Genetic analysis of the presumptive blood from Louis XVI, king of France Carles Lalueza-Fox1, Elena Gigli1, Carla Bini4 , Francesc Calafell1,2, Donata Luiselli3, Susi Pelotti4, Davide Pettener3 1 Institut de Biologia Evolutiva, CSIC-UPF, Dr. Aiguader 88, 08003 Barcelona, Spain 2 CIBER Epidemiología y Salud Pública (CIBERESP), Spain 3 Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy Area di Antropologia, 4 Dipartimento di Medicina e Salute Pubblica, Sezione di Medicina Legale, Università di Bologna, Via Irnerio 49, 40126 Bologna, Italy Keywords: Louis XVI, identification, ancient DNA, mitochondrial DNA, Ychromosome, eye colour Abstract A text on a pyrographically decorated gourd dated to 1793 explains that it contains a handkerchief dipped with the blood of Louis XVI, king of France, after his execution. Biochemical analyses confirmed that the sample contained within the gourd was blood. The mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1) and 2 (HVR2), the Y-chromosome STR profile, some autosomal STR markers and a SNP in HERC2 gene associated to blue eyes, were retrieved, and some results independently replicated in two different laboratories. The uncommon mtDNA sequence retrieved can be attributed to a N1b haplotype, while the novel Y-chromosome haplotype belongs to haplogroup G2a. The HERC2 gene showed that the subject analysed was a heterozygote, which is compatible with a blue-eyed person, as king Louis XVI was. To confirm the identity of the subject, an analysis of the dried heart of his son, Louis XVII, could be undertaken. 1 Introduction Different studies have focused on the ancient DNA analysis of historical individual remains, to gain information regarding their possible identification and also shed new light on historical mysteries. These include the analysis of the remains of the Romanov family[1,2], the putative evangelist Luke [3], the American outlaw Jesse James [4], the heart of Louis XVII, son of Louis XVI, king of France [5], the Italian poet Francesco Petrarca [6], or the astronomer Nicolaus Copernicus [7]. In the aforementioned studies, mummified or skeletal remains have been associated to particular individuals by different means, usually funerary information. In some cases, body remains are not even available and yet, the potential of the paleogenetic techniques can allow us to obtain the genetic profile of some notorious historical characters. After the execution of Louis XVI in January 21st, 1793, eyewitnesses stated that many people from the crowd dipped their handkerchiefs in the king’s blood and kept these objects as mementos [8]. An Italian family has owned for more than a hundred years –as demonstrated by a letter addressed to the director of the Musée Carnavalet in Paris, January 31st, 1900- a dessicated gourd that presumably contained one of these handkerchiefs. The gourd, belonging to the species Cucurbita moschata, measures 23.7 cm in height, 15.2 cm at the base diameter and 7.2 cm at the top diameter, and was originally used as a bottle-gourd for gunpowder. It had been richly decorated with a pyrographic technique (Figures 1 and 2). The portraits of prominent lead actors during the French revolution, including Georges Danton, Jean Paul Marat, Camille Desmoulins, Louis-Sébastien Mercier, Joseph Ignace Guillotin, Maximilien Robespierre, Bernard-René de Launay, Jacques de Flesselles and Joseph Foullon, are depicted, as well as some 2 royalists, including, among others, the king Louis XVI, the queen Marie Antoinette, the Dauphin Louis XVII and the finance minister, Jacques Necker. Perhaps the most interesting information on the origin of this object is depicted in some text boxes between the portraits. In one of these it is stated: “Maximilien Bourdaloue on January 21th, dipped his handkerchief in the blood of the king after his beheading” (Figure 3). In another section it is explained that the author of the gourd’s decoration was Jean Roux, from Paris, and that the work was finished on September 18st, 1793. The final purpose of this object seems to have been economic. In another text box it can be read that the gourd had been crafted as a gift to “the Eagle” (maybe referring to Napoleon) and that a 500 francs profit is expected for it. What looks superficially like a dark and dried substance can be seen within the gourd. A general genetic test on the putative blood remains has been conducted, screening the mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1) and 2 (HVR2), the Y-chromosome STR profile and some autosomal STR markers. Our goal was to explore the genetic homogeneity of the sample and the reproducibility of the