Sweet tooth: DNA profiling of a cranium from an isolated retained root fragment

Abstract Sampling of healthy multi‐rooted teeth is recommended for the genetic identification of human skeletal remains. However, this may not always be possible, as in the reported case consisting of an isolated human cranium found in an aggregate crushing and processing plant in Piedmont, Northwest Italy. The cranium displayed significant weathering, suggesting a post‐mortem interval of several years, and was edentulous with the exception of the apical root fragment of the upper left canine, consequence of an antemortem horizontal fracture. Prolonged decalcification of the root fragment followed by powder‐free DNA extraction from ~10 mg of root tip tissue led to the recovery of >10 ng of high molecular weight human DNA, in comparison with ~0.01 ng of DNA per mg of bone powder obtained from the petrous portion of the temporal bone. Quantity and quality of DNA isolated from apical tooth tissue enabled multiple genotyping, including a reportable female STR profile, mitochondrial DNA analysis, and ancestry‐informative insertion/deletion polymorphisms. Although the cranium remained unidentified after DNA comparisons, our findings confirm that apical tooth tissue is a promising source of DNA, easily obtained through a powder‐free extraction protocol. Results also indicate that root tips should not be overlooked in challenging identification cases, even in the presence of compromised tooth specimens.


| INTRODUC TI ON
Positive identification of human skeletal remains relies on a combination of methods including forensic anthropology, forensic odontology, and DNA profiling. The mineralized extracellular matrix of bone and tooth guarantees prolonged resistance to exogenous factors of DNA degradation, such as microbial nuclease action and non-enzymatic degradation, making genetic analysis often successful even in the presence of severely compromised skeletal remains [1,2].
A general correlation between bone density (i.e. mineralization extent) and DNA profiling success rate was observed in previous studies [3]. Consequently, compact cortical bone from the shaft of long bones, in particular weight-bearing leg bones, is indicated as the sample of choice in operative guidelines for the identification of human remains [4,5]. Recent studies have also shown that the inner part of the petrous portion of the temporal bone, being among the hardest bone parts in the mammalian body, can represent a valuable alternative target for genetic analysis, when an isolated cranium is the only remnant found of an unidentified individual [6,7].
Thanks to their position within the maxillary bones and their unique composition (low porosity and mineralization), teeth are largely protected from the physical and environmental effects that contribute to the peri-or post-mortem degradation process of nucleic acids. For this reason, they can often provide equivalent if not superior DNA yields compared to compact bone samples [8,9].
Dental pulp is a highly cellular tissue, including mainly odontoblasts and fibroblasts, and represents the most obvious target for forensic DNA typing [10]. However, it has been shown that cellular cementum, predominantly found on the apical segment of the roots and in the furcation area of molar teeth, can also be a valuable source of DNA [11], especially in challenging samples affected by post-mortem cellular degeneration [12,13]. For these reasons, the collection of multi-rooted teeth that provide larger quantities of pulp and cellular cementum is currently recommended in human identification procedures [4,5]. Since dental caries can cause pulp retraction and, when extensive, complete pulp loss [14], current guidelines also underline the importance to preferably select healthy teeth, without signs of disease or dental restoration, for genetic identification [4,5].
Despite these caveats, here we report successful DNA profiling of a cranium using the root apex of a retained maxillary canine root as a DNA source, after unsatisfactory genetic analysis carried out on petrous bone.

| C A S E HIS TORY AND ANTHROP OLOG IC AL FINDING S
In April 2020, an isolated jawless cranium was accidentally found lying on a pile of gravel in an aggregate crushing and processing plant in Piedmont, Northwest Italy ( Figure 1). The cranium displayed significant weathering compatible with an exposure to the environment of a few years (score 13 according to [15]) ( Figure S1).
Cranial features were consistent with female sex as confirmed by statistical comparisons of the cranial measurements with samples from Fordisc 3.0. [16] Age assessment according to Meindl and Lovejoy's technique [17] suggested the decedent was middle-aged (mean age of 52.5 ± 13.5 years). With regard to the determination of ancestry, craniometric evaluation based on 22 measurements performed with Fordisc 3.0 [16] indicated that the individual had the smallest Mahalanobis distance to the Asian female reference group centroid. However, the obtained posterior probability (0.427) and typicality probability (0.001) were too low to support population attribution.
The odontological assessment of the upper jaw ( Figure 2) showed it was edentulous, except for the upper left canine (2.3), which consisted of a retained root without the crown due to a horizontal fracture. The root did not present any dental treatment, as confirmed by the periapical X-ray taken using X-ray portable device combined with a radiovideography sensor. The magnified observation of the root showed rounded-off edges indicative of post-traumatic remodeling and repair [18], demonstrating that the fracture was not perior post-mortem In the subsequent days, further research in the area where the cranium was found led to the discovery of a fragmented left human femur ( Figure S1), displaying the same severe weathering process affecting the cranium, compatible with years of exposure to an outdoor environment.

| DNA extraction and quantitation
In order to prevent contamination, prior to DNA extraction, the outer layers of petrous bone and femur diaphysis were mechanically removed with a rasp. DNA was then isolated from compact bone using PrepFiler BTA Forensic DNA Extraction kit (Thermo Fisher   Genetics (ISFG) [21,22].

