The Boy in
the Temple:
Genetic Analysis of a
Royal Mystery
by
Cheryld L. Emmons
Division of Biology
Alfred University, Alfred, NY
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
Part I - Background
Time: early 1790s
Place: France
Situation: social and political upheaval
The Royal Family of France
• Louis XVI: King of France and Navarre
– (23 August 1754 – 21 January 1793)
• Marie-Antoinette: wife of Louis XVI
– (2 November 1755 – 16 October 1793)
• Their children
–
–
–
–
Marie-Thérèse-Charlotte
Louis-Joseph-Xavier-François (died before the revolution)
Louis-Charles (the future King Louis XVII of France)
Sophie-Hélène-Béatrix (died in infancy)
Marie-Antoinette with Marie-ThérèseCharlotte (left),
Louis-Joseph-Xavier-François (right), and
Louis-Charles (baby)
King Louis XVI
3
The events
• August 1792
– the royal family was imprisoned in the tower of the
Temple of Paris.
• January 1793
– Louis XVI was found guilty of treason and sent to the
guillotine.
• July 1793
– Louis-Charles was entrusted to the care of a Temple
commissioner and removed from the tower.
• October 1793
drawn by Le Petit, engraved by
Larbalestier 1853
– Marie-Antoinette was found guilty of treason and sent
to the guillotine.
• 1795
– Marie-Thérèse-Charlotte released from prison
– Reported death of Louis-Charles.
4
The controversy
• Louis-Charles’ body was never positively
identified by any family member prior to
burial.
• Rumors of a substitute who died and the
escape of Louis-Charles spread.
• Claimants came forward including Karl
Wilhelm Naundorff, who was able to convince
many in France (but not Marie-ThérèseCharlotte) that he was the lost prince.
• What analysis could be done to determine if
Naundorff truly was Prince Louis-Charles
(Louis XVII), son of King Louis XVI and Queen
Marie-Antoinette of France?
Louis-Charles, the future Louis
XVII. By Marie Louise Élisabeth
Vigée-Lebrun.
Karl Wilhelm Naundorff
5
House of Bourbon
House of Habsburg
PART II:
BIOLOGICAL SAMPLING
FOR DNA ANALYSIS
6
Activities
Pedigree Preparation
Alleles Identical By Descent
Prepare a Pedigree
• European royal families have vast pedigrees
that are fairly well documented.
• Produce a pedigree for this large, extended
(and somewhat inbred) family. Place stars (*)
next to the individuals who were included in
the DNA study.
8
Generation
I
Francis I
Maria Theresa
Ferdinand IV
Louis XVI
II
III
Johanna-Gabriella
Franz II
Maria-Josepha
Maria-Carolina
Louis Phillipe
Maria-Teresa
IV
Dom Pedro I
Marie-Antoinette
Maria –Amelie Marie-Theresa Louis-Charles
Charlotte
Leopold I
Maria-Leopoldinia Francois Ferdinand Helena Louise-Marie
V
VI
VII
VIII
Robert
Charlotte
Francisca
Francoise
Marie d’Orleans
Valdemar
Rene
Margaret
Anna
Andre
9
Alleles
Identical By
Descent
A*A
AA
A*A
AA
AA
A*A
A*A
A*A
A*A*
10
Inbreeding Coefficient
• Probability that an individual inherits two identical alleles
from a single ancestor
• The individual is said to be homozygous by descent
1
F=
2
n
• where n = number of ancestors in the loop
• If the common ancestor is inbred, then you must multiply
the coefficient by (1+FA), where FA is the coefficient of
inbreeding for common ancestor A.
• If there are multiple loops then you add up the F values for
each loop.
n
Fx = S
1
2
1 + FA
11
1
F=
2
n
A1*A1
A2A3
n = number of ancestors in
the loop
A1*A3
A1A1
A1*A1
F = (1/2)5 = 1/32 = 0.03125
A2A3
A1*A2
A1*A3
A1*A*1
12
Abbreviated pedigree for Francoise
Generation
I
Francis I
Maria Theresa
Ferdinand IV
Louis XVI
II
III
Johanna-Gabriella
Franz II
Maria-Josepha
Maria-Carolina
Louis Phillipe
Maria-Teresa
IV
Marie-Antoinette
Maria –Amelie
Dom Pedro I
Maria-Leopoldinia Francois Ferdinand Helena Louise-Marie
V
VI
Francisca
Francoise
N=6
F = (1/2)6 = 1/64 = 0.0156
13
Abbreviated pedigree for Marie d’Orleans
Generation
I
Francis I
Maria Theresa
Ferdinand IV
Louis XVI
II
III
Johanna-Gabriella
Franz II
Maria-Josepha
Maria-Carolina
Louis Phillipe
Maria-Teresa
IV
Marie-Antoinette
Maria –Amelie
Dom Pedro I
Maria-Leopoldinia Francois Ferdinand Helena Louise-Marie
V
VI
VII
Robert
Francisca
Francoise
Marie d’Orleans
Two paths:
to Maria-Carolina
to Maria-Amelie
F = (1/2)8 + (1/2)5 =
0.0352
14
Part III DNA Analysis
Biological samples were collected and
analyzed
• Naundorff
– Hair and right humerus were removed from his coffin
• Relatives of Marie Antoinette
– Hair samples from a rosary kept by Marie Theresa, verified
by documentation
– Blood samples from living descendants
• No samples were available from Louis XVI or any of his
relatives
• What DNA sequences could provide useful information
concerning the claim being made?
