DNA sequencing links aged hair shafts to the last imperial rulers of Russia

 by Nikki Matta | Helraiser

History has its eyes on important figures, and without a doubt, the Romanov Family is not an exception. The end of the Romanov dynasty sparked a new chapter not only to Russia but also, to the world. The Russian Revolution of 1917 introduced the communist and socialist ideas that  eventually formed a new economic system known as Socialism, where different countries followed through.

The key players to the end of the Romanov dynasty were the Bolshevik revolutionaries. Various accounts and reports reveal that the Romanov family were held prisoners and executed in July 1918. After execution, the fallen bodies were quickly buried in a “superficial and hastily dug” pit around the execution site. Due to the turmoil brought about by the sudden changes in the political and social climate in Russia, it was only in 1991 that the first skeletons of the Romanovs were recovered. The recovery of skeletons from the execution site was temporarily identified to be of the Romanov family, in particular, the Tsar, Tsarina, three of five children of the royal family, and four family retainers. The bodies were subjected to DNA testing using mitochondrial DNA analysis, and the identity was confirmed to be members of the Romanov family after comparing the DNA profile of the remains to the DNA profile of Prince Philip, Duke of Edinburgh, who is a living grandnephew of Tsarina at that time. In the succeeding years, more recoveries were made, and consequently, identities were determined and confirmed through the integration of anthropological, mitochondrial, and nuclear DNA analyses.

One of the most recent recoveries related to the Romanov family came from Loreille et al.’s study published in January 2022. In their study, they subjected the hair shafts found from the artifacts associated with the Romanov family to an improved DNA Extraction protocol and Illumina sequencing, a type of Next-Generation Sequencing (NGS) technique. The latest artifacts from the study was a Karl Fabregé pendant and a framed photograph of Queen Louise of Hesse-Kassel. The Karl Fabregé pendant contained a photograph of the Empress Alexandra Feodorovna Romanov and a lock of light-colored hairs assumed to be from the Empress. The framed photograph of Queen Louise also contained locks of light hair in between the glass of the frame and the photograph itself. These available samples from the artifacts were the ones analyzed to confirm if the hairs belong to a member of the Romanov family.

However, studies have also reported on the challenge of using aged and degraded samples for mtDNA analyses. For example, one study has established that the likelihood to obtain hypervariable regions for mtDNA profile decreases from 90% to almost only 60% when a hair sample is aged around 0-6 years old and when the sample is greater than 20 years old, respectively. Additionally, another paper reports that the average mtDNA that can be collected in human hair samples that are aged at 50-100 years old is only around 61 bp. So, an increased sample age means an increased difficulty to obtain DNA information that may be useful for further analyses.

Loreille and colleagues used Next-Generation Sequencing to develop DNA data from the DNA samples present in the recently-obtained Romanov family artifacts. In order to maximize the DNA extraction in these aged samples, their team developed a modified protocol for the extraction. The hair samples were handled in a laboratory for low-quality and low-quantity DNA samples, with limited access to personnel. In handling the samples, they ensured that the personnel wore Personal Protective Equipment with single-use garments and goggles. Double-gloving was also required during handling. The reagents used for the process were all irradiated before they were introduced to the samples, as well as the plastic consumables so that the risk of contamination would be minimized. The modified DNA extraction protocol is as follows: the sample (aged rootless hair shaft) is decontaminated by using a detergent (5% Terg-a-zyme solution) and was subjected to sonication. After decontamination, lysis/digestion of the samples were carried out by using digestion buffer and proteinase K, with addition of irradiation in a cross-linker. The samples were then incubated in a thermomixer so they may be fully digested. Two purification methods were applied: a silica-based method and a magnetic bead-based purification. The silica-based was applied to the multiple-hairs sample, while the magnetic bead-based was for the single-hair sample. The main modification in the protocol is the magnetic bead-based purification applied to the single-hair samples since the silica-based purification is the common protocol for the process. DNA is then quantified using fluorometric methods with addition of the determination of dsDNA through dsDNA high sensitivity assay kit. The DNA library was prepared using the NEBNext Ultra II DNA library prep kit. There is a step for hybridization capture so that the mitochondrial DNA may be enriched through amplification and purification with AMPure XP beads. The quantification from this step was done through Qubit Fluorometer, and a NGS kit, the MiSeq FGx Sequencing System, was used to sequence the small mtGenome of the samples.

Both the hair samples obtained from the pendant and the framed photograph produced mitochondrial and nuclear DNA results. In addition, the biological sex of the sample source was also determined. Relatedness estimates were only available for the hair samples obtained from the pendant because the nuclear reads of the hairs obtained from the framed photograph were insufficient for a successful assessment.

The hairs from the pendant revealed a mitochondrial genome profile that is similar to the haplogroup of Empress Alexandra’s maternal lineage, which is publicly available in GenBank. The Nuclear DNA information of the samples from the pendant was derived from the shotgun sequence library, and it was confirmed that the hair samples was a human DNA with 98.9% belonging to the nuclear genome. Using the shotgun sequence library, the biological sex of the sample source was also determined, and the Rx confidence interval revealed that the sex for the sample source is a female, therefore eliminating that the possible source of the sample is from the Romanov son, Tsarevich Alexis. Finally, the relatedness estimates for the hair sample in the pendant showed allele mismatch proportions that reveal that the samples come from one source, which is a single individual only.

For the hair samples obtained from the framed photograph, the mitochondrial DNA results showed a haplogroup profile similar to the publicly available sequence of Tsar Nicholas Romanov. There is a noticeable inconsistency due to a presence of a point heteroplasmy from the sequence of Tsar Nicholas, which cannot be pointed in the hair sample from the framed photograph since it produced homoplasmic points in the cytosine, and it was assumed that this may be from hydrolytic deamination, a common form of damage in ancient molecules. For the nuclear DNA data, only the read and DNA length was determined from the sample. These data were insufficient for relatedness analysis, but the biological sex was still be determined from the results of the nuclear DNA, and so the confidence interval based on the nuclear DNA of the hair sample from the framed photograph revealed that the biological sex of the sample source is female, confirming the possibility that the hair is sourced from Queen Louise.

In conclusion, Loreille et al.’s work showed that minimal, aged, and degraded samples such as hairs may be used for forensic casework. This study shows that DNA information can be successfully obtained in samples with limited quantity because they may still produce considerable DNA information after extraction. Loreille et al.’s study highlights the importance of an improved protocol in handling such types of samples. In addition to developing a protocol to extract and amplify DNA from limited, aged, and degraded samples, the methods and results of this study have substantial contributions for forensic casework especially for cases like missing persons where there is very limited sample availability, given that there are alternatives for familial reference information for identity-linking analyses.

The information presented on the Romanov Family case was derived from the scientific paper entitled "Improved DNA Extraction and Illumina Sequencing of DNA Recovered from Aged Rootless Hair Shafts Found in Relics Associated with the Romanov Family", published under Genes which can be accessed at http://dx.doi.org/10.3390/genes13020202.