Feb 19 2018

Harder, Better, Faster, Stronger: The Petrous Bone as a Source of DNA for STR Typing and MPS Analysis

The human body of an adult individual is composed of approximately 206 bones. All of the bones in the adult skeleton consist of two structural components that are either spongy or compact. Generally, compact and dense bones are known to preserve DNA better than brittle bones. Therefore, the identification of skeletal remains in forensic genetics is accomplished by the analysis of the densest bones in the available cohort.

Studies of distinct scientists agree that particularly long bones such as humeri and femora, but also teeth are suitable for molecular identification of skeletal remains [Edson et al., 2004, Alt et al., 2014]. In cases where no long bones or teeth are available, the specimens for analysis must be chosen wisely.

written by: galina Kulstein, institute of legal medicine, university hospital of ulm, Germany

Often, cranial bones are accidentally found during investigations. Cranial bones consist of various elements such as the occipital, the parietal and the temporal bone. During a comparative examination in 2009, Edson et al. revealed that distinct elements of the skull have different success rates for STR profiling.

While the typing success of the parietal, occipital and frontal portions are between 52% and 65%, temporal bones showed the highest success rate of approximately 90% [Edson et al., 2009]. Concomitantly, recent observations of scientists from the field of ancient DNA analysis confirmed these findings and showed that the petrous part of the temporal bone harbors high DNA amounts, even in specimens from the Neolithic period [Pinhasi et al., 2015, Gamba et al., 2014].

The petrous bone is a pyramid-shaped part of the temporal bone, which is located at the base of the skull, between the sphenoidal and occipital bones. It presents a base, an apex and distinct surfaces and houses the components of the inner ear. Petrous comes from the Latin word ‘petrosus’, which means ‘stone-like, hard’. Its name says it all: due to its responsibility to protect the inner part of the ear, the petrous bone is one of the densest bones in the human body.

In order to test the potential of the petrous bone for the analysis of skeletal remains in forensic casework, we performed a comprehensive analysis of capillary electrophorese (CE)- and Massively Parallel Sequencing (MPS)-based analysis of petrous bone specimens. For examination, eight specimens were isolated from skulls that were collected during casework. After isolation, bones were decontaminated via bleaching and ground. Approximately 200 mg of fine bone powder were used for simultaneous decalcification and lysis and subsequent extraction procedure.

After isolation, DNA quantification was accomplished with a novel assay that is able to assess human and male DNA as well as to monitor inhibition and degradation of the extracted DNA. The surveillance of quantity and quality of DNA extracted from skeletal remains is important to ensure an efficient amplification.

DNA concentrations of samples from petrous bones ranged between 5.9 pg/µL and 120.7 pg/µL. No inhibition occurred in those samples and degradation indices were between 3 and 150, showing that enhanced DNA degradation indeed occurred in some of the samples.

Despite measurable degradation, we were able to obtain full DNA profiles from all samples that we could report to the database. STR profiling of full profiles was successful for CE-based as well as for MPS-based analysis.

Both approaches showed concordant results with a similar level of sensitivity. Moreover, MPS-based analysis was accomplished with a multiplex assay that consists of 229 markers. In addition to autosomal STRs, the kit enables the simultaneous analysis of gonosomal STRs and phenotype- well as ancestry-informative SNPs.

In most cases, the conventional STR typing provides sufficient discrimination power. However, the analysis of skeletal remains could frequently benefit from the simultaneous detection of supplementary markers. Additional information can be used to narrow down the assignment of potential missing persons to skeletal remains making it possible for criminal investigations to evolve from the “passive comparison” into the “active search” stage [Yang et al ., 2014].

Hereafter, considering this remarkable STR typing results, our work supports the hypothesis that DNA analysis from dense petrous bone is likely to provide reportable DNA profiles. Therefore, we recommend to use the petrous bone for DNA analysis in casework where only cranial bones are available [Kulstein et al., 2017].

This work would have been inconceivable without Gaby Kottmair who isolated the petrous bones from the skulls and the team of the DNA department of the Bavarian State Criminal Police Office, particularly Thorsten Hadrys and Angelika Fürst.

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References

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Gamba C, Jones ER, Teasdale MD, Mclaughlin RL, Gonzales-Fortes G, Mattiangeli V, Domboróczki L, Kóvári I, Pap I, Anders A, Whittle A, Dani J, Raczky P, Higham TFG, Hofreiter M, Bradley DG, Pinhasi R (2014) Genome flux and stasis in a five millennium transect of European prehistory, Nat Commun 5. doi:10.1038/ncomms6257

Yang Y, Xie B, Yan J (2014) Application of next-generation sequencing technology in forensic science. Genomics, Proteomics, Bioinformatics 12:190–197. doi:10.1016/j.gpb.2014.09.001

Edson S, Ross JP, Coble MD, Parsons TJ, Barritt SM (2004) Naming the dead—confronting the realities of rapid identification of degraded skeletal remains. Forensic Sci Rev 16:64–89

Edson SM, Christensen AF, Barritt SM, Meehan A, Leney MD, Finelli LN (2009) Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains. Forensic Sci. Int. Genet 2:269–270. doi:10.1016/j.fsigss.2009.09.029

Alt KW, Brandt G, Knipper C, Lehn C (2013) Empfehlungen für die Probenentnahme in der forensischen Anthropologie – Untersuchung von DNA und Stabilisotopen. Rechtsmedizin 24:179–185. doi:10.1007/s00194-014-0950-9

Kulstein GK, Hadrys T, Wiegand P (2017) 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. Int. J. Leg. Med. doi:10.1007/s00414-017-1653-z