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Amorim A, Fernandes T, Taveira N. 2019. Mitochondrial DNA in humanidentification: a review. PeerJ 7:e7314 https://doi.org/10.7717/peerj.7314

Mitochondrial DNA in human identification: a review

António Amorim 1 , Teresa Fernandes 2 , Nuno Taveira Corresp. 3, 4

1 Instituto Nacional de Medicina Legal e Ciências Forenses, Lisbon, Portugal2 Biologia, Universidade de Évora, Évora, Portugal3 Instituto Universitário Egas Moniz (IUEM), Almada, Portugal4 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

Corresponding Author: Nuno TaveiraEmail address: ntaveira@ff.ul.pt

Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies,

especially related to the lack of recombination, to a high copy number, and to matrilineal

inheritance. mtDNA typing based on sequences of the control region or full genomic

sequences analysis is used to analyze a variety of forensicsamples such as old bones,

teeth and hair, as well as other biological samples where the DNA content is low.

Evaluation and reporting of the results requires careful consideration of biological issues as

well as other issues such as nomenclature and reference population databases. In this

work we review mitochondrial DNA profiling methods used for human identification and

present their use in the main cases of human identification focusing on the most relevant

issues for the forensic and medico-legal areas.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27500v1 | CC BY 4.0 Open Access | rec: 24 Jan 2019, publ: 24 Jan 2019

1 Mitochondrial DNA in Human Identification: a review

2 Authors

3 António Amorim1, Teresa Fernandes2, Nuno Taveira3,4*

4

5 Affiliations

6 1Instituto Nacional de Medicina Legal e Ciências Forenses, Lisboa, Portugal

7 2Universidade de Évora, Évora, Portugal

8 3Instituto Universitário Egas Moniz (IUEM), Almada, Portugal

9 4Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de

10 Lisboa, Portugal

11

12 * Corresponding author:

13 Nuno Taveira

14 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,

15 Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal

16 ntaveira@ff.ul.pt

17

18

19

20 Abstract

21 Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies,

22 especially related to the lack of recombination, to a high copy number, and to matrilineal

23 inheritance. mtDNA typing based on sequences of the control region or full genomic sequences

24 analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well

25 as other biological samples where the DNA content is low. Evaluation and reporting of the

26 results requires careful consideration of biological issues as well as other issues such as

27 nomenclature and reference population databases. In this work we review mitochondrial DNA

28 profiling methods used for human identification and present their use in the main cases of human

29 identification focusing on the most relevant issues for the forensic and medico-legal areas.

30

31

32 Introduction

33 Human genetic identification for medico-legal or forensic purposes is achieved through the

34 definition of genetic profiles. A genetic profile or the genetic fingerprint of an individual is the

35 phenotypic description of a set of genomic loci that are specific to that individual. In accordance

36 with international recommendations, particularly with recommendations of the European DNA

37 Profiling Group (EDNAP), currently, only genetic profiles obtained from autosomal short

38 tandem repeats (STR) should be used for genetic fingerprinting. However, in a considerable

39 number of situations of medico-legal and forensic identification, autosomal DNA is highly

40 degraded or isn’t available at all. In these cases the study of mitochondrial DNA (mtDNA) for

41 human identification has become routine (1–5).

42 Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies,

43 especially related to the lack of recombination, to a high copy number, and to matrilineal

44 inheritance. mtDNA typing based on sequences of the control region or full genomic sequences

45 analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well

46 as other biological samples where the DNA content is low. Evaluation and reporting of the

47 results requires careful consideration of biological issues as well as other issues such as

48 nomenclature and reference population databases. In this work we review mitochondrial DNA

49 profiling methods used for human identification and present their use in the main cases of human

50 identification focusing on the most relevant issues for the forensic and medico-legal areas.

51

52 Survey methodology

53 We systematically searched in PubMed for papers in English describing 1) mitochondrial DNA

54 biology and genetics, 2) mitochondrial DNA typing guidelines, 3) mitochondrial DNA

55 nomenclature, 4) mitochondrial DNA sequencing methodologies, 5) mitochondrial DNA

56 population data and databases and 6) mitochondrial DNA in human identification and forensics.

57 Our search wasn’t refined by publishing date, journal or impact factor of the journal, authors or

58 authors affiliations. In addition, we used Guideline documents from the International Society for

59 Forensic Genetics available at https://www.isfg.org/.

60

61 Mitochondrial DNA biology and genetics

62 Mitochondria are cellular organelles that contain an extrachromosomal genome, which is both

63 different and separate from the nuclear genome. The mitochondrial DNA (mtDNA) was first

64 identified and isolated by Margit Nass and Sylvan Nass in 1963, who studied some

65 mitochondrial fibers that according to their fixation, stabilization and staining behavior, appeared

66 to be DNA related (6). However, the complete sequence of the first mtDNA was only published

67 and established as the mtDNA Cambridge Reference Sequence (CRS) eighteen years later, in

68 1981 (7).

69 Essentially, the mtDNA is a 5 mm histone-free circular double-stranded DNA molecule, with

70 around 16,569 base-pairs and weighting 107 Daltons (8). mtDNA strands have different densities

71 due to different G+T base composition. The heavy (H) strand encodes more information, with

72 genes for two rRNAs (12S and 16S), twelve polypeptides and fourteen tRNAs, while the light

73 (L) strand encodes eight tRNAs and one polypeptide. All the 13 protein products are part of the

74 enzyme complexes that constitute the oxidative phosphorylation system. Other characteristic

75 features of the mtDNA are the intronless genes and the limited, or even absent, intergenic

76 sequences, except in one regulatory region.

