Keywords

Mitochondria: Structure, Physiology, and Functions

Mitochondria are ovoid or sausage shaped double-membranous cytoplasmic cell organelle ranging 0.5–1.0 μM in diameter, operative towards the function of energy-transduction, imperative for various cellular metabolism. Its double-membranous wall comprises of an outer semipermeable layer that isolates the organelle from cytosol and an inner impermeable layer. The inner layer can be further bifurcated into an outer side facing and running parallel to outer membrane and an inner side which is invaginated into several folds called cristae that outlines the matrix (M-side) of mitochondria. Both the layers are separated by means of intermembrane space of width about 60–80Å, also known as the C-side. At molecular level, both the layers have tail-to-tail arrangements of phospholipids bilayer along with embedded protein molecules. This bilayer assembly exists in liquid-crystalline state with one fraction of phospholipid molecule of hydrophobic (water-repellent) nature and another fraction being hydrophilic (water-attracting) nature (Demirel and Gerbaud 2019). Inner side of cristae enclosing the matrix is known to be the house of various enzymatic reaction and is embedded with majority of the electron transport chain (ETC) complexes, enzymatic components of citric acid cycle, and ATP synthase dimmers (McCarron et al. 2013; Glancy et al. 2020). Mitochondrial genome along with essential components required for its replication, transcription, and translation are present within the matrix region (Shoubridge 2004).

Functionally, mitochondria are involved in myriad of regulatory mechanisms that differs from organ-to-organ. Primarily the high pH of matrix ranging around 8 provides a transmembrane electrochemical gradient that helps in production of cellular energy in the form of ATP (adenosine triphosphate) (Kühlbrandt 2015). The crista lumens are the sites of electron carriers protein cytochrome c and ROS (reactive oxygen species) production, which are essential components of electron transport chain and are also involved in cell apoptosis (Plecitá-Hlavatá and Ježek 2016; Pearce et al. 2017). Mitochondria are also engaged in protein import and calcium influx for several mechanisms, such as tricarboxylic acid cycle, urea cycle, oxidative phosphorylation , and biosynthesis of heme, iron-sulfur cluster, cholesterol, glucose, etc. (Shoubridge 2004; Pearce et al. 2017; Spinelli and Haigis 2018; Demirel and Gerbaud 2019).

Mitochondrial Genome

Mitochondria possess a form of autonomously replicating genome originated from (eu)bacterial domain of life. The mitochondrial genomes (mt-genome) are structurally distinct from that of nuclear genome and are present in double-stranded circular conformity. Human mitochondrial DNA (mtDNA) is the covalently bonded double-stranded circular structure of 16,569 bp in length. Number of copies of mtDNA per cell ranges from 100 to 1000, depending on the cell-specific functions. Human mitochondrial proteome comprises of ~1500 proteins, majority of which are encoded by nuclear DNA while mt-genome expresses a subset of 13 polypeptides component of respiration chain, translated from 11 mRNAs along with 22 tRNAs, and 2rRNAs (Gray et al. 2001; Pearce et al. 2017) (Table 1). Based upon the G-C content gradient, the coding regions of mitochondria are classified as G nucleotide-rich heavy strand (H-strand) and C nucleotide-rich light strand (L-strand). Transcriptional promoters of these strands are located in the noncoding region of mtDNA, known as the displacement loop (D-loop) (Anderson et al. 1981; Taanman 1999). Graphical representation of mitochondrial genome is demonstrated in Fig. 1. D-loop or the control region is a metabolically active region of length 1.1 kb and is unsusceptible to the formation of polypeptide chain. D-loop consists of highly polymorphic regions as compared to the rest of the genome, known as the hypervariable (HV) regions 1 and 2 (Horai and Hayasaka 1990).