results, but also to characterize the putative genetic profile of Louis XVI for possible future comparisons, for instance with his son, Louis XVII, through the analysis of his presumed dried heart. Material and Methods DNA Extraction Several samples were scraped from the inside of the gourd and send to two different ancient DNA laboratories in Bologna and Barcelona for genetic analysis. In 3 Barcelona, DNA was extracted following a protocol described elsewhere [9] n short, 50 mg of sample were incubated overnight at 50oC in a lysis solution (0.5% SDS, 50 mM TRIS, and 1 mg/mL of proteinase K in H2O). Subsequently the DNA was extracted with phenol-chloroform and concentrated using a Centricon-30 filter column (Millipore) up to a 30 μl volume. In Bologna, the gourd sample was extracted using the QIAmp DNA Micro Kit (QIAGEN, Hilden, Germany), following the manufacturer’ instructions. Standard precautions to avoid contamination in ancient DNA were adopted during the experimental procedures [10,11] Confirmatory blood test A chromatographic method based on silica paper was applied on three different powder samples of few milligrams: a sample diluted in 10 µl of sterile water, a sample diluted in 20 µl of 5% ammonia and incubated at room temperature for 30 min [12] and a third sample left overnight in 200 µl of lysis buffer and proteinase K provided in QIAmp DNA Micro Kit (QIAGEN) [12]. After a incubation at 120°C for 10 min, an alcoholic benzidine spray reagent in conjunction with a hydrogen peroxide spray was used to detect blue spots, that correspond to blood residues [12]. Mitochondrial DNA In Barcelona, the mitochondrial DNA (mtDNA) hypervariable region 1 (HVR1) was amplified by polymerase chain reaction (PCR) in two overlapping fragments (with the L16,055-H16,218 and L16,209-H16,378 primers, numbered according to the Cambridge Reference Sequence), along with extraction and negative controls to monitor against possible contamination. The amplification was based in a two-step PCR, as described in Krause et al. [13]. The reaction was performed in a total volume of 20 µl, 4 containing: 5µ of DNA extract, 1X PCR buffer, 2,5 mM MgCl2, 500 mM of each dNTP, 2U of AmpliTaq Gold DNA Polymerase (Applied Biosystems), 150 nM of each primer in the first multiplex step and 1.5 µM of each primer in the second step. The annealing temperature used was 50oC. The PCR products were visualised in a 1% low-melting point agarose gel, excised from it under UV lights and purified using a silica-binding method. Subsequently, they were cloned into bacteria using TOPO-TA cloning kit (Invitrogen); inserts of the right size were sequenced in a ABI3730 capillary sequencer (Applied Biosystems) following manufacturer’s instructions. Forty-six clones were generated for the mtDNA HVR1. In Bologna, the mtDNA HVR1 and HVR2 were amplified in two fragments (with the L15,997-H16,401 and L29-H408 primers). The PCR was performed in a reaction volume of 25 µl containing: 10 ng of genomic DNA, 1X PCR buffer, 1.5 mM MgCl2, 200 mM of each dNTP, 1.5 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems) and 2 µM of each primer. The amplification was carried out for 35 cycles: 3 min at 94°C, 1 min at 94°C, 30 sec at 56°C and 1 min at 72°C, with a final extension of 10 min at 72°C. PCR products were ran on a 2% agarose gel stained with ethidium bromide. Amplicons were purified with ExoSAP-ITTM reagent (USB Corporation, Cleveland, OH) according to the manufacturer’s protocol. Sequencing was performed using BigDye Terminator Cycle Sequencing Ready reaction Kit v 1.1 (Applied Biosystems) and sequence products were purified by filtration devices Performa DTR Gel Filtration Cartridges (Edge BioSystems Inc., Gaithersburg, MD). Data were analysed with Sequencing Analysis Software v 3.4 and sequences were aligned to the Cambridge Reference Sequence with the Navigator 1.01 Software (Applied Biosystems). 5 Y-STR loci In both laboratories, a Y-STR genotype was performed on the sample by generating a 17 loci Y-STR profile (DYS19, DYS189I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385I/II, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458, DYS635 and Y GATA H4) with the AmpFlSTR Yfiler™ PCR amplification kit (Applied Biosystems, Foster City, CA), following manufacture’s instructions. All YSTR amplification products were analyzed on a ABI PRISM 3100 Genetic Analyzer machine (Applied Biosystems). The size of each fragment was calculated automatically with the GeneMapper software version 3.2 and the alleles assigned by comparison to an internal size ladder included in the Y-filer kit. In Bologna, eight Y-SNPs located