Double mutations causing haemophilia B: a double whammy!

Correspondence Virchow’s triad I read with interest the recent Historical Review by Bagot and Arya (2008), which revisits the origins of Virchow’s Triad. I applaud the authors’ effort to impart a broader understanding of the use of this term and, to a similar end, I wish to offer the following: While others may have preceded him, I believe that Dr Charles A. Owen deserves important recognition for pointing-out that Virchow did not, himself, propose a triad for venous thrombosis. Owen (2001) reported this in his text: A History of Blood Coagulation; this preceded my own presentation (Dickson, 2003) and reports by others with an interest in this subject (Brotman et al, 2004; Malone, 2005). Finally, I believe that it is perhaps most accurate to suggest that the triad was misattributed to Virchow, rather than misinterpreted by our predecessors. Ultimately, a more comprehensive review of the German medical literature may one day hold important clues to the origins of this enigmatic eponym (Dickson, 2004). Brendan C. Dickson References Bagot, C.N. & Arya, R. (2008) Virchow and his triad: a question of attribution. British Journal of Haematology, 143, 180–190. Brotman, D.J., Deitcher, S.R., Lip, G.Y. & Matzdorff, A.C. (2004) Virchow’s triad revisited. Southern Medical Journal, 97, 213–214. Dickson, B.C. (2003) Venous thrombosis: on the history of Virchow’s triad. Clinical and Investigative Medicine, 26, 201–202. (Abstract). Dickson, B.C. (2004) Venous thrombosis: on the history of Virchow’s triad. University of Toronto Medical Journal, 81, 166–171. Malone, P.C. (2005) Further reflections on Virchow’s triad. Southern Medical Journal, 98, 125. Owen, C.A. (2001) A History of Blood Coagulation (ed. by W.L. Nichols & E.J.W. Bowie), pp. 169–180. Mayo Foundation for Medical Education and Research, Rochester. Keywords: venous medicine. thrombosis, coagulation, history of First published online 17 February 2009 doi:10.1111/j.1365-2141.2009.07617.x Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. E-mail: [email protected] Double mutations causing haemophilia B: a double whammy! Haemophilia B is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor IX gene (F9). The latest update of the haemophilia B Mutation Database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase. html) lists a total of 2891 patient entries, with 962 unique molecular events and the remainder being reported mutations. About 96% of the mutations leading to haemophilia B have been found to occur in the 2Æ2 kb of F9 that includes the promoter region, coding sequences and splice junctions (Roberts, 1993). The database also reveals 32 double mutations and 1 triple mutation in haemophilia B patients. One hundred and three haemophilia B patients were referred to our Comprehensive haemophilia Care Centre at Mumbai for F9 mutation analysis. Informed consent was obtained from all the patients and the study was approved by the Institute ethics committee. The clinical details involving number of bleeds, site of bleeding, clinical presentation, history of transfusion and other details were recorded in a clinical proforma. Factor IX coagulant activity (FIX: C) was determined by one-stage assay (Dacie & Lewis, 1991) and factor IX antigen (FIX: Ag) by enzyme-linked immunosorbent assay using commercial kits (Diagnostica Stago, Asniers, France). DNA analysis was done by polymerase chain reaction (PCR) using appropriate sets of primers (Hinks et al, 1999) in a PTC 225 thermal cycle (MJ Research, Boston, MA) followed by conformation sensitive gel electrophoresis (CSGE) as previously described (Williams et al, 1998; Jayandharan et al, 2005). The samples showing altered mobility were subjected to DNA sequencing in an automated sequencer ABI prism 310 Gene X using both forward and reverse primers. Haplotypes were constructed using the five polymorphic markers of F9: Dde I, Hha I, Mse I, Sal I and Mnl I (Peake et al, 1993). All sequence variants in F9 are reported using the standard sequence nomenclature described by the Human Genome Variation Society (HGVS). Sequence variants have also been checked manually by identifying the base position on the cDNA ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 145, 424–437 433 Correspondence Patient Exon Nucleotide change Amino acid change CRM status 1. 