Table 1 Information of mt-genome, its encoding regions for 13 proteins, 2 rRNAs, and 22 tRNAs
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Fig. 1
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Graphical representation of human mitochondrial genome

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Hypervariable region 1 (HV1) sequencing from nucleotide 16024–16365 and hypervariable region 2 (HV2) sequencing from nucleotide 73–340 are the regions of significance in terms of forensic interests. Relatively small stretch, highly polymorphic nature, and inter-person discriminatory power of these hypervariable regions add to their utility in forensic caseworks. Interpopulation variability in terms of mitochondrial genome has been widely reported in evolutionary and phylogenetic studies. In addition to these, hypervariable region 3 (HV3) with nucleotide 438–576 along with complete sequence of D-loop are found to be the sites of evolutionary mutational hotspots and are highly significant in determination of haplotype and ancestral origin of an individual (Hwa et al. 2012). Studies have revealed escalation of mtDNA mutation to a rate of 10–17 folds as compared to the nuclear DNA induced by exposure to oxidative damages as a result of chronic exposure to mutagenic products such as ROS formed from oxidative phosphorylation, nonfunctioning of defensive protein molecules, and incompetent genomic repair mechanisms. Accumulation of such oxidative damages leads to mitochondrial dysfunction or oxidative stress and is found to be associated with onset of senescence and ageing-related mechanisms (Reid 2018).

Inheritance of Mitochondrial Genome

In general terms, an individual keeps up only a single type of mitochondrial DNA (maternally inherited) regardless of the presence of several folds of mitochondrial DNA copies present in each cell, maintaining the condition of homoplasmy or identical genotype within entire mitochondrial genome (Fig. 2). Both the features of uniparental inheritance as well as homoplasmy are responsible for pertinence of mtDNA in forensic caseworks. However, the condition of heteroplasmy can raise complication in data interpretation. Heteroplasmy is the condition of existence of more than one genotype of mtDNA within an individual or tissue. This may arise due to pathogenic mutations resulting in transmission of both wild type as well as mutant type of genome. Such heteroplasmic genome may carry DNA molecules of varying length (length heteroplasmy) or different nucleotide sequence (point heteroplasmy) (Reid 2018).

Fig. 2
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Hypothetical pedigree illustrating the maternal inheritance of mtDNA. Squares represents male and circles represent females. Different colors represent unique type of mtDNA

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Conventionally, the transmission of mitochondrial DNA was supposed to be carried out strictly along the matrilineal descent without undergoing any chromosomal recombination. However, events of biparental inheritance have also been documented in recent years (Schwartz and Vissing 2002; Luo et al. 2018) posing a challenge to the concept of central dogma and uniparental inheritance based premises of evolutionary and phylogenetic studies in forensic science. According to some theories, paternal mitochondria borne on sperms penetrate the cytoplasm of oocyte after fertilization and transiently coexists within the zygote along with bulk of maternal mitochondria. Later, these paternal mitochondria are eliminated during cell divisions by some mechanisms and are never transmitted to the offspring. Several hypotheses including two major hypotheses of “simple dilution model” and “active degradation model” have been laid to justify the mechanism for elimination of paternal mitochondria (Sato and Sato 2013). However, high-depth sequencing of mtDNA extending to several million folds have deplored the hypotheses of simple dilution. Besides, autophagal lysis of paternal mitochondria has also been reported in C. elegans (Sato and Sato 2012).

Despite such eliminating mechanisms and lysosomal pathways, chances of paternal mtDNA transmission via paternal leakage persist. Theoretically, stockpiling of small degree of male fertility-impairing mutations might be responsible for such genetic acclimatization that would necessitate paternal leakage (Vaught and Dowling 2018). Existence of such mechanisms demands analysis of paternal mtDNA transmission in cases with sporadic mitochondrial mutations and occurrence of heteroplasmy.

Application of Mitochondrial DNA Analysis in Forensic Caseworks

Mitochondrial DNA analysis has been employed to several forensic cases related to historical individual identification, genealogy, and phylogeny. Average mtDNA copy number ranging from hundreds to several thousands in somatic cells presents the possibility of extracting sufficient amount of DNA from degraded or compromised samples encountered in forensic caseworks where nuclear DNA cannot be recovered in sufficient quantities to be typed. mtDNA analysis is considered to be useful in a range of biological materials such as hair, bones, teeth, whose nuclear DNA quantity may be low depending upon the circumstances. Manner of mtDNA inheritance is another beneficial characteristic useful in individual identification where no direct relative is available to use as reference sample. A distant relative, matrilineally related person to be identified can be used as reference to establish maternal link between the two for possible identification.

