The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

Assessing the preservation of biogenic strontium isotope ratios (87 Sr/86 Sr) in the pars petrosa ossis temporalis of unburnt human skeletal remains: A case study from Saba.

RATIONALE: Strontium isotope (87 Sr/86 Sr) analysis of skeletal remains has become a powerful tool in archaeological studies of human migration and mobility. Owing to its resistance to post-mortem alteration, dental enamel is the preferred sampling material used for 87 Sr/86 Sr analysis in bioarchaeological provenance research, although recent studies have demonstrated that cremated bone is also generally resistant to diagenesis. This paper presents the results of a pilot study exploring the potential of unburnt petrous bone (pars petrosa) as a reservoir of biogenic (diagenetically unaltered) strontium, as the otic capsule or bony labyrinth within the petrous bone is extremely dense and is thought to be unable to remodel after early childhood, potentially providing an alternative for dental enamel. METHODS: From an individual from a colonial-era (18th century) site on the island of Saba in the Caribbean for whom previous enamel 87 Sr/86 Sr results had indicated non-local origins, multiple locations (n = 4) on the petrous were sampled and measured for strontium isotope composition. Saba (13 km2 ) has been extensively mapped for baseline strontium isotopes (n = 50) with 87 Sr/86 Sr varying from ca 0.7065 to 0.7090, whereas enamel 87 Sr/86 Sr (n = 3) ranged from 0.7104 to 0.7112. RESULTS: All four petrous 87 Sr/86 Sr ratios (0.7111-0.7122) are consistently and considerably higher than the local bioavailable range, and very similar to the enamel 87 Sr/86 Sr. These results provide initial evidence that unburnt petrous bones may preserve biogenic strontium, at least in this specific burial context. CONCLUSIONS: While more research in diverse burial conditions is needed to validate this observation, if confirmed, it would have broader implications for sample selection strategies in bioarchaeological studies using the strontium isotope method.

Strontium isotope ratios related to childhood mobility: Revisiting sampling strategies of the calcined human pars petrosa ossis temporalis.

RATIONALE: Strontium isotope analysis can be applied to the calcined human otic capsule in the petrous part (pars petrosa ossis temporalis; PP) to gain information on childhood mobility in archaeological and forensic contexts. However, only a thin layer of the otic capsule, the inner cortex, demonstrates virtually no remodelling. This paper proposes an improved sampling method for the accurate sampling of the inner cortex of the otic capsule to ensure that 87 Sr/86 Sr ratios related to early childhood are obtained. METHODS: Calcined rib and diaphyseal fragments and PP from ten cremation deposits are sampled for strontium isotope analysis, whereby our improved sampling strategy is applied to sample the inner cortex of the otic capsule. This allows inter- and intraskeletal 87 Sr/86 Sr comparison within an Iron Age collection from Oss, The Netherlands. RESULTS: Forty percent (4/10) of the calcined PP that were evaluated for this study show marked differences in 87 Sr/86 Sr (0.00035-0.00065) between the inner cortex and the bone sample surrounding this layer, the external cortex that has higher remodelling rates. Differences in 87 Sr/86 Sr between various skeletal elements also aided in the identification of the minimum number of individuals. CONCLUSIONS: Our study demonstrates the problematic nature of the external cortex and stresses the need for a precise sampling method of the correct areas of the otic capsule. This can only be obtained by cutting the calcined PP midmodiolarly to enable adequate combustion degree assessment, and the correct identification and sampling of the inner cortex of the otic capsule.

The petrous bone: Ideal substrate in legal medicine?

