Emmons Division of Biology Alfred University Alfred NY NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE Genetic Analysis of a Royal Mystery the Temple Time early 1790s Place France ID: 932786
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Slide1
The Boy in
by
Cheryld L. EmmonsDivision of BiologyAlfred University, Alfred, NY
NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
Genetic Analysis of a Royal Mystery
the Temple:
Slide2Time: early 1790s
Place: France
Situation: social and political upheaval
Part I - Background
Slide3The 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 childrenMarie-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-
Thérèse
-Charlotte (left),
Louis-Joseph-Xavier-François (right), and Louis-Charles (baby)
King Louis XVI
3
Slide4The events
August 1792the royal family was imprisoned in the tower of the Temple of Paris.January 1793Louis 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 1793Marie-Antoinette was found guilty of treason and sent to the guillotine.1795Marie-Thérèse-Charlotte released from prisonReported death of Louis-Charles.drawn by Le Petit, engraved by Larbalestier 1853
4
Slide5The 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-Thérèse-Charlotte) 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
Slide6Part
II:Biological Samplingfor dna
analysis6House of Bourbon
House of Habsburg
Slide7Activities
Pedigree PreparationAlleles Identical By Descent
Slide8Prepare 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
Slide9Robert
Franz II Louis Phillipe
Francis I
Maria Theresa
Johanna-Gabriella Maria-Josepha Maria-Carolina Marie-Antoinette
Ferdinand IV Louis XVI
Maria-Teresa Maria –Amelie Marie-Theresa Louis-Charles
Charlotte
Anna Andre
Margaret
Marie
d’Orleans
Rene
Valdemar
Maria-
Leopoldinia
Francois Ferdinand
Helena
Louise-Marie
Dom Pedro I
Francisca
Leopold I
Charlotte
Francoise
Generation
I
II
III
IV
V
VI
VII
VIII
9
Slide10A*A
AA
A*A
A*A
AA
AA
A*A
A*A
A*A*
Alleles Identical By Descent
10
Slide11Inbreeding Coefficient
Probability that an individual inherits two identical alleles from a single ancestorThe individual is said to be homozygous by descentwhere 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.
F =
12
n
F
x
=
1
2
n
S
1 + F
A
11
Slide12A
1
*A
1
A
2
A
3
A
1
*A
2
A
1
*A
3
A
2
A
3
A
1
A
1
A
1
*A
1
A
1
*A
3
A
1
*A*
1
n
= number of ancestors in the loop
F = (1/2)
5
= 1/32 = 0.03125
F =
1
2
n
12
Slide13Abbreviated pedigree for Francoise
Francis I
Maria Theresa
Johanna-Gabriella Maria-Josepha Maria-Carolina Marie-Antoinette
Ferdinand IV Louis XVI
Maria-Teresa Maria –Amelie
Franz II Louis Phillipe
Maria-
Leopoldinia
Francois Ferdinand
Helena
Louise-Marie
Dom Pedro I
Francisca
Francoise
Generation
I
II
III
IV
V
VI
N = 6
F = (1/2)
6
= 1/64 = 0.0156
13
Slide14Abbreviated pedigree for Marie d’Orleans
Francis I
Maria Theresa
Johanna-Gabriella Maria-Josepha
Maria-Carolina
Marie-Antoinette
Ferdinand IV Louis XVI
Maria-Teresa
Maria –Amelie
Franz II Louis Phillipe
Marie
d’Orleans
Maria-
Leopoldinia
Francois Ferdinand
Helena
Louise-Marie
Dom Pedro I
Francisca
Robert
Francoise
Generation
I
II
III
IV
V
VI
VII
Two paths:
t
o Maria-Carolina
t
o Maria-Amelie
F = (1/2)
8
+ (1/2)
5
= 0.0352
14
Slide15Part III DNA Analysis
Slide16Biological samples were collected and analyzed
NaundorffHair and right humerus were removed from his coffinRelatives of Marie AntoinetteHair samples from a rosary kept by Marie Theresa, verified by documentation
Blood samples from living descendantsNo samples were available from Louis XVI or any of his relativesWhat 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
Slide17Sequences used by Jehaes et al
. 1998Sequence from the X-Y homologous amelogenin geneMitochondrial sequencesAnderson consensus sequence
17
Slide18Amelogenin gene
Located on the X and Y chromosomes at Xp22.1-Xp22.3 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
Slide19Cytoplasmic Inheritance of mtDNA
37 genesInherited from mother only
19
Slide20Cytoplasmic Inheritance of
mtDNA
d-loop containing HVR1 and HVR212S rRNA
16S rRNA
origin
1 16,569
HVR2 at HVR1 at
73 – 340 16,364-16024
HaeIII
restriction site at 16519
Human Mitochondrial DNA
16569
bp
Cyt
b
ATPase
COXI
COXII
COXIII
ND1
ND2
ND3
ND4
ND5
ND6
ND4L
HyperVariableRegion1 (HVR1) and HyperVariableRegion2 (HVR)
HaeIII
Restriction Site
Polymorphism
between
HyperVariableRegion1
and
HyperVariableRegion2
20
Slide21Anderson 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
Slide22Discussion 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
Slide23Compare the sequences presented in Table 1 from Jehaes et al. (1998)
Slide24CQ#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? They can be different
They should be mostly the sameThey should all be the same
24
Slide25CQ#2: Are the sequences presented in this study consistent with the pedigree of relationship among these individuals?
Definitely, yesMaybe
Definitely, no
25
Slide26CQ#3: If Naundorff was the son of
Marie-Antoinette as he claimed, what do we expect to see when comparing their mtDNA sequences?They can be different
They should be mostly the sameThey should be exactly the same
26
Slide27CQ#4: Do the mtDNA
sequences support Naundorff’s claim?
Definitely, yesMaybeDefinitely, no
27
Slide28CQ#5: Based on this evidence, was Naundorff
truly Louis XVII?Yes
NoMaybe
28
Slide29Part 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
Slide30Results
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
Slide31Evaluate the DNA Results
Slide32CQ#6: If the boy was the son of Marie-Antoinette
, what do we expect to see when comparing their mtDNA sequences?They can be different
They should be mostly the sameThey should be exactly the same
32
Slide33Compare 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
Slide34CQ#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? Yes
NoMaybe
34
Slide35CQ#8: Considering all of the evidence, is there conclusive proof that the heart is from Louis XVII?
Yes
No
35
Slide36Discussion 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
Slide37Image 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.antique-prints.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