Human Identification from Degraded Compromised Samples

Skeletal remains are often the only source of DNA for human identification in cases of mass disasters and missing person identification. Human bodies found in such cases are generally exposed to harsh environments with high temperature and humidity, traumatic events, and long range of time span. Bones and teeth are the only source of DNA available after enduring such severe conditions. However, exposure to ultraviolet rays, humidity, low pH, and enzymatic reaction results in degradation of nuclear DNA in such samples. Linear structure of nuDNA and the chromatin structure may have a deteriorating impact on nuDNA but have no such relation with mtDNA (Foran 2006). High copy number per cell and ability of mtDNA to resist degradation are the grounds on which mitochondrial DNA profiling yields better results compared to nuclear DNA profiling analysis in case of hard tissue. Similarly, genomic content in hairs is comparatively low due to catabolic breakdown of nucleic acid in the course of keratinization process. In hair samples, retrieval of mtDNA is much greater due to its high copy number than nuclear DNA in terms of quantity and quality, even from the shafts of telogen or rootless hairs (Melton et al. 2012).

Skeletal remains of American soldiers excavated from tombs of Vietnam and returned to US government after Vietnam War in 1984 were identified by the application of mtDNA analysis where nuclear DNA analysis, HLA-DQ alpha, and VNTR analysis remain unsuccessful in positive identification (Holland et al. 1993).

mtDNA proved to be an efficient evidence in the trial of Tennessee Murder Case (1996) in a US courtroom for convicting 27-year-old Paul Ware with the charges of rape and murder. This was the first case of utilizing mtDNA as evidence in any courtroom. No exchange of biological fluids or evidence were found on the body of victim and suspect Ware, and only the circumstantial evidences presented against Ware were insignificant. However, during the autopsy, a red hair was found inside the throat of the victim. On reanalysis of the crime scene, several red hairs were found in the bed. One-one hair, each from the throat and bed were processed for mtDNA analysis. As a reference, mtDNA was also extracted from saliva of the suspect and blood of the victim. On comparison of the questioned sample with reference samples, both the questioned samples were found to have same source and their mtDNA sequence had an exact match with the suspect. The sequence from the samples did not match with the victim’s source (Davis 1998).

A human identification case of highly decomposed body in Rio de Janeiro was resolved by implementation of MPS techniques on mtDNA fragments. The body was found in the seacoast of Rio de Janeiro in 2015. The investigation brought forth the alleged mother, but the advancing stage of decomposition of the body raised complication on processing routine STR profiling for its identification. Samples for DNA testing (both STR profiling and mtDNA sequencing) were acquired from the bone fragments of the body and buccal swabs of the alleged mother. STR analysis of bone samples generated partial profile due to sample degradation resulting in inconclusive results. However, sequence of mtDNA genome resulted in full profile of haplotype. Final haplotype generated from the bone of the body and buccal swab of alleged mother is represented in Table 2. Sequences generated from both the samples were fully concordant and thus matrilineage relation between the two cannot be excluded. As per the phylogeographic analysis based on online softwares EMPOP and HAPLOGREP, haplogroup of son/mother belonged to L3b1a (Bottino et al. 2021).

Table 2 Haplotype generated from the casework samples
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Age Estimation Using mtDNA Analysis

Several works over last two decades suggest that accumulation of mtDNA degradation and mutation contribute to physiological mechanisms that result in ageing and age-related diseases owing to enhanced oxidative damages caused by ROS generation as well as deteriorated repair mechanisms and characterized lesser replication extent in comparison with nuclear DNA (Fig. 3). Age-dependent mutations are frequently observed in postmitotic tissues that have high demands of energy. Clinical manifestations related with neurological, cardiac, hepatic, osteologic, and renal dysfunctions are common repercussion resulting from age-dependent mtDNA mutations. Accumulation of several mutations including deletion, duplication, and point mutation has been reported to have increased frequency with increasing age. Among these mutations, 4977-bp deletion is the most common age-related mutation in mitochondrial genome and is employed for age estimation at time of death. Correlation between 4977 deletion and ageing has been successfully demonstrated in several studies and is found effective in discriminating elder person and young person in cases of mass disasters (Meissner et al. 1999; Trifunovic 2006). Besides this, 3715- and 6278-bp deletions in skin samples of ageing people have been reported to possess similar frequency as that of 4977-bp deletion (Eshaghian et al. 2006). Point mutation at 186-bp transiting from A to G at significant levels has been identified in skeletal tissue of ageing individuals. However, coexistence of mutant type (186G) along with wild type (186A) resulting in heteroplasmy possesses difficulty in deciphering mtDNA damages and their extent.