77 The mitochondrial D-loop is a triple-stranded region found in the major non-coding region

78 (NCR) of many mitochondrial genomes, and is formed by stable incorporation of a third 680

79 bases DNA strand known as 7S DNA (9). The origin of replication is located at the non-coding

80 or D-loop region, a 1121 base pairs segment that is located between positions 16,024 and 516,

81 according to the CRS numeration (7) (Figure 1). The D-loop region, also comprehends two

82 transcription promotors, one for each strand. Nucleotide positions in the mtDNA genome are

83 numbered according to the convention presented by Anderson et al. (7), which was slightly

84 modified by Andrews et al. (10). More precisely, the numerical designation of each base pair is

85 initiated at an arbitrary position on the H strand, which continues thereafter and around the

86 molecule for approximately 16,569 base pairs.

87 The apparent lack of mtDNA repair mechanisms and the low fidelity of the mtDNA polymerase

88 lead to a significant higher mutation rate in the mitochondrial genome, when compared to the

89 nuclear genome. For example, Sigurğardóttir and collaborators, estimated the mutation rate in

90 the human mtDNA control region to be 0.32x10-6/site/year (10) which compares to

91 0.5 × 10−9/site/year in the nuclear genome (12). Most of the sequence variation between

92 individuals is found in two specific segments of the control region, namely in the hypervariable

93 region 1 (HV1, positions 16,024 to 16,365) and in the hypervariable region 2 (HV2, positions 73

94 to 340) (13). A third hypervariable region (HV3, positions 438 to 574), with additional

95 polymorphic positions can be useful in the resolution of indistinguishable HV1/HV2 samples

96 (14). The small size and relatively high inter-person variability of the HV regions are very useful

97 features for forensic testing purposes.

98 A mitochondrion contains 2 to 10 copies of mtDNA and each somatic cell can have up to 1,000

99 mitochondria (15,16). Hence, when the amount of the extracted DNA is quite small or degraded,

100 it is more likely that a DNA typing result can be obtained by typing the mtDNA than by typing

101 polymorphic regions that are found in nuclear DNA.

102 Contrarily to the nuclear DNA, the mtDNA is exclusively maternally inherited, which justifies

103 the fact that, apart from mutation, mtDNA sequence of siblings and all maternal relatives is

104 identical (17–19). This specific characteristic can be very helpful in forensic cases, such as in the

105 analysis of the remains of a missing person, where the known maternal relatives can provide

106 some reference samples for a direct comparison to the mtDNA type. Due to the lack of

107 recombination, maternal relatives from several generations apart from the source of evidence (or

108 biological material) can be used for reference samples (17–19).

109 The haploid and monoclonal nature of the mtDNA in most individuals simplifies the process of

110 interpretation of the DNA sequencing results. Still, it is possible to find heteroplasmy at

111 occasional cases (20–25). A person is considered as heteroplasmic if she/he carries more than

112 one detectable mtDNA type. There are two classes of heteroplasmy, related to length

113 polymorphisms and to point substitutions. Only the latter is important for forensic human

114 identification. Most forensic laboratories worldwide do not report length polymorphisms and the

115 guidelines on human identification with mtDNA do not point them as mandatory information

116 (2,4). Furthermore, the information of length polymorphisms has no impact in haplogroups’

117 definition.

118 Heteroplasmy manifests itself in diverse ways (26). An individual may show more than one

119 mtDNA type in a single tissue. An individual may be heteroplasmic in one tissue sample and

120 homoplasmic in another one. Finally, an individual may exhibit one mtDNA type in one tissue

121 and a different type in another tissue. Of the three possible scenarios, the last one is the least

122 likely to occur. When heteroplasmy is found in the mtDNA of an individual, it usually differs at

123 a single base, in HV1 or HV2.

124 Heteroplasmy was observed at position 16,169 of the mtDNA control region in the putative

125 remains of Tsar Nicholas II of Russia and his brother, the Grand Duke of Russia Georgij

126 Romanov (22,23). Comas et al. (21), in turn, detected heteroplasmy at two distinct positions,

127 16,293 and 16,311, in the mtDNA of an anonymous donor’s plucked hair. Wilson et al. (24)

128 found a family constituted by a mother and two children carrying a heteroplasmic mtDNA at

129 position 16,355 both in blood and buccal swab samples.

130 The existence of heteroplasmic individuals and the limited knowledge about both the mechanism

131 and the rate of heteroplasmy can be issues raised in an attempt to exclude mtDNA evidence from

132 forensic investigations. In the context of forensic analysis, both mtDNA sequences of a reference

133 sample and an evidence sample(s) are compared. If the mtDNA sequences are identical, the

134 samples can’t be excluded since they must have the same origin or derive from the same

135 maternal lineage. Similarly, samples can’t be excluded when heteroplasmy is observed at the

136 same nucleotide positions in both samples. Finally, when one sample is heteroplasmic and the

137 other is homoplasmic but they both share at least one mtDNA species, the samples can’t be

138 excluded since they may have the same origin. Several authors have suggested that samples with

139 mtDNA with one-base difference should be further evaluated, mainly regarding their rate of

140 mutation (1,27–29). When two or more nucleotide differences exist between the two sequences,

141 the overall interpretation is exclusion (4).

