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.
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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