Fig. 3
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Conceptual representation of various mechanisms of mtDNA mutation and its relation with the process of senescence or ageing

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Genealogy Tracing

Maternal inheritance of genetic polymorphism across mitochondrial sequence without undergoing any kind of recombination accounts for the accumulation of these variants along the diverging maternal lineages in forms of phylogenetically related haplotypes. The group of such haplotypes that shares same genetic variants descendant from a common ancestor is known as haplogroups. Gradual mutations in the mitochondrial genome accumulated over time has led to the clustering of individuals into discrete haplogroups. Mitochondrial Eve (mt-Eve), also known as matrilineal most recent common ancestor (or mt-MRCA), is considered as the female biological ancestor of all humans inhabited in African subcontinent over 150,000–200,000 years before present (YBP). Mt-Eve diverged into seven major lineage specific groups – L0, L1, L2, L3, L4, L5, and L6 around 100,000 YBP. On facing harsh climatic changes and interstadial phases, a part of the population belonging to L3 group migrated from Africa to inhabitate other parts of the world resulting in generation of two macro-haplogroup – M defining the population that migrated to Asia and N went to Eurasia, Asia, and America. More than 30 subclades of haplogroup M is present in Asian continent that includes subclade A, B, C, D, G, and F. Haplogroup N served as the root for group R, that resulted in European haplogroups H, I, J, K, T, U, V, W, X around 45,000 YBP. HV, U, and JT are the major macro-haplogroups from European continent. Indian subcontinent and south-east Asian continents constitutes of M, N, and R. These subclades are further divided into many sub-haplogroups. At present, the global mtDNA haplogroup tree consists of more than 4000 discrete haplogroups (Mancuso et al. 2011; Chinnery and Gomez-Duran 2018).

MtDNA analysis for genealogical tracing was successfully employed to authenticate the identity of last Tsar Nicolas Romanov II and his family. Tsar Romanov II, his wife Tsarina Alexandra and their children Maria, Tatiana, Anastasia, Olga, and Alexei along with three servants and a doctor were killed during the Bolshevik Revolution of 1918. Nine sets of skeletal remains were excavated in 1991 in a mass grave with suspicion of them belonging to Romanov Family. Remains of Tsarina Alexandra and three children were identified by comparison of mtDNA sequence with the biological sources of a known maternal descendant – Prince Phillip, Duke of Edenburg. MtDNA sequence obtained from skeletal remains suspected to be that of Tsar matched with two of his maternal relatives with an uninterrupted maternal descendancy of Nicolas’s maternal grandmother – Duke of Fife and Princess Xenia Cheremeteff Sfiri except at L16169 showing heteroplasmic state of C/T. For obtaining a complete authenticity of the remains, mtDNA analysis was performed on the excavated remains of Nicolas’s brother – Duke of Russia Georgij Romanov (death in 1889). Comparison of mtDNA sequence from suspected Nicolas and his brother confirmed the presence of heteroplasmy (Gill et al. 1994). Remains of two children excavated in 2007, presumed to be of Tsar’s two children, Anastasia (female, 18–23 years old) and Alexei (male, 10–14 years old), were subjected to mtDNA sequencing and its comparison to the mtDNA sequence generated from the remains of Alexandra and her three daughters. Thus, mtDNA sequencing played a significant role in identification of Romanov Family and tracing maternal descendancy (Coble et al. 2009; Rogaev et al. 2009). Pedigree chart demonstrating the matrilineage phylogeny of Tsar Nicholas and Tsarina Alexandra is given in Fig. 4.

Fig. 4
figure 4

Pedigree demonstrating the maternal lineage of Tsar Nicolas and Tsarina Alexandra showing maternal relationships of Tsar with Duke of Fife, Princess Xenia Cheremeteff Sfiri, Georgij Romanov, and maternal relationships of Tsarina Alexandra and her children with Prince Phillip

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Mitochondrial DNA Analytical Methodologies in Forensic Caseworks

MtDNA analysis for any forensic casework, either involving human identification or genealogical assessment, requires processing of the questioned sample (Q) collected from the scene of crime along with the processing of a known sample (K) collected from the maternal relative and their comparison. Contamination-free environment is the major requirement of mtDNA analysis, as high copy number of mtDNA than nuDNA within the sample makes it more susceptible to external contaminations. Figure 5 describes the complete procedure of mtDNA data generation, comparison, and interpretation. Procedure follows general step of DNA extraction, its quantitation, amplification of control region, sequencing of amplified product, and data interpretation.

