The ‘Royal Disease’

Authors


Peter Green, Department of Medical and Molecular Genetics, Kings College London, London, UK.
Tel.: + 20 71883707; fax: + 20 71882585.
E-mail: peter.green@genetics.kcl.ac.uk

Abstract

See also Ramírez L, Altisent C, Parra R, Vidal F. The ‘royal disease’ mutation in a Spanish patient. This issue, pp 2316–7.

Hemophilia is probably one of the best known single-gene disorders in the eyes of the public, not because of its frequency (others are more common), but because it occurred in the royal families of Europe. Queen Victoria was a carrier of the disease and had a son, Prince Leopold, who suffered from it (Fig. 1). At least two of her daughters were also carriers and passed it on to their children. In total, at least nine male descendants of Queen Victoria had hemophilia (there may have been more: the disease was an embarrassment to the monarchy, who generally kept as quiet as possible about it, and some that died in infancy may not have been reported). By far the most well-known hemophiliac is Alexei, only son of Tsar Nicholas II and Tsarina Alexandra, grand-daughter of Queen Victoria. It is also well known that Alexei and his family met their violent end during the Russian revolution in 1918, when they were all executed.

Figure 1.

 Hemophilia in the royal family. Selected descendants of Queen Victoria are shown, including all known hemophiliacs and carriers. Obligate carriers are those with a dot in the circle.

In recent years, the remains of the Tsar, Tsarina and their five children have been identified by DNA sequencing of mitochondrial DNA extracted from bone fragments [1,2]. However, the type of hemophilia in the family has remained a mystery since the last known hemophiliac of royal descent (Prince Waldemar of Prussia) died in 1945 – some 7 years before hemophilia A and B were distinguishable [3]. The last obligate carrier descended from Queen Victoria died in 1981 (Princess Alice, daughter of Prince Leopold), just a year before the first hemophilia gene was cloned [4]. Although it is possible that the mutation could have been passed down through a female line in Spanish descendants, this is unlikely.

It took a revolution in DNA sequencing technology to allow further investigation. Next-generation sequencing was the key that allowed researchers to at last use the vanishingly small traces of genomic DNA found in bone remains to screen for a possible causative mutation in the factor (F)VIII and FIX genes [5]. A priori, one might expect the disease to be hemophilia A, as it is about five times more common than hemophilia B, and so is statistically more likely to have been the cause. Furthermore, as about half of all hemophilia A cases are attributable to a large inversion [6], this might be expected to be the prime suspect. This inversion could not be identified during next-generation sequencing protocols carried out by Rogaev et al., as only the exonic regions were sequenced, and the inversion breakpoints are within intronic repeats. In the end, however, it turned out to be a mutation just inside an intron of FIX that creates a novel acceptor splice site, thereby confounding normal splicing. This effect was at first predicted from the sequence, but was difficult to prove, as the FIX gene is expressed almost exclusively in the hepatocytes. The authors sought to confirm that this was indeed the pathogenic mutation: first, they demonstrated that the mutation caused mis-splicing in an exon trap assay (although this was in COS cells); and second, they noted that the mutation has been reported three times previously (twice by researchers in the USA, and once from Spain). One of the US cases was shown to be a novel mutation arising in the maternal grandfather’s line [7,8]. The fact that it was shown to be novel is strong evidence that the mutation is causative (as a newly arising normal variant would be highly unlikely). This brings us to the Spanish case first reported by Vidal et al. in 2000 [9] and updated in a letter in this issue [10]. This living patient with severe hemophilia B has the same ‘royal’ mutation in the FIX gene (IVS3-3A>G). The first question that springs to mind is ‘has this been inherited in some way from the royal family?’ The question is appropriate, as the Spanish royal family also suffered from the stigma of the disease; two sons of King Alfonso XIII of Spain inherited hemophilia through his wife, Queen Victoria Eugenie (‘Ena’, who was a grand-daughter of Queen Victoria). However, Ramirez et al. have gone to some lengths to show that the mutation in their family has arisen de novo in the germline of the maternal grandfather of the patient or else as a somatic mutation in the patient’s mother.

Was the ‘Royal Disease’ the severe form of hemophilia? Certainly, it would appear so from contemporary reports on spontaneous bleeds in affected individuals. Now that we have a living patient with the same mutation, it can also be seen that this mutation results in < 1% FIX coagulant activity, which fits with the modern definition of severe hemophilia B.

In total, then, there appear to be at least four separate occurrences of this mutation, for two of which the origin has been pinpointed. Furthermore, in all likelihood, the royal mutation arose in the germline of Queen Victoria’s father, Prince Edward. So it is fair to say that the ‘Royal Disease’ has been diagnosed as hemophilia B and the pathogenic mutation pinpointed as IVS3-3A>G, which appears to create a novel acceptor splice site, thereby introducing a frameshift and predicting a truncated inactive protein. The only remaining question is ‘does this really happen in hepatocytes in vivo?’ However, in reality, there are no further suspects. Case closed.

Disclosure of Conflict of Interests

The author states that he has no conflict of interest.

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