Over the last few years, palaeogenomic studies of the petrous bone (the densest part of the temporal bone) have shown that it is a source of DNA in both larger quantities and of better quality than other bones. This dense bone around the otic capsule has therefore been called the choice substrate in palaeogenomics. Because the practice of forensic genetics responds to different imperatives, we implemented a study aimed at (i) understanding how and why the petrous bone is an advantageous substrate in ancient DNA studies and (ii) establishing whether it is advantageous in forensic STR typing. We selected 50 individual skeletal remains and extracted DNA from one tooth and one petrous bone from each. We then amplified 24 STR markers commonly used in forensic identification and compared the quality of that amplification using the RFU intensities of the signal as read on the STR profiles. We also performed histological analyses to compare (i) the microscopic structure of a petrous bone and of a tooth and (ii) the microscopic structure of fresh petrous bone and of an archaeological or forensic sample. We show that the RFU intensities read on STR profiles are systematically higher in experiments using DNA extracted from petrous bones rather than teeth. For this reason, we were more likely to obtain a complete STR profile from petrous bone material, increasing the chance of identification in a forensic setting. Histological analyses revealed peculiar microstructural characteristics (tissue organization), unique to the petrous bone, that might explain the good preservation of DNA in that substrate. Therefore, it appears that despite the necessity of analysing longer fragments in forensic STR typing compared to NGS palaeogenomics, the use of petrous bones in forensic genetics could prove valuable, especially in cases involving infants, toothless individuals or very degraded skeletal remains.

Endoscopic Endonasal Transpterygoid Approach Using Pedicled Nasoseptal Flap for Petrous Apex Cholesterol Granuloma With Intraoperative Video.

With the development of minimally invasive endoscopic surgery and image-guided navigation systems, the endoscopic endonasal approach to petrous apex lesions has become more favorable. In this case, a petrous apex cholesterol granuloma was successfully treated with an endoscopic endonasal transpterygoid approach. A navigation system was used, and a contralateral nasoseptal flap and silicone tube stent were applied to maintain the cholesterol granuloma drainage pathway. The authors describe the endoscopic endonasal transpterygoid approach using a pedicled nasoseptal flap for petrous apex cholesterol granuloma and provide an intraoperative video and a literature review.

Isolating the human cochlea to generate bone powder for ancient DNA analysis.

The cortical bone that forms the structure of the cochlea, part of the osseous labyrinth of the inner ear, is now one of the most frequently used skeletal elements in analyses of human ancient DNA. However, there is currently no published, standardized method for its sampling. This protocol describes the preparation of bone powder from the cochlea of fragmented skulls in which the petrous pyramid of the temporal bone is accessible. Using a systematic process of bone removal based on distinct anatomical landmarks and the identification of relevant morphological features, a petrous pyramid is cleaned with a sandblaster, and the cochlea is located, isolated, and reduced to a homogeneous bone powder. All steps are carried out in dedicated ancient DNA facilities, thus reducing the introduction of contamination. This protocol requires an understanding of ancient DNA clean-room procedures and basic knowledge of petrous pyramid anatomy. In 50-65 min, it results in bone powder with endogenous DNA yields that can exceed those from teeth and other bones by up to two orders of magnitude. Compared with drilling methods, this method facilitates a more precise targeting of the cochlea, allows the user to visually inspect the cochlea and remove any residual sediment before the generation of bone powder, and confines the damage to the inner ear region and surface of the petrous portion of fragmentary crania.

Neither femur nor tooth: Petrous bone for identifying archaeological bone samples via forensic approach.

One of the major challenges of molecular biology in anthropological analysis is the identification via DNA typing of bone or teeth samples that can be collected from archaeological site in order to investigate kinship relationships. Due to the difficulties of isolating and analysing DNA from such samples, several efforts have been made to solve these problems, but less work has been conducted to identify the proper type of bone samples for the DNA analysis. Therefore, following the promising results obtained from the DNA analysis of petrous bones by different groups of researchers, for the first time, here we investigated the possibility of using petrous bones as skeletal elements useful for short tandem repeat (STR) typing via capillary electrophoresis technique in ancient bone samples. In order to compare the results from petrous bone, femur and tooth samples, a total of 39 skeletal elements were collected from 13 different individuals excavated from Italian archaeological sites, dating from the sixth to seventh century C.E. The DNA was extracted, quantified, and subsequently amplified using two STR multiplex kits. The presence of a good amount of genetic material, despite high degradation, allowed us to quantify and subsequently identify STR profiles via CE analysis from ancient petrous bones that were complete for four out of thirteen samples and higher than 11 autosomal loci for all samples. Our results indicated that petrous bone is the best skeletal element with regard to DNA conservation and is a valuable element from which it is possible to obtain a complete STR profile also when analysing ancient bones. The STR results showed the possibility to use the petrous bones for identification and matching purposes in cases in which the biological material is poor and highly degraded such as in archaeological studies. Therefore, STR typing could represent a time-saving and cheap chance to verify kinship relationships in archaeological sites and evaluate sex when skeletal material is not suitable for morphometric estimate as in case of infants.