142 Heteroplasmy at one nucleotide position is more frequently observed in hair samples, mainly due

143 to genetic drift and to bottlenecks which occur due to the hair follicle’s semiclonal nature (5,30–

144 32). Hence, if an evidentiary hair sample contains one of the two heteroplasmic lineages that are

145 observed in a reference sample, or vice versa, then the interpretation of exclusion may be

146 incorrect. In this case, typing additional hairs may be required to solve the problem (5).

147 As it was previously pointed out, the mitochondrial genome is maternally inherited. Even though

148 the sperm contains a few mitochondria in the neck and in the tail region, the male mitochondrial

149 genome is destroyed either during or shortly after the fertilization. More precisely, sperm

150 mitochondria disappear in the early embryogenesis, namely by selective destruction, inactivation

151 or dilution (33–35). Nevertheless, there are a few examples of paternal inheritance of the

152 mitochondrial genome in animals (36), and by that reason and despite the limited evidence for

153 paternal inheritance of the mitochondrial genome in humans, the courtroom can use such

154 possibility to exclude the use of mtDNA evidence.

155

156 Mitochondrial DNA Nomenclature

157 Even though the process of naming mtDNA sequences seems simple and obvious, it is crucial to

158 properly consider the nomenclatures, since complications might arise. Considering that listing

159 more than 600 bases in order to describe the results from a new HV1 and HV2 sequence would

160 be unpractical, an alternative approach was developed which essentially identifies and reports the

161 differences relative to a reference sequence (Cambridge Reference Sequence or CRS sequence)

162 (7). For example, if the bases beyond the position 16,192 were out of the register by one base

163 due to the insertion of a C the mutation is designated as 16,192.1 C. If two Cs were inserted, they

164 would be designated as 16,192.1 C and 16,192.2 C. Regarding deletions, these are recorded by

165 the number of the base(s) that is missing, with respect to the CRS (i.e., 249 D or 249-). The bases

166 that cannot be unambiguously determined are coded by using an N. The guidelines that are used

167 to record all the sequence’s differences have been described by several authors (1,2,4,29), and

168 are used by the forensic community in general.

169 In forensics, the origin of the evidentiary sample is unknown. Consequently, and to be fair to the

170 defendant, it is important to list any sequences in a reference mtDNA database that are identical

171 to the evidentiary mtDNA sequence, especially when estimating how rare or common the

172 evidentiary mtDNA sequence is. Currently, most ambiguities in the alignment/nomenclature

173 arise due to insertions and/or deletions (indels). Hence, Wilson et al. (37) developed an approach

174 to attempt to standardize all mtDNA alignments which is based on a phylogenetic context, and

175 gives differential weighting to transversions, transitions, insertions and deletions.

176 The nomenclature approach mentioned above aims the standardization of reference mtDNA

177 databases, which is why recommendations are provided to address several scenarios (1,2,4,29).

178 The recommendations are as follows: a) characterize the variant(s), namely by using the least

179 number of differences (i.e., insertions, substitutions, deletions) from the reference sequence; b) if

180 there is more than one alignment, each one having the same number of differences when it

181 comes to the reference sequence, indels must be prioritized; c) indels must be placed at 3’ end,

182 especially with respect to the light strand (if possible, deletions and/or insertions must be

183 combined); and d) gaps are combined only when they can be placed at the 3’ end, while

184 maintaining the exact same number of differences from the reference sequence. The bases that

185 are designated with an N should not affect these recommendations. These recommendations are

186 hierarchical, which is why the first recommendation takes precedence, being immediately

187 followed by the remaining ones, that are, and to some degree, arbitrary. The last recommendation

188 is a clarification, since it aims to facilitate the alignment of all interpretations. Lastly, it is

189 relevant to point out that several examples have been identified, where alternative alignment

190 strategies are possible, being adequately described elsewhere in the literature (37).

191

192 Mitochondrial DNA Typing Guidelines

193 In 2014 the DNA Commission of the International Society of Forensic Genetics (ISFG)

194 published updated guidelines and recommendations concerning mitochondrial DNA typing.

195 These guidelines referred to good laboratory practices, targeted region, amplification and

196 sequencing ranges, reference sequence, alignment and notation, heteroplasmy, haplogrouping of

197 mtDNA sequences, and databases and database searches. In Table 2 we present the 16

198 recommendations of ISFG. Overall, these are the main guidelines concerning the application of

199 mtDNA polymorphisms in human identification, which are regularly revised and published by

200 the International Society of Forensic Genetics (1,2,4,29).

201

202 Mitochondrial DNA Sequencing Methodologies

203 In 1977 Sanger presented the first DNA sequencing technology (38), also called the chain

204 termination method and now known as first generation sequencing. The incorporation of ddNTPs

205 in newly synthesized DNA strands results in termination of the elongation process and

206 correspondent knowledge about the specific nucleotide present at the sequence at each position.

207 Sanger sequencing method can produce reads from 25 up to 1200 nucleotide positions, allowing

208 the read of a maximum of 96 kb nucleotides in 2 hours.