| DNA typing
STR profiles and mtDNA haplotypes obtained from bone and tooth samples were compared against the laboratory staff elimination database to rule out potential contamination.

| G ENE TI C RE SULTS
Mean concentration of total DNA in the two petrous bone ex- Comparisons between DNA profiles obtained from the root apex and femur extracts (mean total DNA yield 0.044 ng/µl) showed multiple genotype incompatibilities, among them a X-Y genotype at Amelogenin locus for the femur sample, thus excluding that cranium and femur could belong to the same individual ( Figure S2).  (Figure 3).

| DISCUSS ION
Despite the recommendation to analyze healthy, multi-rooted teeth in order to increase the success in DNA analysis [4,5,28], there is a growing interest in the study of restored and/or decayed teeth as a source of DNA. This is due to the fact that intact teeth may not be always available in real forensic investigations, as in the present case where an isolated cranium with only a radicular residue of the upper left canine was found.
It was previously shown that teeth with caries, extracted following orthodontic or periodontal disease treatment, can still contain enough pulp tissue to ensure successful STR typing [29]. Pulp, however, undergoes a quick loss of structural integrity post-mortem, with nuclear DNA yields rapidly declining beyond a four-month postmortem interval (PMI) [30].
On the contrary, successful DNA profiling from cementocytes is often possible even after prolonged PMI and in challenging postmortem conditions [12], even in single-rooted teeth with comparatively smaller pulp volume [13]. Moreover, cellular cementum is largely unaffected by dental caries and periodontal disease [10].
Experimental studies have shown that suitable DNA yields for identification purposes can be obtained from cementum of teeth that | 1977 underwent root canal treatment and restoration [31], or affected by periodontitis and pulpal/periapical disease [14,31]. Accordingly, DNA kinship analysis on single-rooted restored teeth, collected from exhumed human remains buried for 46 years, was recently reported [32].
DNA is routinely isolated from cementocytes after pulverization of whole teeth or root segments [33]. This approach, however, presents several disadvantages [10]: It requires dedicated and expensive instrumentation; the heat generated in the crushing process can affect DNA integrity; and the presence of large amounts of acellular material increases the risk of contamination, dilutes DNA content, and interferes with the following extraction steps.
An effective alternative is represented by powder-free methods in which, after prolonged decalcification of whole teeth, demineralized root tips are collected and submitted to standard forensic DNA extraction protocols designed for soft tissue samples [34,35]. Preferential targeting of root tips is justified by the higher thickness and cellularity of cementum expected in apical radicular portions [36]. Such methods, besides being simple and inexpensive, are also minimally destructive, so that relevant teeth parts can be preserved for forensic odontology, and the tested samples returned to families after identification. This report indicates that a similar approach can be applied not only to restored teeth with long PMI [32], but also to isolated root fragments. In particular, the root apex was confirmed as an optimal target for DNA isolation, with DNA yields rapidly declining in the adjacent cervical radicular segments.
In this case study, the retained root emerged above the alveolar crest and was therefore an obvious target for molecular analysis.
It can be assumed that the upper canine received trauma which caused the transverse fracture of the crown leaving the root in situ, followed by incomplete healing and interposition of soft tissue [37].
In such traumatic injuries, the apex of the root is usually unaffected and there is no damage to cementum and periodontal ligament [38].
At the periphery of the fracture line, together with remodeling and resorption of the edges, the formation of new cementum may occur, which may even join the two fragments to some extent [37]. In the present case, however, no detectable DNA could be isolated from the coronal end of the root fragment.
Transverse root fracture is an infrequent type of traumatic dental injury, with prevalence between 0.5 and 7% in permanent teeth and highest incidence in the maxillary anterior region [39]. Root fractures, however, can also occur during teeth extraction. In such cases, root tips can successfully remain in situ with normal healing taking place together with the formation of a cementum layer on the dentine allowing bone deposition, thus enclosing the root fragment within bone [40].

| CON CLUS ION
Joint anthropological, odontological, radiological, and genetic investigations indicated that the recovered skeletal remains consisted of a commingling of human specimens, with cranium and femur belonging to two different individuals. While the cranium remains unidentified, it was shown that a single fractured dental root, despite long-term (exceeding a few years) PMI and tissue modifications due to mechanical damage, can provide high molecular weight DNA at concentrations suitable with forensic analysis of STRs, mtDNA, and AIM-Indels. A powder-free protocol previously applied with success to healthy and restored post-mortem teeth was used to isolate DNA from the demineralized root tip. The method is straightforward, compared to more complex procedures adopted for other skeletal material, its major drawback being the need for prolonged decalcification, making it less suitable for high priority cases that require immediate processing of samples. Obviously, results from this case report cannot be generalized and need to be supplemented in the future by larger comparative studies of DNA yield from intact/damaged post-mortem teeth and other cranial elements in different taphonomic conditions. If confirmed, persistence of high-quality genetic material in apical tooth tissue, even in conjunction with trauma, disease, and dental work, will provide forensic investigators with an alternative and easily accessible DNA source, not to be overlooked in the analysis of challenging human remains.