• Genetically, what do the individuals with stars on the
pedigree have in common?
16
Sequences used by Jehaes et al. 1998
• Sequence from the X-Y homologous
amelogenin gene
• Mitochondrial sequences
• Anderson consensus sequence
17
Amelogenin gene
• Located on the X and Y
chromosomes at Xp22.1Xp22.3 and Yp 11.2
• Differences between the
X chromosome (AMELX)
and Y chromosome
(AMELY) versions enable
it to be used in sex
determination of
unknown human
samples.
http://ghr.nlm.nih.gov/gene
18
Cytoplasmic Inheritance of mtDNA
• 37 genes
• Inherited from mother only
19
Cytoplasmic Inheritance of mtDNA
d-loop containing
HVR1 and HVR2
•
•
HyperVariableRegion1 (HVR1) and
HyperVariableRegion2 (HVR)
HaeIII Restriction Site Polymorphism
12S rRNA
between HyperVariableRegion1 and
HyperVariableRegion2
16S rRNA
origin
1 16,569
HVR2 at
73 – 340
HVR1 at
16,364-16024
ND1
Cyt b
ND6
ND5
Human Mitochondrial DNA
16569 bp
ND2
ND4
ND4L
ND3
COXIII
HaeIII restriction site at 16519
COXI
COXII
ATPase
20
Anderson Reference (consensus)
sequence
• aka the Cambridge Reference Sequence of the
16,569-base pair human mitochondrial
genome determined by Anderson et al. (1981)
and revised by Andrews et al. in 1999
• This reference sequence may be used for
baseline comparisons of individuals to
determine relationships.
21
Discussion Questions
• What information would the XY amelogenin
sequence provide for each individual sample?
• Why was mtDNA the appropriate DNA to use for
this analysis?
• Why are mtDNA sequences used rather than
autosomal DNA sequences?
• In general, could Y chromosome sequences
provide any further information? If so, what type
of information? Is that information critical to this
case?
22
Compare the sequences presented
in Table 1 from Jehaes et al. (1998)
CQ#1: Johanna-Gabriela (JG), Maria-Josepha (MJ),
Marie-Antoinette (MA), Anna (A) and Andre (AB) can all
trace their maternal lineage back to Maria Theresa.
What do we expect to see when comparing their
mtDNA sequences?
A. They can be different
B. They should be mostly the same
C. They should all be the same
33%
33%
m
e
sa
th
e
lb
e
m
d
ey
sh
ou
l
Th
Th
ey
sh
ou
l
d
al
be
be
ey
ca
n
Th
os
t ly
di
ffe
r
t..
en
t
33%
24
CQ#2: Are the sequences presented in this study
consistent with the pedigree of relationship
among these individuals?
33%
33%
,n
o
ly
ite
M
ay
be
33%
De
fin
De
fin
ite
ly
,
ye
s
A. Definitely, yes
B. Maybe
C. Definitely, no
25
CQ#3: If Naundorff was the son of MarieAntoinette as he claimed, what do we expect to
see when comparing their mtDNA sequences?
33%
ac
t ly
ex
be
sh
ou
l
ey
Th
Th
ey
Th
sh
ou
l
d
d
ey
ca
n
be
be
m
os
t ly
di
ffe
r
...
en
t
A. They can be
different
B. They should be
mostly the same
C. They should be
exactly the same
33%
...
33%
26
CQ#4: Do the mtDNA sequences
support Naundorff’s claim?
33%
33%
,n
o
ly
ite
M
ay
be
De
fin
De
fin
ite
ly
,
ye
s
A. Definitely, yes
B. Maybe
C. Definitely, no
33%
27
CQ#5: Based on this evidence, was
Naundorff truly Louis XVII?
33%
33%
33%
ay
be
M
No
Ye
s
A. Yes
B. No
C. Maybe
28
Part IV: Was the boy who died in 1795
Louis-Charles (proclaimed Louis XVII
upon the death of Louis XVI) or a
substitute?
• As was the custom for royalty, the heart was
removed and preserved.
• In 1999 this tissue was sampled and the DNA
was analyzed and compared to the previous
analysis.