As solid as a rock-comparison of CE- and MPS-based analyses of the petrosal bone as a source of DNA for forensic identification of challenging cranial bones.

Short tandem repeat (STR) typing from skeletal remains can be a difficult task. Dependent on the environmental conditions of the provenance of the bones, DNA can be degraded and STR typing inhibited. Generally, dense and compact bones are known to preserve DNA better. Several studies already proved that femora and teeth have high DNA typing success rates. Unfortunately, these elements are not present in all cases involving skeletal remains. Processing partial or singular skeletal elements, it is favorable to select bone areas where DNA preservation is comparably higher. Especially, cranial bones are often accidentally discovered during criminal investigations. The cranial bone is composed of multiple parts. In this examination, we evaluated the potential of the petrous bone for human identification of skeletal remains in forensic case work. Material from different sections of eight unknown cranial bones and-where available-additionally other skeletal elements, collected at the DNA department of the Institute of Legal Medicine in Ulm, Germany, from 2010 to 2017, were processed with an optimized DNA extraction and STR typing strategy. The results highlight that STR typing from the petrous bones leads to reportable profiles in all individuals, even in cases where the analysis of the parietal bone failed. Moreover, the comparison of capillary electrophorese (CE) typing to massively parallel sequencing (MPS) analysis shows that MPS has the potential to analyze degraded human remains and is even capable to provide additional information about phenotype and ancestry of unknown individuals.

A minimally-invasive method for sampling human petrous bones from the cranial base for ancient DNA analysis.

Ancient DNA (aDNA) research involves invasive and destructive sampling procedures that are often incompatible with anthropological, anatomical, and bioarcheological analyses requiring intact skeletal remains. The osseous labyrinth inside the petrous bone has been shown to yield higher amounts of endogenous DNA than any other skeletal element; however, accessing this labyrinth in cases of a complete or reconstructed skull involves causing major structural damage to the cranial vault or base. Here, we describe a novel cranial base drilling method (CBDM) for accessing the osseous labyrinth from the cranial base that prevents damaging the surrounding cranial features, making it highly complementary to morphological analyses. We assessed this method by comparing the aDNA results from one petrous bone processed using our novel method to its pair, which was processed using established protocols for sampling disarticulated petrous bones. We show a decrease in endogenous DNA and molecular copy numbers when the drilling method is used; however, we also show that this method produces more endogenous DNA and higher copy numbers than any postcranial bone. Our results demonstrate that this minimally-invasive method reduces the loss of genetic data associated with the use of other skeletal elements and enables the combined craniometric and genetic study of individuals with archeological, cultural, and evolutionary value.

Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum.

Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements.

Searching for the mother missed since the Second World War.

The aim of the study was to perform the genetic identification of a human cranium from a Second World War gravesite in Slovenia and find out if it belonged to the mother of a woman used as a family reference. Both genetic and anthropological examinations were carried out. The genetic examination was performed on 2 molars and petrous bone. Prior to DNA isolation 0.5 g of tooth and bone powder was decalcified. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The nuclear DNA of the samples was quantified and short tandem repeat (STR) typing performed using two different autosomal and Y-STR kits. Up to 22.4 ng DNA/g of powder was obtained from samples analyzed. We managed to obtain nuclear DNA for successful STR typing from the left second molar and from the petrous bone. Full autosomal genetic profile including amelogenin locus revealed the male origin of the cranium that was further confirmed by the analyses of Y-STRs. The same conclusions were adopted after the anthropological analysis which identified the cranium as that of a very young Caucasoid male. The male origin of the cranium rejected the possibility of motherhood for the compared daughter. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and Y-STRs of all persons that had been in contact with the analyzed cranium and no match was found.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Strontium isotope signals in cremated petrous portions as indicator for childhood origin.