209 Since 2005 a wide number and variety of new sequencing methods have been developed and

210 launched on the market (39). Second generation sequencing methods do not allow to read the

211 complete sequence of an individual genome at one-time step but only small DNA fragments each

212 time, from 35 up to 75 nucleotide positions using the SOLiD technology (39–41), to 100 up to

213 1,000 nucleotide positions using the 454 Pyrosequencing technology (39,42). Depending on the

214 technology, these methods allow sequence reads from 60 to 80 million nucleotide positions in 2

215 hours or 6 billion nucleotide positions in 1- 2 weeks as (43,44).

216 Third generation sequencing technologies were introduced in the market in 2010 and allow

217 massive parallel analysis combined with reading in real time (39). With these methods it is

218 possible to read between 8,000 to 200,000 nucleotide positions at one-time, and they allow

219 sequence reads from 100,000 up to 4 Tb nucleotide positions in less than 48 hours (45,46). Many

220 different methodologies designed to attend to different priorities had been proposed. Some

221 methodologies are focused on the number of reads, other’s give priority to results accuracy and

222 other’s attend to the range of the reads. Pacific Biosciences (PacBio) introduced equipment’s

223 designed to produce low number of reads with high accuracy (45,47), such as PacBio RSII (48),

224 while Oxford Nanopore Technologies (ONT) designed methodologies to produce high number

225 of reads with lower accuracy, such as ONT MinION (46), and Illumina gives priority to short-

226 read with high accuracy technologies and present’s a ‘post-light’ methodology with directly

227 sensing non-optical sequencing, such as Illumina HiSeq (49,50). These massive parallel

228 sequencing (MPS) technologies have been quickly applied in a wide range of areas where

229 forensics is included (39). Personal Genome Machine (PGM) first introduced the Ion Torrent

230 methodology for sequence complete mtGenomes in forensic context (51). Nevertheless,

231 regarding to mtDNA analysis for forensic human identification, and according to current

232 international guidelines (2,4), Sanger sequencing still continues to be an adequate method and is

233 used in most casework laboratories worldwide (52). However, if guidelines on human

234 identification with mtDNA come to establish as mandatory the inclusion of information of the

235 coding region, the benefits of MPS could be explored to type whole mtDNA genomes. In 2015,

236 Magalhães and collaborators presented the results of Ion Torrent PGM NGS technology applied

237 to whole mtDNA molecule sequencing. Although it proved to be sensitive and accurate at

238 detecting and quantifying mixture and heteroplasmy, there were some problems in the coverage

239 of the mtDNA genome with some regions presenting extreme strand bias, and presenting false

240 positives mostly generated by alignment problems in the analysis algorithms (53). Woerner and

241 collaborators presented, in 2018, the evaluation of the Precision ID mtDNA Whole Genome

242 Panel on two massively parallel sequencing commercial systems - Ion S5 System (Thermo

243 Fisher Scientific) and MiSeq FGx Desktop Sequencer (Illumina) (54). According to their

244 conclusions, Ion and MiSeq methodologies provide consistent mtDNA haplotypes estimation.

245 Beyond this study many other studies on the use of MPS technologies for forensic genetics and

246 mtDNA analysis have been published (55,56,65–67,57–64). However, further validation studies

247 and specialized software functionality tailored to forensic practice should be produced in order to

248 facilitate the incorporation of NGS processing into standard casework applications (68,69).

249

250 Mitochondrial DNA Population Data and Databases

251 When two mtDNA sequences, one from an evidence sample and another from a reference

252 sample, cannot be excluded as being originated from the exact same source, it is necessary to

253 convey some information concerning the rarity of the mtDNA profile. The current practice is to

254 count how many times a specific sequence is observed within a population database(s) (70).

255 Overall, the population databases that are used in forensics comprehend several convenience

256 samples, representing the major population groups of the potential contributors in terms of

257 evidence.

258 Phylogenetic methods have been used in order to identify the main human haplogroups, as well

259 as the most important SNPs that define such groups. Regarding the construction of a

260 phylogenetic tree of mtDNA sequences, there are several alternative methods, which include

261 neighbor-joining, maximum likelihood, minimum spanning networks, maximum parsimony and

262 Bayesian methods (71). Overall, the large majority of individuals from African populations, and

263 specially from sub-Saharan African populations, are categorized into one of the main haplogroup

264 lineages that diverged from macro-haplogroup L - L0, L1, L2, L3, L4, L5 and L6 - (72–79). On

265 the other hand, more than 90% of the individuals of the European and USA Caucasian

266 populations are categorized into 10 main haplogroup lineages - H, I, J, K, M, T, U, V, W and X -

267 (5,76,77,80,81). Concerning to African-American populations, the most commonly observed

268 haplogroups are L2a, L1c, L1b and L3b (75). The main haplogroups found in individuals from

269 Asian populations are haplogroups M and N (82,83).

270 The first database for human mtDNA was Mitomap in 1995 (84). In 1996, this database

271 developed into an online database, www.mitomap.org, containing published human mtDNA

272 variation along with geographic and disease specific variants. Currently, Mitomap is manually

273 curated, frequently updated and a functionally rich resource, presenting high-quality human

274 mtDNA data for clinicians, investigators and geneticists (84). Mitomap has three main categories

275 for usage. It contains some background information regarding the human mitochondrial DNA,

276 such as the general representation of mtDNA, haplogroups and their frequencies and illustrations

277 of mtDNA, among others. Furthermore, users can also find information about other mtDNA-

278 specific databases, tools and useful resources.

