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Abstract

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  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References

See also Green P. The ‘Royal Disease’. This issue, pp 2214–5.

A recent article has unraveled the uncertainty around the ‘royal disease’, a blood disorder transmitted from Queen Victoria to European royal families, by proving that it is a severe form of hemophilia B (HB) caused by a mutation that creates an abnormal splicing site in the F9 gene [1]. The authors convey their understandable interest in previously described patients with the ‘royal disease’ mutation [2,3]. There are now no living descendants of Queen Victoria with hemophilia; hence, patients with an identical mutation would represent excellent mimics of the phenotypic and molecular characteristics of the disease.

The Spanish patient with this mutation [2] is a 12-year-old boy with sporadic hemophilia, factor (F) IX levels always below 1%, and a first episode of hemarthrosis at 8 months of age. He has been receiving prophylactic treatment twice a week, beginning when he was 3 years old. His severe HB symptoms are exclusively attributable to the only alteration found, IVS3-3A > G, which supports the hypothesis that this mutation alone may be responsible for the disease that affected many members of the European courts during the 19th and 20th centuries.

In an attempt to determine the origin of the IVS3-3A > G mutation, that is, when the mutation arose de novo in this family, we investigated its genetic transmission in the patient’s relatives by direct sequencing of F9 and by X chromosome linkage studies. It was not possible to obtain a sample from the patient’s grandfather, who is not hemophilic. The mutation was found in the patient’s mother, but not in his grandmother or aunt (Fig. 1B). Because the aunt has all the polymorphic markers seen in the mother and she does not carry the mutation, a de novo event is highly probable, on the condition that both women have the same father. To rule out the possibility of a non-paternity event, 15 autosomal STR loci in the grandmother and her daughters were genotyped for kinship confirmation (Fig. 1C). Statistical analysis to test the cumulative likelihood ratio (CLR) of maternity/paternity vs. maternity/non-paternity yielded a value above 670, which is high enough to guarantee full sibship. Based on these studies, we can confidently conclude that the mutation is associated with the X chromosome inherited from the patient’s grandfather and is a consequence of a de novo somatic mutation that occurred in the patient’s mother or a germinal mosaicism in his grandfather. In either case, it would be a completely independent molecular event from that causing HB in the descendants of Queen Victoria.

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Figure 1.  (A) Partial nucleotide sequence of the boundary between intron 3 and exon 4 of F9 corresponding to the patient, his mother (carrier) and his grandmother (non-carrier). The rectangular box outlines the position of mutation IVS-3A > G, which is identical to the mutation described in Queen Victoria’s descendants. (B) Pedigree chart showing transmission of the putative mutation responsible for HB and the five polymorphic markers tested. A multiplex fluorescent PCR described by our group [6] comprising four X chromosome short tandem repeats (STR13, STR22, DXS1073, DXS1108), commonly used for tracing of F8 inheritance, was applied to genomic DNA samples. Study of these extragenic STRs and one intragenic F9 SNP (rs6048), informative in this family, indicates that the mutation was linked to the grandfather-inherited X-chromosome. (C) Tabulated results of 15 autosomal STR loci (AmpFLSTR Identifiler kit, Applied Biosystems, Foster City, CA, USA). Genetic profiles were obtained using an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems). Data collection and analysis were performed using GeneMapper software, version 3.7 (Applied Biosystems). Genotypes were in agreement with Mendelian inheritance between the sisters and mother. Identifiler genotypes were used with the EasyPat program [7] to compute the cumulative likelihood ratio (CLR) of maternity/paternity vs. maternity/non-paternity, which was calculated to be 6.71575 × 102 (a CLR of ≥ 100 is the standard to establish paternity). Results obtained using Familias software version 1.81 (Norwegian Computing Center, Oslo Norway) [7] were of equivalent statistical significance (posterior probability: 0.99822).

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Whatever the genetic origin of the mutation, what is more relevant in our view is accessibility to a patient with the ‘royal disease’ mutation, which provides a valuable opportunity to follow the clinical evolution of a disease that will predictably imitate the clinical evolution of affected members of the European royal families, as well as the possibility to carry out functional studies to corroborate and extend the experiments of Rogaev et al. [1]. Along this line, although we performed F9 mRNA studies from the patient’s lymphocytes, we have been unable to confirm or rule out the creation of a novel functional splice acceptor site. The results were inconclusive (data not shown) because of the well-documented difficulty of studying F9 expression when lymphocytes are the source of mRNA [4,5]. Our current technical challenge is to circumvent this inconvenience through transdifferentiation of the patient’s mature cells into hepatic lineage using induction of pluripotent stem cell technology and establish an in vitro model of the ‘royal disease’.

Disclosure of Conflict of Interests

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  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References

The authors state that they have no conflict of interest.

References

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  2. Abstract
  3. Disclosure of Conflict of Interests
  4. References