29
Results
• Analysis of the X-Y homologous amelogenin
gene indicated that the heart was from a
male.
• An anatomical report estimated the age of the
child to be between 5 and 12 years.
– Louis-Charles would have been 10 at the time of
his reported death.
30
Evaluate the DNA Results
CQ#6: If the boy was the son of MarieAntoinette, what do we expect to see when
comparing their mtDNA sequences?
A. They can be different
B. They should be mostly the same
C. They should be exactly the same
33%
33%
...
ac
t ly
be
sh
ou
l
ey
Th
Th
ey
Th
ey
sh
ou
l
d
d
ca
n
be
be
m
ex
os
t ly
di
ffe
r
...
en
t
33%
32
Compare Marie-Antoinette’s HVR2
sequence to the sequence from her
sister Johanna-Gabriela.
Why could Marie-Antoinette’s
sequence be different from her sister’s
but not from her son’s?
33
CQ#7: Are the nucleotide substitutions found in
members of the Habsburg family and the heart tissue
of the boy consistent with the boy being related to this
family through cytoplasmic inheritance?
ay
be
33%
M
Ye
s
A. Yes
B. No
C. Maybe
33%
No
33%
34
CQ#8: Considering all of the evidence,
is there conclusive proof that the heart
is from Louis XVII?
50%
No
50%
Ye
s
A. Yes
B. No
35
Discussion Questions
• Who else could the DNA sample have been
from?
• Would you want any further information to
make a more conclusive decision? If so, what
information?
• If not, what is the most concrete evidence in
this case?
36
Image Credits
•
•
•
•
•
•
•
•
•
•
•
•
Slide 1: Painting of Tour du Temple, circa 1795j, painter unknown. Public domain, downloaded from
https://commons.wikimedia.org/wiki/File:Tour_du_Temple_circa_1795_Ecole_Francaise_18th_century.jpg.
Slide 2: Image from a battle during the French Revolution from http://resourcesforhistoryteachers.wikispaces.com/Key+Concept+5.3
(Contributions to http://resourcesforhistoryteachers.wikispaces.com/ are licensed under a Creative Commons Attribution Share-Alike
2.5 License.)
Slide 3: Marie-Anotinette and children and Louis XVI from http://en.wikipedia.org/wiki/Louis_XVI_of_France (Permission is granted to
copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later
version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts.)
Slide 4: The Temple http://www.antique-prints.de/shop/catalog.php?cat=KAT32&product=P004294 (http://www.antiqueprints.de/shop/catalog.php?page=use_of_images For non commercial illustration purposes, they are free of charge.
ex: universities, librairies, museums, research instituts, and related publications use. We would also very much appreciate a link or the
mention of our name.) The execution of Louis XVI from
http://en.wikipedia.org/wiki/Guillotine#/media/File:Execution_of_Louis_XVI.jpg
Slide 5: Louis-Charles from http://en.wikipedia.org/wiki/Louis_XVI_of_France and Karl Wilhelm Naundorff from
http://en.wikipedia.org/wiki/Karl_Wilhelm_Naundorff#/media/File:Naundorff1.jpg (Permission is granted to copy, distribute and/or
modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free
Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts.)
Slide 6: : House of Bourbon crest from http://en.wikipedia.org/wiki/House_of_Bourbon; House of Habsburg crest from
https://commons.wikimedia.org/wiki/File:Familienwappen_Habsburg-Stroehl.jpg.
Slide 9: Habsburg family pedigree created by author (Cheryld Emmons) from historical information gathered from multiple websites
and textbooks.
Slides 10 - 12: Alleles Identical By Descent graphics produced by author (Cheryld Emmons)
Slides 13 - 14: Abbreviated family pedigrees created by author (Cheryld Emmons).
Slide 18: Idiogram of X and Y chromosomes from http://http://ghr.nlm.nih.gov/dynamicImages/chromomap/chr-X.jpeg and
http://ghr.nlm.nih.gov/dynamicImages/chromomap/chr-Y.jpeg (Government information at NLM Web sites is in the public domain.
Public domain information may be freely distributed and copied, but it is requested that in any subsequent use the National Library of
Medicine (NLM) be given appropriate acknowledgement.)
Slide 19: Mitochondrial Inheritance from US National Library of Medicine
http://ghr.nlm.nih.gov/handbook/illustrations/patterns?show=mitochondrial (Government information at NLM Web sites is in the
public domain. Public domain information may be freely distributed and copied, but it is requested that in any subsequent use the
National Library of Medicine (NLM) be given appropriate acknowledgement.)
Slide 20: Mitochondrial chromosome map re-drawn by author (Cheryld Emmons) from Mitomap:
http://www.mitomap.org/pub/MITOMAP/MitomapFigures/mtDNAMorbidMap.pdf All site content, except where otherwise noted, is
licensed under a Creative Commons Attribution 3.0 License.
37