279 Mitomap stores the annotated listing of the mtDNA variants from both healthy individuals and

280 patients. The frequencies of the variants are calculated from human mitogenomes retrieved from

281 the GenBank. Therefore, users can retrieve information about the loci, the nucleotide change, the

282 codon position and the number, among others, and download the most important data in different

283 file formats.

284 Mitomap contains the Mitomaster analysis tool, currently providing the Application

285 Programming Interface for it. The main function of this tool is to allow the identification of

286 polymorphic positions, the calculation of variant statistics and the assignment of haplogroups to

287 complete or partial mitogenomes. Such query might be performed by recurring to mtDNA

288 sequences, to GenBank identifiers or to single nucleotide variants (85).

289 Another database for human mtDNA is the EDNAP Mitochondrial DNA Population Database

290 (EMPOP, www.empop.org) (86). In its early stages, EMPOP was designed and envisioned to

291 serve as a reference population database, specifically to be used in the evaluation of the mtDNA

292 evidence around the world, aiming to provide the highest quality mtDNA data. The architecture

293 of this online database and its analysis tools, which are also provided via the website, have

294 evolved over the last few years, even though the main emphasis of the EMPOP database remains

295 to be mtDNA data quality. Therefore, and as a direct consequence, EMPOP not only serves as a

296 reference population database, but also as a quality-control tool for scientists in forensic genetics,

297 as well as in other disciplines. Finally, and even though there is a significant number of high-

298 quality reference population databases for forensic comparisons, EMPOP is the most

299 comprehensive resource, especially from the standpoint of the populations that are represented in

300 such database (4).

301 EMPOP uses SAM, a string-based search algorithm that converts query and database sequences

302 into alignment-free nucleotide strings and thus guarantees that a haplotype is found in a database

303 query regardless of its alignment. SAM-E, an updated version of SAM that considers block

304 InDels as phylogenetic events, is used currently. At EMPOP, the tool haplogroup browser

305 represents all the established Phylotree haplogroups in convenient searchable format and

306 provides the number of EMPOP sequences assigned to the respective haplogroups by estimating

307 mitochondrial DNA haplogroups using a maximum likelihood approach EMMA (87). For

308 multiple possible haplogroups, most recent common ancestor (MRCA) haplogroups are

309 provided.

310 In order to facilitate a better use of known mtDNA variation, van Oven & Kayser, in 2008, have

311 constructed an updated comprehensive phylogeny of global human mtDNA variation -

312 PhyloTree -, based on both coding and control region mutations (78). The complete mtDNA tree

313 includes previously published as well as newly identified haplogroups, is continuously and

314 regularly updated, and is available online at http://www.phylotree.org. In figure 2 we present the

315 basic structure of the PhyloTree, which is divided into 25 subtrees accessible through

316 http://www.phylotree.org. At EMPOP the geographical haplogroup patterns are provided via

317 maps to visualize and better understand their geographical distribution (Figure 3).

318 From another perspective, ethical and legal problems may arise in the implementation of mtDNA

319 databases. The informative potential which the analysis of mtDNA entails can generate privacy

320 questions (88,89). Mitochondrial diseases affect between 1 in 4,000 and 1 in 5,000 people. In

321 most people, primary mitochondrial disease is a genetic condition that can be inherited.

322 Information about the mitochondrial genome composition may therefore enable the identification

323 of the current or future state of health of an individual. For this reason, the analysis of mtDNA

324 must be carried out only on non-coding regions, which have not been associated with any kind of

325 disease or phenotypical information.

326

327 Mitochondrial DNA in Medico-Legal Human Identification

328 At this section we present some selected published cases of human identification with mtDNA.

329 Table 1 summarizes the selected published cases.

330 In 1991, Stoneking and collaborators presented the first report of successful application of the

331 mtDNA typing to a case that involved the individual identification of skeletal remains (90). This

332 was the case of a 3-year-old child disappeared from her parents’ house in October of 1984. In

333 March of 1986, the skeletal remains of a human child were found in the desert, 2 miles away

334 from the parents’ residence. Using hybridization with 23 sequence-specific oligonucleotide

335 probes (SSO) targeting nine regions of HV1 and HV2 on the control region, they found that the

336 skeletal sample and the mother shared the same mtDNA types, corroborating that those skeletal

337 remains were of the missing child. Moreover, they anticipated that the mtDNA typing would be

338 valuable not only in linking biological remains to missing individuals, but also in the analysis of

339 material in sexual assault cases.

340 In July of 1990, the body of a female, in a quite advanced state of decomposition, was discovered

341 in an open field. Despite being impossible to identify the remains by analyzing the individual’s

342 clothes and fingerprints, her dentition was consistent with old dental records of a missing person

343 from the same region. Some fragments of the heel bone and fibula, plus samples of the hair and

344 skin, were provided for the DNA analysis, as well as a blood sample from a putative sister of the

345 deceased. In 1992, Sullivan and collaborators attempted the identification of the highly

346 decomposed remains of the corpse, amplifying and directly sequencing 2 hypervariable segments

347 within HV1 and HV2 in the mtDNA (91). No statistical value was given to the evidence, since

348 no database of the British population sequences were available at that time. Still, no differences

349 were found between both sequences, the blood of the putative sister and the bone of the corpse,

350 indicating they were sisters.