in the basal branches of the phylogenetic tree defining the major clades were additionally typed by minisequencing analysis using primer sequences published elsewere [14]. Autosomal STR loci In Bologna, a fluorescent multiplex polymerase chain reaction (PCR) was performed to amplify 15 tetranucleotide STR loci included in the AmpFlSTR Identifiler PCR amplification Kit, (Applied Biosystems): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S357, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA and amelogenin. The amplified products were separated with the ABI PRISM 310 Genetic Analyzer (Applied Biosystems) using GeneScan 500 LIZ as internal size standards and allelic ladders provided by the manufacturer. Results were analyzed using GeneScan Analysis software 3.7. To exclude possible contamination during sampling and analysis, DNA samples from a buccal swab of the gourd holder and the male laboratory technicians were also investigated. 6 HERC2 gene The king Louis XVI had blue eyes, as it can be seen in different portraits, including those painted by Antoine-François Callet in 1786 (Musée Carnavalet, Paris) or by Joseph-Siffred Duplessis in 1777 and 1785 (Musée National du Chateau de Versailles). Therefore, a further test for providing support to the attribution of the sample to Louis XVI is to check the SNP (rs12913832) that influences the blue eye colour in modern humans and that is located in the 86 exon of the HERC2 gene. The conserved region around rs12913832 is a regulatory region that controls the expression of the OCA2 gene [15]. The rs12913832*G allele inhibits the expression of OCA2, particularly within the iris melanocites, leading to blue eye colour [16,17]. In Barcelona, a couple of primers (Forward 5’-TGTCTGATCCAAGAGGCGAG-3’ and Reverse 5’GATGATAGCGTGCAGAACTTG-3’) were designed to amplify the HERC2 rs12913832 SNP in a short 67 bp amplicon, a size appropriated for degraded DNA. The amplification conditions were the same used previous studies [13], with 50ºC of annealing temperature. Results All presumptive blood samples obtained from inside the gourd were positive to the confirmatory blood tests performed. At the mtDNA HVR1, the majority of the cloned sequences (87%) showed a rare N1b haplotype, with the substitutions 16093T16145G-16176(G)-16223C. The same results were found in Bologna by direct sequencing, along with another substitution (16390G), not included in the amplicon generated in Barcelona. The substitutions found at the mtDNA HVR2(73G, 151T, 152C, 189G, 194T, 195C, 263G and 315.1C), are consistent with those from the HVR1. The resulting mtDNA haplotype is very rare and currently only found in two other 7 European individuals from Turkey and the Caucasus, among a database of 20,960 individuals gathered from the literature. In Barcelona, another residual haplotype was observed in four out of 25 clones (16%) for the 16055-16218 fragment and in two out of 21 clones (9.5%) for the 1620916378 fragment. The haplotype 16093-16192-16224 belongs most likely to the K haplogroup, and it is not contained in the current European database, although is phylogenetically close the 16093-16192-16224-16311 haplotype found in 2 out of 20,960 individuals. It does not correspond to any of the laboratory researchers involved in this study and it is not a known contaminant at the Institute of Evolutionary Biology in Barcelona, where none of the researchers (N=38) belong to the K haplogroup. The Y-filer profile gave positive results, which is indicative that the sample corresponds to a male. This was confirmed also by the amelogenin test in the AmpFlSTR Identifiler PCR amplification Kit. The AmpFlSTR Yfiler™ profile was repeated three times in Barcelona and once in Bologna. All genotypes were concordant between and within laboratories (Table 1). The Y-chromosome haplotype corresponds to a G2a haplogroup, an attribution confirmed by the additional Y SNPs typed in Bologna: M52A, M216G, M174A, M181T, M201T, M91A, M96G and M214A. To determine the significance of this finding a searched was performed at the YHRD database () and no matches were found among 21,800 haplotypes, including 6,382 from Eurasia. The autosomal STR profile of the sample gourd (Table 2) did not match the profiles obtained for the gourd holder and the laboratory researchers. Fourteen clones were generated for the rs12913832 SNP from the HERC2 gene. Six of them showed a G (variant associated to blue eyes colour) while eight showed an A (associated to brown colour). The approximately 50% ratio suggests that this individual was a G/A heterozygote. 