The Boy in by Cheryld L. - PowerPoint Presentation

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:

Slide2

Time: early 1790s

Place: France

Situation: social and political upheaval

Part I - Background

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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 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

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The 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

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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-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

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Part

II:Biological Samplingfor dna

analysis6House of Bourbon

House of Habsburg

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Activities

Pedigree PreparationAlleles Identical By Descent

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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

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Robert

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

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A*A

AA

A*A

A*A

AA

AA

A*A

A*A

A*A*

Alleles Identical By Descent

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Inbreeding 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

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A

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

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Abbreviated 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

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Abbreviated 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

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Part III DNA Analysis

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Biological 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

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Sequences used by Jehaes et al

. 1998Sequence from the X-Y homologous amelogenin geneMitochondrial sequencesAnderson consensus sequence

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Amelogenin 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

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Cytoplasmic Inheritance of mtDNA

37 genesInherited from mother only

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Cytoplasmic 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

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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

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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

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Compare the sequences presented in Table 1 from Jehaes et al. (1998)

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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? They can be different

They should be mostly the sameThey should all be the same

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CQ#2: Are the sequences presented in this study consistent with the pedigree of relationship among these individuals?

Definitely, yesMaybe

Definitely, no

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CQ#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

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CQ#4: Do the mtDNA

sequences support Naundorff’s claim?

Definitely, yesMaybeDefinitely, no

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CQ#5: Based on this evidence, was Naundorff

truly Louis XVII?Yes

NoMaybe

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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.

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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

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Evaluate the DNA Results

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CQ#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

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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?

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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? Yes

NoMaybe

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CQ#8: Considering all of the evidence, is there conclusive proof that the heart is from Louis XVII?

Yes

No

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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

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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.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

.

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