351 Perhaps the most well-known lineage study using mtDNA sequencing is related to the

352 identification of Tsar Nicholas II’s bones. Gill and collaborators, in 1994 (22), and Ivanov and

353 collaborators, in 1996 (23), compared the sequences of HV1 and HV2 fragments of the mtDNA

354 obtained from the putative bones of the Tsar with those of Tsar living maternal relatives,

355 Countess Xenia Cheremeteff-Sfiri and the Duke of Fife. It was found that the sequences were

356 very similar, corroborating the hypothesis that the bone remains were of Tsar Nicholas II.

357 In a distinct scenario, Deng et al. (92) used direct sequencing of the HV1 and HV2 fragments of

358 the mtDNA control region to identify Tsunami victims in Thailand in 2004. This tsunami killed

359 nearly 5,400 people in Southern Thailand, including foreign tourists and local residents. They

360 succeeded in obtaining fully informative results for mtDNA markers (HV1 and HV2) from 258

361 tooth samples with a success rate of 51% (258/507).

362 More recently, in 2010, Ríos and collaborators (93) used direct sequencing of the HV1 and HV2

363 fragments of the mtDNA control region to identify human skeletal remains that were exhumed

364 from a mass grave from the Spanish Civil War (1936-1939). There was a match between the

365 mtDNA profiles of the biologically youngest skeleton and the sister of the youngest person that

366 was presumptively known to be buried in the grave, allowing the identification of that person.

367 Also in 2010, Piccinini and collaborators (94) attempted to identify the remains of a famous

368 World War One Italian soldier that was killed in a battle along the Italian front in 1915. Like

369 previous studies, they used the direct sequencing of the HV1 and HV2 fragments of the mtDNA

370 control region to define single mtDNA haplotypes. The availability of the offspring maternal

371 lineage allowed the mtDNA analysis, which presented a clear exclusion scenario: the remains did

372 not belong to the supposed war hero.

373 In 2012, a skeleton was excavated at the site of the Grey Friars friary, in Leicester, which is the

374 last-known resting place of King Richard III (95). To determine if the remains belonged to King

375 Richard III, the HV1, HV2 and HV3 regions of the mtDNA of the skeletal remains and of the

376 living relatives of King Richard III were sequenced and compared. There was a perfect match

377 between the sequences indicating that the remains belong to King Richard III.

378 The communist period in Poland during 1944-1956 resulted in the death of more than 50,000

379 people, who were buried in secret. One mass grave was found at the cemetery Powazki Military,

380 in Warsaw, Poland. In 2016, Ossowski and collaborators (96) identified 50 victims, specifically

381 by using autosomal, Y-STR and direct sequencing of the HV1 and HV2 fragments of the

382 mtDNA control region.

383 In 2016, among the first studies on human identification with mtDNA using massive paralell

384 sequencing, Park and collaborators proposed a protocol that includes the study of ten regions of

385 mtDNA for the identification of historical human remains with forensic genetic markers (97).

386 They studied a 140-year-old human skeletal remains discovered at a historical site in Deadwood,

387 South Dakota, United States. The remains were in an unmarked grave and there were no records

388 available regarding the identity of the individual. The mtDNA profiles of the unidentified

389 skeletal remains obtained with their method were consistent with H1 haplogroup. This

390 haplogroup is the most common in Western Europe. The ancestry-informative nuclear SNPs also

391 studied in this case indicated a European background. These genetic results are consistent with

392 the findings of previous anthropological report which determined that the Deadwood

393 unidentified skeletal remains belong to a male of European ancestry.

394 In 2017, the victims’ remains from the World Trade Center terrorism act, which occurred in

395 September 11 of 2001, were still being identified by using the mtDNA sequencing technology,

396 among other techniques, with protocols and guidelines as recommended by the International

397 Society for Forensic Genetics (1,2,4,29,98).

398

399 Conclusions

400 Over the last 25 years, mtDNA typing has been widely used around the world to solve several

401 human identifications related issues in violent crimes, lesser crimes, acts of terrorism, mass

402 disasters and missing persons’ cases.

403 Forensic DNA methods are constantly questioned in terms of their admissibility for several years

404 now, and we foresee that this scenario is likely to continue in the future. Some of the most well-

405 known challenges to the mtDNA analysis are focused on the issue of the heteroplasmy as well as

406 on recombination and paternal leakage.

407 Over the last decades progress in mtDNA typing was overwhelming, going from the examination

408 of small fragment in a matter of days to sequencing multiple mtDNA genomes in a couple of

409 hours using a point of care sequencing machine.

410 An individual’s mtDNA genome can tell us much about his/her ancestors. Even though many

411 would readily accept that there are good reasons for researchers to determine information about

412 an unknown suspect’s potential ancestral background, many still might find the potential to

413 determine genetic dispositions to certain disorders as being unacceptable. Hence, new

414 technologies must be wisely used and for the reasons that they are intended, considering their

415 specific focus and contribution within the field of forensic and medico-legal human

416 identification.

417

418 Acknowledgements

419 None to report.420

421

422 References

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659 human mtDNA control region sequences detected by enzymatic amplification and

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662 automated sequencing of mitochondrial DNA. Int J Legal Med. 1992;105(2):83–6.

663 92. Deng YJ, Li YZ, Yu XG, Li L, Wu DY, Zhou J, et al. Preliminary DNA identification for

664 the tsunami victims in Thailand. Genomics, Proteomics Bioinforma. 2005;3(3):143–57.

665 93. Ríos L, García-Rubio A, Martínez B, Alonso A, Puente J. Identification process in mass

666 graves from the Spanish Civil War II. Forensic Sci Int. 2010;219(1–3).