Fig. 5
figure 5

Analytical process of evaluating mtDNA sample. Q represents the questioned sample that is collected from the crime scene and K represents the known or reference sample collected from the maternal relative of suspected individual

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Data Interpretation

Cambridge Reference Sequence(s)

Complete sequencing of mtDNA was determined for the first time in 1981 in MRC Laboratory, Cambridge, England (Anderson et al. 1981). The proposed sequence, known as “Anderson” sequence or Cambridge Reference Sequence (CRS) (GenBank accession: M63933), was considered as a reference sequence for comparison and interpretation of newly analyzed sequence over the years. Reports of any analyzed mtDNA sequence were generated in term of variation in the sequence as compared to the light strand (L-strand) of original Anderson sequence. Later in 1999, the source of DNA used for generating CRS was re-sequenced by Andrews et al. (1999) and came up with 18 rectified or corrected nucleotide positions including loss of one cytosine base at position 3106. The corrected sequence was designated as revised CRS (or rCRS) and acknowledged as new standard for comparison purposes. Differences observed between CRS and rCRS are listed in Table 3. Sequence comprised in rCRS belongs to an individual from European descent belonging to haplogroup H2a2 (Bandelt et al. 2014).

Table 3 Nucleotide difference between original CRS and rCRS sequence
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Several web-based bioinformatics software are available that provide a platform for management and analysis of mtDNA sequence conveniently. “mtDNAmanager” is one such interface that allows computational analysis of mtDNA control regions and identify its probable haplotype (Lee et al. 2008). BLAST or basic local alignment search tool helps in comparison and identification of mtDNA sequence and thereby is applicable for determining evolutionary sequence between the sequences. SeqScape, DNAStar’sSeqMan Pro, Variant Reporter are some of the popular web-based interfaces for mtDNA sequence analysis. GeneMarker HTS, Converge Forensic Analysis Software, mitoAnalyzer, mtDNA-Server facilitates mtDNA analysis based on next-generation sequencing (NGS) platforms with the ability to identify minor heteroplasmic variants, phylogenetically correct SNP and INDEL base calls, and correct haplogroup prediction.

Nomenclature of mtDNA Sequence

Reporting of mtDNA sequence demands a minimum data format in the form of difference with reference to rCRS. Any observed difference is noted in the format of listing nucleotide position followed by the base present at that point. Presence of ambiguous base is marked by N. Heteroplasmy at any nucleotide is coded in the form of X/Y where X is the wild type base and Y is the mutant type base. In case of insertion of a base in a sequence as against rCRS, the nucleotide position is denoted by noting the site followed by a point and number of insertions followed by the base. Deletions are coded as “-” or “D” or “d” or “del” after the nucleotide position.

mtDNA Sequencing Result Interpretation

Comparison of questioned (Q) sample with known (K) samples for all 610 nucleotides (16024–16365, 73–340) is essential for conclusion of the results. On account of Q-K sequence comparison, the interpretation of mtDNA sequencing result can be categorized into three classes – exclusion, failure to exclusion, and inconclusive.

  • Exclusion: Two or more nucleotide difference between questioned and known sample with no evidence of heteroplasmy.

  • Failure to exclusion: (i) Fully concordant Q-K pair, (ii) one heteroplasmic base in both the samples at the same position, (iii) one heteroplasmic base in one sample, not observed in the other sample, with a common nucleotide present in both samples, (iv) two heteroplasmic bases at the same position in both samples, and (v) one heteroplasmic base at the same position, one base showing heteroplasmy in one sample but not in the other, with a common nucleotide in each.

  • Inconclusive: Difference of one nucleotide between Q-K pair, with no signs of heteroplasmy and one heteroplasmic base at the same position, one different base at another position with no evidence of heteroplasmy.

Human Mitochondrial DNA Sequence Database

DNA database of any population is essential for estimation of expected haplotype frequency of the given samples. Collection of high-quality genetic information from a large number of maternally unrelated individuals offers reliable frequency estimation for a random match. Several databases of mtDNA sequences have been compiled globally that focuses on sequence information as well as phylogenetic information. Some of the popular databases include:

  • MITOMAP – MITOMAP is a human mitochondrial genome database developed in 1996 that focuses on collecting information related with mtDNA polymorphisms and mutation in humans. It involves real-time sequencing method (Kogelnik et al. 1996).

