N. Lannoy and C. Hermans FIX VIII FVIII FIX X X. Fig. 1. II Yekaterinburg

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1 N Lannoy and C Hermans Centre de Génétique humaine; and Service d hématologie, Cliniques universitaires Saint-Luc, Brussels, Belgium 20 1 VIII FVIII A IX FIX B 1991 II FIX 4 B 20 II FIX X VIII FVIII IX FIX X X FVIII FIX Correspondence: Professor Cedric Hermans, MD, MRCP (UK), PhD, Haemostasis and Thrombosis Unit, Haemophilia Clinic, Division of Haematology, St-Luc University Hospital, Avenue Hippocrate 10, 1200 Brussels, Belgium Tel: ; fax: ; cedrichermans@uclouvainbe Haemophilia (2010), 16, Blackwell Publishing Ltd Fig XIII II Yekaterinburg 20

2 A B Albert of Saxe-Coburg I 1 2 II Edward VII Alice 4 5 Louis IV of Hesse Alfred 6 Helen 7 Louise 8 Arthur 9 Leopold Queen 10 Helena of Waldeck 11 Beatrice III English Royal Family Elisabeth Irène Henri de Ernest Frédéric Alexandra Prusse Nicolas II (tsar) Alexandre Alice (Espagne) 12 Henry of Battenberg Léopold Maurice IV Waldemar Henri Alexis Maurice Ruprecht Alphonse Beatriz Maria Cristina Juan Gonzalo Hemophilic male Carrier female Normal female Prussian Royal Family Russian Imperial Family Spanish Royal Family Normal male Elisabeth II Juan Carlos Fig 1 Queen, grandmother of Europe XIII XIII XIII II II A B Beatriz Fig 1 IV-14 IV II Yekaterinburg 21

3 Full Translation: N Lannoy and C Hermans DNA DNA 2 DNA DNA DNA DNA mtdna 2 Gill 1 DNA ,000 DNA mtdna II 3 Fig 2 II 2 DNA II 1 II 2 II = 3 mtdna II DNA II C/T T II mtdna Louise of Hesse-Kassel Queen Grand Duke George Czar Nicolas II Czarina Alexandra Duke of Fife Xenia Sfiris Olga Tatiana Maria Alexis Anastasia Prince Philip Duke of Edinburgh Study of mitochondrial DNA markers by Gill et al [1] allowing identification of the imperial couple and their three children Study of mitochondrial DNA markers by Gill et al [1] in the Romanov family and confirmed by AFDIL [3] Study of mitochondrial DNA markers by Ivanov et al [2] to establish the authenticity of the remains of Czar Nicolas II Study of mitochondrial DNA markers by AFDIL [3] allowing identification of the Grand Duchess Maria and the Czarevitch Alexis Fig 2 Mitochondrial DNA study in the Romanov family 22

4 A B II Evgeny Rogaev DNA Russian Academy of Sciences University of Massachusetts Armed Forces DNA Iden ti fica tion Laboratory Gregor Mendel Institute mtdna DNA Y Edouard Rossel II DNA DNA Rogaev Russian Academy of Medical Sciences 5 DNA 117 bp Illumina Genome Analyzer FVIII 26 FIX 8 32 PCR 180 FVIII 22 1 DNA FIX 4 3 bp A > G - IVS3-3A > G Fig 3 DNA RNA RNA mrna 5,500 mrna 9998 B FIX 1 B X X 23

5 Full Translation: N Lannoy and C Hermans (a) (c) (b) Fig 3 Presence of a mutation at the splice acceptor site of exon 4 (a) Schematic representation of the eight exons of the F9 gene, as well as the localization of primers having allowed the amplification of exons coding for the gene (b) Detail of the intron/exon junction of exon 4 In position 3 as compared to the beginning of the exon 4 sequence on the 5 3 fragment, in the DNA of Czarevitch Alexis, the A nucleotide normally present has been replaced by a G This substitution creates a new cryptic splice acceptor site which produces an aberrant frameshift transcript that results in a stop 11 codons downstream (c) Among the Imperial coupleõs four daughters, the IVS-3A > G genetic anomaly is found at the heterozygote state only in Anastasia, confirming that she was a haemophilia carrier DNA FIX B References 1 Gill P, Ivanov PL, Kimpton C et al Identification of the remains of the Romanov family by DNA analysis Nat Genet 1994; 6: Ivanov PL, Wadhams MJ, Roby RK, Holland MM, Weedn VW, Parsons TJ Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II Nat Genet 1996; 12: Report_26_Feb_09 AFDIL_pdf Accessed February 26, Rogaev EI, Grigorenko AP, Moliaka YK et al Genomic identification in the historical case of the Nicholas II royal family Proc Natl Acad Sci USA 2009; 106: Rogaev EI, Grigorenko AP, Faskhutdinova G, Kittler EL, Moliaka YK Genotype analysis identifies the cause of the royal disease Science 2009; 326: 817 [Epub 8 October 2009] 6 =SUSTITUTION Accessed March 23, version 13,