667 94. Piccinini A, Coco S, Parson W, Cattaneo C, Gaudio D, Barbazza R, et al. World war one

668 Italian and Austrian soldier identification project: DNA results of the first case. Forensic

669 Sci Int Genet. 2010;4(5):329–33.

670 95. King TE, Fortes GG, Balaresque P, Thomas MG, Balding D, Delser PM, et al.

671 Identification of the remains of King Richard III. Nat Commun. 2014;5:1–8.

672 96. Ossowski A, Diepenbroek M, Kupiec T, Bykowska-Witowska M, Zielińska G,

673 Dembińska T, et al. Genetic Identification of Communist Crimes’ Victims (1944–1956)

674 Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military

675 Cemetery in Warsaw, Poland. J Forensic Sci. 2016;61(6):1450–5.

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677 comprehensive forensic genetic marker analyses for accurate human remains

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679 9).

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682

683

684

685 Figure and Table legends

686 Figure 1 – The human mitochondrial DNA genome with genes and control regions labeled. Adapted from

687 Shokolenko et al., 2014 (11)

688

689 Figure 2 - Phylogenetic tree of global human mitochondrial DNA variation. mtDNA tree Build 17 (18

690 Feb 2016). Available at http://www.phylotree.org/tree/index.htm. mtDNA-MRCA- Most recent common

691 ancestor of mtDNA.

692

693 Figure 3 - Representation of the geographical origin of mtDNA haplogroups and main mutations that are

694 at the origin of each haplogroup. Adapted from Kivisild et al., 2015 (83).

695

696 Table 1 - Selected published cases of human identification with mtDNA.

697

698 Table 2 - Guidelines of the DNA Commission of the International Society of Forensic Genetics,

699 2014

Table 1(on next page)

Selected cases of human identification with mtDNA

Selected published cases of human identification with mtDNA

1 Table 1 - Selected published cases of human identification with mtDNA

Reference/Year Studied samples

mtDNA studied regions

Used methodologies

Reference samples Results

Stoneking M, Hedgecock D, Higuchi RG, Vigilant L, Erlich HA. Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes. Am J Hum Genet. 1991;48(2):370–82.

Skeletal remains of a human child, found in 1986

HVI, HVII PCR for amplification

Hybridization with oligonucleotide probes for sequence determination

Parents of a 3-year-old child disappeared from home in 1984

Identical mtDNA sequence in skeletal remains and sample of the 3-year-old child mother

Positive ID

Sullivan KM, Hopgood R, Gill P. Identification of human remains by amplification and automated sequencing of mitochondrial DNA. Int J Legal Med. 1992;105(2):83–6.

Body of a female, in an advanced state of decomposition discovered in 1990

HVI, HVII PCR for amplification

Sanger sequencing

Blood sample from a sister of a deceased female at the same region

No differences were observed between the corpse and blood from the putative sister

Positive ID

Gill P, Ivanov PL, Kimpton C, Piercy R, Benson N, Tully G, et al. Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 1994;6(2):130–5.

Nine skeletons found in a grave in Ekaterinburg, Russia, 1991

HVI, HVII PCR for amplification

Sanger sequencing

Blood sample from Gt. Gt. Grandson of Louise of Hesse-Cassel and from Gt. Gt. Gt. Granddaughter of Louise of Hesse-Cassel

Exact sequence between putative Tsarina Alexandra and putative three children. Exact mtDNA results between putative Tsar Nicholas II and two living maternal relatives of the Tsar

Ivanov PL, Wadhams MJ, Roby RK, Holland MM WV& PT. Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet. 1996;(12):417–20.

Skeleton of putative Tsar Nicholas II

HVI, HVII PCR for amplification

Sanger sequencing

Skeleton of Grand Duke of Russia Georgij Romanov (Tsar’s brother)Blood sample from Countess Xenia Cheremeteff-Sfiri (maternal Tsar’s relative)

Establishment of the authenticity of the remains of Tsar Nicholas II

Deng YJ, Li YZ, Yu XG, Li L, Wu DY, Zhou J, et al. Preliminary DNA identification for the tsunami victims in Thailand. Genomics, Proteomics Bioinforma. 2005;3(3):143–57.

258 tooth samples from killed people at the 2004 Southeast Asia Thailand

HVI, HVII PCR for amplification

Sanger sequencing

200 relatives of the tsunami victims

200 tsunami victims have been identified, including both Thai nationals and foreign tourists from several nations

Tsunami

Ríos L, García-Rubio A, Martínez B, Alonso A, Puente J. Identification process in mass graves from the Spanish Civil War II. Forensic Sci Int. 2010;219(1–3).

Skeletal remains exhumed from a mass grave from the Spanish Civil War (1936–1939)

HVI, HVII PCR for amplification

Sanger sequencing

Sister of the youngest person presumptively known to be buried in the grave

Match between mtDNA profiles of the biologically youngest skeleton and the sister of the youngest person presumptively known to be buried in the grave

Piccinini A, Coco S, Parson W, Cattaneo C, Gaudio D, Barbazza R, et al. World war one Italian and Austrian soldier identification project: DNA results of the first case. Forensic Sci Int Genet. 2010;4(5):329–33.