2. 3. 4. 5. 6. 7. 8. b, c e, h g, h g, h h h h h g.A6349C, g.T6696A g.A17705G, g.A31142G g.T30046C, g.A31142G g.T30046C, g.A31142G g.C31133T, g.A31142G g.A31160C, g.G31172A g.A31160C, g.G31172A g.A31124G, g.A31142G p. p. p. p. p. p. p. p. CRM) CRM) CRM) CRM) CRM) CRM) CRM) CRM+ N-9H, p. Y45N Q97R, p. K341E L198S, p. K341E L198S, p. K341E R338Stop, p. K341E N347T, p. A351T N347T, p. A351T T335A, p. E341K Clinical classification Severe Severe Mild Mild Severe Severe Severe Severe Table I. Double mutations detected in haemophilia B patients. The novel mutations are highlighted in bold. p. R338 and p. K341 were fairly conserved residues, suggesting their functional importance (Fig 1), which agreed fairly well with the prediction software. Although most patients with haemophilia B have one mutation, about 1% was reported to have double mutations (Farsani et al, 2008). Even in the factor IX mutation database, only 32 double mutations have been reported out of 2891 patient entries (1Æ1%). It is probable that the figure is under estimated as it is conventional for many laboratories to stop scanning the gene once they detect the causative mutation, especially in larger genes like F8 and F9. The high prevalence of double mutations in the present study is possibly due to the fact that the entire coding region was scanned and studied for the presence of mutations. None of these single nucleotide changes were detected in the control population, ruling out the possibility of them being subtle polymorphic changes. The cumulative impact of mutations on the phenotype is evident at least in a few cases in the mutation database. For instance, patients carrying either G30992C or C30970G mutations have a moderate deficiency of factor IX clotting activity, ranging from 3 to 6%. But the presence of two mutations in the same patient has shown a severe phenotype. A similar observation was also made in our cases. Both g.T6696A sequence for each gene. Previously reported single nucleotide polymorphisms were checked in the ‘Ensembl database’ (http://www.ensembl.org/). The functional importance of each of the mutations was confirmed by computational analysis using the panther software (http://www.pantherdb.org/). For studying the conservation status of the mutated residues, the multiple sequence alignment software, ClustalW2 was used (http://www.ebi.ac.uk/clustalw/) which produces biologically meaningful multiple sequence alignments of divergent sequences. Double mutations were detected in eight (1 related) of 103 patients (7Æ8%) screened for the presence of mutations, of which five were novel (Table I). None of the controls showed the presence of these molecular lesions. All the patients with double mutations were severely affected with <1% factor IX levels except the two siblings whose FIX: C levels were in the mild range of 5Æ6–12%. Among these eight patients, five had the same second molecular defect (1 related) i.e., g.A31142G in exon h of F9. The mothers of all the eight patients carried the same double mutation. All 8 patients except the siblings showed different haplotypes. Clustal analysis by multiple sequence alignment showed that p. L198, p. T335 and p. A351 residues were highly conserved in the vertebrates whereas Y45N Homo_sapiens_Human Pan_troglodytes_Chimpanzee Canis_lupus_familiaris_Dog Mus_musculus_Mouse Bos_taurus_Cattle Gallus_gallus_Chicken Q97R L198S T335A R338X K341E N347T A351T EFWKQYVDGDQ……QFCKNS……LNG-KV……TCL……RSTKFTIYNNMFCAGFHEG EFWKQYVDGDQ……QFCKNS……LNG-KV……TCL……RSTKFTIYNNMFCAGFHEG EFWKQYVDGDQ……QFCKLG……LNG-KV……TCL……RSTKFTIYNNMFCAGFHEG EFWKQYVDGDQ……QFCKNS……LNG-EI……TCL……RSTTFTIYNNMFCAGYREG WFVPVLTDTDL……IFLRSS……LHG-EI……TCL……RSTKFSIYSHMFCAGYHEG EFWKIYIDGDQ……HFCRHD……LVDSRG……TCL……KSTSTTILHSMFCAGYTAG * * * * : . * . . *** :**. :* *****: * Consensus symbols denoting the degree of conservation: * indicates that the residues or nucleotides in that column are identical in all sequences in the alignment. : indicates that conserved substitutions have been