specific variation rate at position 16183 in association with an uninterrupted polyC-stretch was also observed in a germline transmission study involving 248 pedigrees with up to four generations [31]. While no lengthening was observed in a total of 802 transmissions, of which 440 were within families with 16189C, the shortening of the polyA-stretch occurred in a pedigree with 16189Y. In the present study, variation was only observed at position 16183 and not at position 16182. The absence of variation was most probably related to a lower variation rate at position 16182 compared to 16183. Apparently more hairs should be studied in order to obtain a variation rate at position 16182. While variation was observed at position 16183 in some hairs of individuals with an uninterrupted HV1 polyC-stretch, no variation was observed in previous data from 310 hairs of 10 individuals P1-P10 [19]. All of them had an interrupted polyC-stretch in HV1 either by T16189 (Persons P1-P3, P6-P11) or by 16186 T (Person P4) and all of them showed A16182 A16183. These findings confirm the association between variation at position 16183 and the presence of an uninterrupted HV1 polyC-stretch.
4. Conclusions
Base status at positions 16182 and 16183 in buccal cells
Table 2 The average percentage (with standard deviation) of hairs with a particular base status at positions 16182 and 16183 compared to their respective buccal cells reference profile. All profiles contained an uninterrupted polyC-stretch in HV1.
S. Desmyter, et al.
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a) a suspect of a crime shows haplotype 16519C 263G 315.1C in his buccal cells, and a hair strand collected from the hand of the victim shows haplotype 16093C 16519C 263G 315.1C b) a suspect of a crime shows haplotype 16093C 16519C 263G 315.1C in his buccal cells, and a hair strand collected from the hand of the victim shows haplotype 16519C 263G 315.1C
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In both cases, the suspect cannot be excluded as the donor of the hair strand. But taking into account what we have described in this study, scenario b) is more probable than scenario a) if the hair strand really belongs to that suspect. Nevertheless, the results presented here are only based on Sanger sequencing, but other more sensitive techniques, such as massive parallel sequencing, could shed more light on the real base-content at these positions. Sanger sequencing has a limited capacity to detect heteroplasmies and therefore, some samples described here as homoplasmic, could contain some level of heteroplasmy. For both positions 16093 and 16183, the variation rates obtained show inter-individual differences (e.g. T16093 varies from 0% in individual P11 to 25% in individual X2). The current data set is too small to evaluate if the observed differences between individuals are statistically significant. Therefore, more research would be needed in order to use inter-individual variation in casework interpretation. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2019.102142. References [1] M.M. Holland, T.J. Parsons, Mitochondrial DNA sequence analysis - validation and use for forensic casework, Forensic Sci. Rev. 11 (1999) 21–50. [2] A. Carracedo, W. Bär, P. Lincoln, W. Mayr, N. Morling, B. Olaisen, P. Schneider, B. Budowle, B. Brinkmann, P. Gill, M. Holland, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Forensic Sci. Int. 110 (2000) 79–85. [3] W. Bär, B. Brinkmann, B. Budowle, A. Carracedo, P. Gill, M. Holland, P.J. Lincoln, W. Mayr, N. Morling, B. Olaisen, P.M. Schneider, G. Tully, M. Wilson, DNA commission of the international society for forensic genetics: guidelines for mitochondrial DNA typing, Int. J. Legal Med. 113 (2000) 193–196. [4] G. Tully, W. Bär, B. Brinkmann, A. Carracedo, P. Gill, N. Morling, W. Parson, P. Schneider, Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles, Forensic Sci. Int. 124 (2001) 83–91. [5] B. Budowle, M.W. Allard, M.R. Wilson, R. Chakraborty, Forensics and mitochondrial DNA: applications, debates, and foundations, Annu. Rev. Genomics Hum. Genet. 4 (2003) 119–141, https://doi.org/10.1146/annurev.genom.4.070802. 110352. [6] A. Salas, H.-J. Bandelt, V. Macaulay, M.B. Richards, Phylogeographic investigations: the role of trees in forensic genetics, Forensic Sci. Int. 168 (2007) 1–13, https://doi.org/10.1016/j.forsciint.2006.05.037. [7] G. Tully, J. Wetton, Mitochondrial DNA: interpretation, in: A. Moenssens (Ed.), Encycl. Forensic Sci. Wiley, Chichester, UK, 2009, pp. 1823–1832. [8] A.W. Röck, A. Dür, M. van Oven, W. Parson, Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA), Forensic Sci. Int. Genet. 7 (2013) 601–609, https://doi.org/10.1016/j.fsigen.2013.07.005. [9] R.M. Andrews, I. Kubacka, P.F. Chinnery, R.N. Lightowlers, D.M. Turnbull, N. Howell, Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA, Nat. Genet. 23 (1999) 147, https://doi.org/10.1038/13779. [10] M. van Oven, M. Kayser, Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation, Hum. Mutat. 30 (2009) E386–394, https://doi.org/ 10.1002/humu.20921. [11] M. Richards, V. Macaulay, E. Hickey, E. Vega, B. Sykes, V. Guida, C. Rengo, D. Sellitto, F. Cruciani, T. Kivisild, R. Villems, M. Thomas, S. Rychkov, O. Rychkov, Y. Rychkov, M. Gölge, D. Dimitrov, E. Hill, D. Bradley, V. Romano, F. Calì, G. Vona, A. Demaine, S. Papiha, C. Triantaphyllidis, G. Stefanescu, J. Hatina, M. Belledi, A. Di Rienzo, A. Novelletto, A. Oppenheim, S. Nørby, N. Al-Zaheri, S. SantachiaraBenerecetti, R. Scozari, A. Torroni, H.J. Bandelt, Tracing European founder lineages in the Near Eastern mtDNA pool, Am. J. Hum. Genet. 67 (2000) 1251–1276. [12] W. Parson, A. Dür, EMPOP–a forensic mtDNA database, Forensic Sci. Int. Genet. 1 (2007) 88–92, https://doi.org/10.1016/j.fsigen.2007.01.018. [13] J.A. Irwin, J.L. Saunier, H. Niederstätter, K.M. Strouss, K.A. Sturk, T.M. Diegoli, A. Brandstätter, W. Parson, T.J. Parsons, Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples, J. Mol. Evol. 68 (2009) 516–527, https://doi.
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