Remains of missing soldiers occasionally found during excavations

HVI, HVII PCR for amplification

Sanger sequencing

Offspring of the italian soldier Libero Zugni Tauro

Both mtDNA and Y-STR data showed clear exclusion scenariosbetween the human remains and the reference samples

King TE, Fortes GG, Balaresque P, Thomas MG, Balding D, Delser PM, et al. Identification of the remains of King Richard III. Nat Commun. 2014;5:1–8.

Skeleton excavated at the presumed site of the Grey Friars friary in Leicester, 2012

Whole mitochondrial genome

PCR for amplification

Massive parallel sequencing

Saliva samples of the modern relatives of Richard III

Positive mtDNA match between the only known female-line of Richard III and studied modern relatives of Richard III

Ossowski A, Diepenbroek M, Kupiec T, Bykowska-Witowska M, Zielińska G, Dembińska T, et al. Genetic Identification of Communist Crimes’ Victims (1944–1956) Based on the Analysis of One of Many Mass Graves Discovered on the Powazki Military Cemetery in Warsaw, Poland. J Forensic Sci. 2016;61(6):1450–5.

Remains of eight people buried in one of many mass graves, which were found at the cemetery Powazzki Military in Warsaw, Poland

HVI, HVII PCR for amplification

Sanger sequencing

Reference material was collected from the closest living relatives of Communist Crimes’ Victims (1944–1956)

Positive mtDNA match between 6 putative victims and 6 living relatives

2

Table 2(on next page)

Table 2

Guidelines of the DNA Commission of the International Society of Forensic Genetics, 2014

1 Table 2 - Guidelines of the DNA Commission of the International Society of Forensic Genetics, 2014

Addressement Recommendation Statement

Recommendation #1

Good laboratory practice and specific protocols for work with mtDNA must be followed in accordance with previous guidelines

Recommendation #2

Negative and positive controls as well as extraction reagent blanks must be carried through the entire laboratory process

Recommendation #3

Reported consensus sequences must be based on redundant sequence information, using forward and reverse sequencing reactions whenever practical

Recommendation #4

Manual transcription of data should be avoided and independent confirmation of consensus haplotypes by two scientists must be performed

General recommendations/ good laboratory practice

Recommendation #5

Laboratories using mtDNA typing in forensic casework shall participate regularly in suitable proficiency testing programs

Targeted region, amplification and sequencing ranges

Recommendation #6

In population genetic studies for forensic databasing purposes, the entire mitochondrial DNA control region should be sequenced.

Reference sequence Recommendation #7

MtDNA sequences should be aligned and reported relative to the revised Cambridge Reference Sequence (rCRS, NC001807), and should include the interpretation range (excluding primer sequence information)

Recommendation #8

IUPAC conventions using capital letters shall be used to describe differences to the rCRS and (point heteroplasmic) mixtures. Lower case letters should be used to indicate mixtures between deleted and non-deleted (inserted and non-inserted) bases. N-designations should only be used when all four bases are observed at a single position (or if no base call can be made at a given position). For the representation of deletions, ‘‘DEL’’, ‘‘del’’ or ‘‘S’’ shall be used

Alignment and notation

Recommendation #9

The alignment and notation of mtDNA sequences should be performed in agreement with the mitochondrial phylogeny (established patterns of mutations). Tools to assist with the notation of mtDNA sequences are available at http:// empop.org/

Recommendation #10

In forensic casework, laboratories must establish their own interpretation and reporting guidelines for observed length and point heteroplasmy. The evaluation of heteroplasmy depends on the limitations of the technology and the quality of the sequencing reactions as well as the experience of the laboratory. Differences in both PHP and LHP do not constitute evidence for excluding two otherwise identical haplotypes as deriving from the same source or same maternal lineage

Heteroplasmy

Recommendation #11

For population database samples, length heteroplasmy in homopolymeric sequence stretches should be interpreted by calling the dominant variant, which can be determined by identifying the position with the highest representation of a non-repetitive peak downstream of the affected stretch

Haplogrouping of mtDNA sequences Recommendation #12

MtDNA population data should be subjected to analytical software tools that facilitate phylogenetic checks for data quality control. A comprehensive suite of QC tools is provided by EMPOP

Recommendation #13

The entire database of available sequences should be searched with respect to the sequencing (interpretation) range to avoid biased query results

Recommendation #14

Laboratories must be able to justify the choice of database(s) and statistical approach used in reporting

Recommendation #15

Laboratories must establish statistical guidelines for use in reporting an mtDNA match between two samples

Databases and database searches

Recommendation #16

Highly variable positions such as length variants in homopolymeric stretches should be disregarded from searches for determining frequency estimates. Heteroplasmic calls should be queried in a manner that

does not exclude any of the heteroplasmic variants

2

3

Figure 1(on next page)

Genes and control regions labeled of the human mitochondrial DNA genome

The human mitochondrial DNA genome with genes and control regions labeled. Adapted from

Shokolenko et al., 2014 (11)

Figure 2(on next page)

Phylogenetic tree of global human mitochondrial DNA variation

Phylogenetic tree of global human mitochondrial DNA variation. mtDNA tree Build 17 (18 Feb 2016).

Available at http://www.phylotree.org/tree/index.htm . mtDNA-MRCA- Most recent common ancestor of

mtDNA.

mtDNA-MRCA

Figure 3(on next page)

Geographical origin of mtDNA haplogroups and main mutations that are at the origin of

each haplogroup

Representation of the geographical origin of mtDNA haplogroups and main mutations that are at the origin

of each haplogroup. Adapted from Kivisild et al., 2015 (83) .