  • EMPOP – EDNAP Mitochondrial DNA Population Database (EMPOP) is a collaborative exercise of European DNA Profiling (EDNAP) body operational since 2004. The database aims at determining the uniformity of mtDNA analysis among different laboratories and identifying the possible source of errors (Parson and Dür 2007).

  • HmtDB – It is an open-source genomic resource developed in 2005 that comprise of human mtDNA sequences with information related with population variability. The database is effective for population genetics as well as mitochondrially generated diseases (Attimonelli et al. 2005).

  • MitoVariome – The database was created in 2009 for aiding the works on human evolution and variation. It is a cost-free accessible database in textual and graphical format beneficial for research works related with forensic science, tumors, ageing, and degenerative diseases (Lee et al. 2009).

  • AmtDB – First database of ancient mitochondrial DNA reported in 2019. It is beneficial for tracking human past demographic events (Ehler et al. 2019).

EMPOP: Innovative and Forensically Significant Human mtDNA Database

In 2004, European DNA Profiling (EDNAP) group collaborative exercised on the development of online database consisting of mtDNA sequence data obtained from various populations known as EDNAP Mitochondrial DNA Population Database (EMPOP). The database aims at determining the uniformity and concordance of mtDNA analysis among different laboratories, supplying of a uniform stage for data interpretation and nomenclature of mtDNA analysis procedure, identifying the possible source of errors and development of a secured IT-based platform for logistic data transport and storage. It involved the usage of PHRED – computer software for computing base calls and assigning them a quality score (Parson et al. 2004). Website of EMPOP – www.empop.org was launched in October 2006. Since then, the database continuously evolved in terms of quality control and feasible results, and has emerged as the globally known repository for mitotyping data. Software based on quasi-median network analysis-based software was developed to review mtDNA data table in order to identify the occurrence of any possible type of error including that of interpretation as well as transcription errors and keep a check on quality assurance. Condition of mixing of HVI and HVII, also known as “artificial recombination,” was one of the common errors rectified with specific tools. Network analysis software package in the name of NETWORK have been introduced for routine evaluation of mtDNA sequence. It makes use of three filters designed for a specific function – EMPOPspeedy for removal of frequent mutation, EMPOPall for deletion of all mutations, and Unfiltered for no removal of mutations (Parson and Dür 2007). SAM, the string-based search software, was introduced in 2011 to harmonize EMPOP searches and rectify the issue of phylogenetic alignment difference in case of database searches that require rCRS comparison (Röck et al. 2011). The issue of alignments in casework mitotypes was resolved but, reporting and alignment ambiguity in population studies still remained an unsolved problem. Latest version of the database EMPOP 4 came up with modified string-based search software – SAM2 that comes with unbiased and harmonized database useful for not only the field of forensic but also for other genetic areas (Huber et al. 2018).

Modifications to the Current Methods of Mitochondrial DNA Analysis and Future Perspective

Screening Approaches for mtDNA Analysis

Methods of mitochondrial DNA analysis currently being utilized for obtaining full sequence information are time-consuming, expensive, and laborious. Consequently, these conventional methods demand utilization of screening assays in order to avoid analysis of complete sequence of the samples that could be excluded. Screening assays often includes physical screening, anthropological screening prior to performing mtDNA analysis in order to eliminate the unnecessary exhibits and concentrate on prime samples. MtDNA variation-based screening is employed to escape complete mtDNA sequencing across HVI and HVII regions. Sequence-specific oligonucleotide (SSO) probe, linear array typing, minisequencing, pyrosequencing, denaturing gradient gel electrophoresis (DGGE) are some of the commonly practiced screening assays performed prior to mtDNA sequencing in forensic caseworks (Butler 2011).

Currently Employed CE Technique Versus Next-Generation Sequencing Approaches

Sanger sequencing, also considered as the first-generation sequencing, was the earliest approach towards determination of nucleotide sequence in a DNA strand. The method involves incorporation of dideoxynucleotides triphosphates (ddNTPs) onto the amplified DNA molecules that results in production of DNA strands of varying lengths. Four types of DdNTPs namely ddATP, ddTTP, ddGTP, and ddCTP were utilized in the method. Sanger sequencing method is one of the widely employed methods for detection of mtDNA mutations. It can generate data of 25–1200 nucleotides. Also, the method is cost-effective and user-friendly. However, detection of low levels of heteroplasmy (<15%) is unachievable, indicating low degree of sensitivity of the sequencing method (Zhou et al. 2020).