8 Discussion The genetic results independently replicated are totally concordant, which points to a reasonably well preserved blood sample belonging to a male. The low frequency of the K haplotype found among the mtDNA clones and the fact that the Y-STR profile results are homogenous among laboratories and replicates point to an old residual maternal contamination in the sample of unknown origin. The fact that in Bologna the K haplotype has not been detected could indicate that the contaminant is not randomly distributed within the gourd or that it corresponds to a DNA background not detected by direct sequencing. All the genetic data seem to suggest that the majoritarily sequences are the endogenous ones. The amplification of the HERC2 gene provides controversial evidence on the physical appearance of the subject studied. Of course, lack of the rs12913832G allele would immediately imply that the subject is not Louis XVI because the presence of this variant is required for blue eyes. However, while most of the rs12913832 heterozygotes have hazel, brown or black eye colour, still about 15.8% of them (in a total sample of 388) have blue eyes [18]. The fact that both his parents, the Dauphin Louis-Ferdinand and Marie-Josèphe of Saxony had brown eyes, as shown in their respective portraits available, makes slightly more probable that Louis XVI was heterozygous at rs12913832, despite having blue eyes. An alternative explanation could be that the A sequences correspond to the contamination background detected in one laboratory at the mtDNA level. However this possibility seems unlikely, partially because of the different sequence ratios observed in both the mtDNA and the nuclear marker, although these extrapolations are sometimes problematic [19]. Additionally, we have no evidence of 9 nuclear DNA contamination since the autosomal STR profile from the sample is not found among the people involved in the study. At present it is not possible to prove genetically that the sample really belongs to the king Louis XVI. One possibility would be to extract a new sample from the dry heart attributed to the Dauphin Louis XVII, son of Louis XVI, preserved at the Basilique Saint-Denis in Paris, and compare both Y-chromosome profiles. Owing to the fact that the Y-chromosome profile found is not present in our current genetic databases such as YHRD, a potential match would directly authenticate the studied blood sample. Acnowledgments This work has been founded by a grant from the Ministerio de Ciencia e Innovación from Spain. References [1] P. Gill, P.L. Ivanov, C. Kimpton, R. Piercy, N. Benson, G. Tully, I. Evett, E. Hagelberg, K. Sullivan, Identification of the remains of the Romanov family by DNA analysis, Nat. Genet. 6(1994) 130–135. 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Genet. 82(2)(2008)424-431. [16] R.A.Sturm, M. Larsson, Genetics of human iris colour and patterns, Pigment Cell Melanoma Res. 22(5) (2009)544-562 [17] H.Eiberg, J. Troelsen, M,Nielsen, A. Mikkelsen, J. Mengel-From, K.W.Kjaer, L. Hansen, Blue eye color in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression, Hum. Genet. 123(2)(2008)177-187. [18] W.Branicki, U. Brudnik, A.Wojas-Pelc, Interactions between HERC2, OCA2 and MC1R may influence human pigmentation phenotype, Ann. Hum. Genet. 73(2)(2009)160-170. 12 [19] R.E. Green, A.W. Briggs, J. Krause, K. Prüfer, H.A. Burbano, M. Siebauer, M. Lachmann, S. Pääbo, The Neandertal genome and ancient DNA autenticity, EMBO J. 28(17)(2009)2494-2502. 13 Table captions Table 1 Y-STR profile for the putative Louis XVI sample, independently replicated in the Bologna and Barcelona laboratories. Table 2 Autosomal STR loci for the putative Louis XVI sample. Figure captions Figure 1 Picture of the gourd containing the presumptive blood of Louis XVI, depicting the portraits of French revolution leaders J. Danton, P. Marat, and C. Desmoulins. Figure 2 Picture of the gourd with the portraits of M. de Launay, Flesselles, and Foullon. Figure 3 Text-box on the gourd explaining –in French- the origin of the blood sample. English translation: “Maximilien Bourdaloue on January 21th, dipped his handkerchief in the blood of the king after his beheading”. 14 Marker Alleles (Bologna) DYS389I 12 DYS389II 30 DYS390 22 DYS456 15 DYS19 15 DYS385 13, 18 DYS458 21 DYS437 15 DYS438 10 DYS448 21 YGATAH4 12 DYS391 10 DYS392 11 DYS393 14 DYS439 12 DYS635 21 Alleles (Barcelona) 12 30 22 15 15 13, 18 21 15 10 21 12 10 11 14 12 21 15 Marker D8S1179 D21S11 D7S820 CSF1P0 D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 D5S818 FGA Amel Alleles (Bologna) 12-14 28-31.2 11-11 9-10 14-18 9-9.3 8-11 11-13 16-22 13-14 15-16 8-11 16-16 12-13 20-21 XY 16