Dental enamel is currently of high informative value in studies concerning childhood origin and human mobility because the strontium isotope ratio in human dental enamel is indicative of geographical origin. However, many prehistoric burials involve cremation and although strontium retains its original biological isotopic composition, even when exposed to very high temperatures, intact dental enamel is rarely preserved in cremated or burned human remains. When preserved, fragments of dental enamel may be difficult to recognize and identify. Finding a substitute material for strontium isotope analysis of burned human remains, reflecting childhood values, is hence of high priority. This is the first study comparing strontium isotope ratios from cremated and non-cremated petrous portions with enamel as indicator for childhood origin. We show how strontium isotope ratios in the otic capsule of the petrous portion of the inner ear are highly correlated with strontium isotope ratios in dental enamel from the same individual, whether inhumed or cremated. This implies that strontium isotope ratios in the petrous bone, which practically always survives cremation, are indicative of childhood origin for human skeletal remains. Hence, the petrous bone is ideal as a substitute material for strontium isotope analysis of burned human remains.

The petrous bone--a new sampling site for identifying early dietary patterns in stable isotopic studies.

Intraskeletal variation in the composition of carbon (delta(13)C) and nitrogen (delta(15)N) stable isotopes measured in collagen is tested from various human bones and dentine. Samples were taken from the femur, rib, and petrous part of the temporal bone from well-preserved skeletons of both adults (n = 34) and subadults (n = 24). Additional samples of dentine from the root of 1st molars were taken from 16 individuals. The skeletal material is from a medieval cemetery (AD 1200-1573) in Holbaek, Denmark. Our results indicate that the petrous bone has an isotopic signal that differs significantly from that of femur and rib within the single skeleton (P < 0.001 and P < 0.001, respectively), with only minor variation seen between femur and rib. On the other hand, there was no significant difference between the petrous bone and the 1st molar. The intraskeletal variation may reflect differences in turnover rate among skeletal elements. The inner periosteal layer of the petrous bone is formed in uterus and does not undergo any further remodelling after the age of 2 years, whereas the rib and femur have a continuous turnover rate of approximately 5 and 10-20 years, respectively. From the results of this study it is believed the petrous bone may be a new useful bone element and a supplement or a proxy for teeth in the analysis of early dietary patterns as it may reflect diet in fetal stages and early years of life.

Primary intra-diploic meningioma in a child.

BACKGROUND: Primary intra-diploic meningiomas are uncommon in childhood and, at the clinical onset, may be confused with other and more frequent bone tumours because they lack specific clinical and radiological characteristics. Surgery is indicated not only to remove the lesion but also to obtain an accurate histological diagnosis. CASE REPORT: We report the case of a young girl who presented with a recently developed subcutaneous hard mass in the left pterional region. Neuroradiological investigations revealed an intra-osseous lytic mass with a sclerotic reaction. Diagnosis was possible only after the total removal of the tumour and its histological examination.

[Infralabyrinthine approach for cholesterol granuloma of the petrous apex]. / Granulomes à cholestérine de l'apex pétreux : intérêt du drainage par voie infra-labyrinthique.

Cholesterol granuloma of the petrous apex are cystic lesions, revealed by otologic and/or cranial nerve palsies, and diagnosed with the help of computed tomography and magnetic resonance imaging. Surgical treatment is not the complete removal of the lesion, but a conservative approach requiring drainage of the cyst and re-establishment of a correct aeration of the cavity. Three cases of cholesterol granuloma of the petrous apex were treated through a transmastoid infralabyrinthine procedure. Through a postauricular approach, a simple mastoidectomy was performed. The third portion of the facial nerve was identified. The posterior and lateral semicircular canals, and the jugular bulb were skeletonized. In two cases, a high diverticulum of the jugular bulb was impacted downwards with wax. The cystic lesion was then opened, and evacuated. The opening must be large to permit a correct aeration of the cavity and prevent stenosis of the drainage site. Hearing and the facial function were preserved in all cases. In conclusion, conservative approach to cholesterol granuloma of the petrous apex provides satisfactory drainage of this intrapetrous deep seated lesion with preservation of hearing and facial nerve function.