Next-generation sequencing (NGS) technique is a class of advanced sequencing technique developed since 2005. These modern approaches are massively parallel sequencing techniques designed to provide enhanced sensitivity and high-throughput in detection of mtDNA mutations as well as heteroplasmy. Roche 454 based on pyrosequencing, Illumina “HiSeq” based on incorporation of fluorescently tagged nucleotides and ABI SOLiD based on ligation are some of the popular NGS platforms for mtDNA analysis. In spite of the rapid advancement, NGS platforms still need upgradation of several forensic-based softwares so as to be employed efficiently in the legal system. Conventional method of Sanger sequencing focusing upon the targeted mitochondrial regions, particularly HVI and HVII are used in most of the forensic science laboratories. In the recent years, the range of targeted sequence or regions have been extended to HVIII regions of D-loop resulting in sequencing of whole control regions (Sinha et al. 2020).

Implementation of whole genome sequencing (WGS) and whole exome sequencing (WES) data for mtDNA analysis can be useful in genomic screening, detection of mutation, as well as assessment of heteroplasmy. WGS and WES are applicable for identifying broad range of variants including single nucleotide polymorphisms (SNPs) (Parsons 2006), insertion/deletion (INDEL) variants (Diroma et al. 2020), structural polymorphisms (Yuan et al. 2020), copy number variants (CNVs) (Longchamps et al. 2020), pseudogenes (Cihlar et al. 2020), as well as heteroplasmy (Li et al. 2010; Cao et al. 2017), and therefore stands out as an effective technique for forensic human identification. In spite of being a time-consuming process, the technique of WGS is gaining attentions due to its advantages of cost-effectiveness and reduction in trial and experimental efforts (Duan et al. 2019).

Hybridization Capture-Based Target Enrichment Coupled with Massive Parallel Sequencing

Degraded biological samples are quite often encountered in forensic caseworks that possess problems in ideal extraction of DNA from such samples, thereby hampering the analytical procedure based on Sanger sequencing. Conventional hybridization capture-based target enrichment combined with NGS technology can facilitate in detection of degraded and decomposed DNA and enhance their yield. Hybridization-based target enrichment is a DNA preparation step performed prior to DNA sequencing to enrich and sequence the part of genome containing the region of interest thereby allowing analysis of DNA fragments as small as 30 bp, often ranging below the PCR threshold. The process involves conversion of DNA fragments into a DNA library with the help of barcoded adapters, immortalization of DNA by PCR primers complementary to the adapter, and hybridization enrichment. Hybridization capture-based target enrichment coupled with massive parallel sequencing has been employed on a wide range of forensic samples such as simulated degraded DNA, telogenic hairs, chemical-exposed DNA, buried skeletal remains, and archeological samples (Young et al. 2019; Sinha et al. 2020).

Conclusion

Mitochondrial DNA analysis has been used for the past three decades in various forensic caseworks that involves human identification. Besides its favorable characteristics of high copy number and susceptibility to external degradations, the technical advancements in mtDNA analysis play an incredible role in enhancing its utility in analysis of variety of samples ranging from tiny degraded fragments of DNA to sequencing of entire mt-genome in short span of time. Over the past two decades, mtDNA analysis has been availed for forensic caseworks related with human identity such as mass disaster, missing-persons identification, decomposed skeletal remains identification, as well as maternal dispute and child swapping cases. Besides individual identification in forensic caseworks, the detailed analysis of mitochondrial genome is effectual in clinical, genealogical, and human evolutionary studies. In spite of several justified explanations for collecting detailed history of suspected offender related with his phylogeny or health conditions, many researchers contemplate it as unethical to unveil someone’s genetic structure that reveals his personal information. Therefore, the advancing technologies related with mtDNA analysis must be mindfully utilized for expected purposes. Besides ethical complications, technical and structural drawbacks such as condition of heteroplasmy, paternal leakage, nuclear pseudogene, and sample mixture raise complication in analysis and interpretation of mitochondrial DNA sequences, which needs to be addressed by further technical advancement. Ability to recognize heteroplasmy and coherent explanation of biparental inheritance by recombination are some of the major challenges that must be focused in the interest of mtDNA analysis to emerge as an important alternative for forensic purposes.