observed, according to the colour table above. . indicates that semi-conserved substitutions were observed. AVFPMILW Red Small (small + hydrophobic (incl.aromatic -Y)) DE Blue Acidic RK Magenta Basic STYHCNGQ Green Hydroxyl + Amine + Basic - Q Others Grey Fig 1. CLUSTAL 2Æ0Æ10 multiple sequence alignment of coagulation factor IX. 434 ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 145, 424–437 Correspondence and g.A31142G mutations individually have exhibited a milder phenotype; however, the presence of a second mutation has resulted in a severe phenotype. There are several instances of patients carrying the same mutation showing a wide range of factor IX levels. For instance, the G10430A mutation in exon d of F9 is a common mutation observed in all the patients with mild to moderate factor IX deficiency belonging to the Gujarati community from Western India (Quadros et al, 2007). There are several entries in the database of this mutation with F IX: C levels ranging between <1 and 36%. Whether the patients with severe phenotype have an additional mutation needs to be studied. Whether the presence of double mutations is a double set back to haemophilia patients in view of the clinical severity is still not very clear. Detection of two mutations will also have strong impact in genetic diagnosis of the affected families. There will be a chance of misdiagnosis if only one of the base changes is identified as the mutation in the index case without confirming the deleterious effect of this change. The same will be tracked in subsequent generations whereas the actual disease causing mutation will be missed if the remaining part of the gene is not scanned. In the absence of functional studies of the affected protein, the query will always remain as which variant is deleterious and implicated in the disease pathology. Until more patients with more than one mutation are reported and studied in great detail, the clinical significance of these mutations will remain unresolved. Kanjaksha Ghosh Shrimati Shetty Leera Quadros Bipin Kulkarni National Institute of Immunohaematology, KEM Hospital, Parel, Mumbai, India. References Dacie, J.V. & Lewis, S.M. (1991) Practical Hematology, 7th edn. Churchill Livingstone, UK. Farsani, M.A., Rastegar, G., Kazemi, A., Ala, F., Mohammadi, M., Ravanbood, S. & Allahbakhshian, A. (2008) Mutation detection in promoter region of coagulation factor IX in haemophilia B patients in Iran. Haematologica, 93(Suppl. 1), 553. Abstract 1465. Hinks, J.L., Winship, P.R., Makis, M., Preston, I.R. & Goodeve, A.C. (1999) A rapid method for haemophilia B mutation detection using CSGE. British Journal of Haematology, 104, 915–918. Jayandharan, G.R., Shaji, R.V., Baidya, S., Nair, S.C., Chandy, M. & Srivastava, A. (2005) Molecular characterization of factor IX gene mutations in 53 patients with haemophilia B in India. Thrombosis and Haemostasis, 94, 883–886. Peake, I.R., Lillicrap, D.P., Bouljenkov, V., Briet, E., Chan, V., Ginter, E.K., Kraus, E.M., Ljung, R., Mannucci, P.M., Nicolaides, K. & Tuddenham, E.G.D. (1993) Hemophilia: strategies for carrier detection and prenatal diagnosis. Bulletin of the World Health Organization, 71, 429–458. Quadros, L., Ghosh, K. & Shetty, S. (2007) A common G10430A mutation (Gly 60 Ser) in the factor IX gene describes the presence of moderate and mild hemophilia B in the majority of the Gujarati population. Annals of Hematology, 86, 377–379. Roberts, H.R. (1993) Molecular biology of hemophilia B. Thrombosis and Haemostasis, 70, 1–9. Williams, I.J., Abuzendah, A., Winship, P.R., Preston, F.E., Dolan, G., Wright, J., Peake, I.R. & Goodeve, A.C. (1998) Precise carrier and prenatal diagnosis in families with hemophilia A. Use of conformation sensitive gel electrophoresis for rapid mutation screening and polymorphism analysis. Thrombosis and Haemostasis, 79, 723–726. Keywords: haemophilia B, double mutations, conformation sensitive gel electrophoresis. First published online 19 February 2009 doi:10.1111/j.1365-2141.2009.07618.x E-mail: [email protected] Late onset hypogonadism: an alternate cause for night sweats in the haematology clinic We report two cases of septuagenarian men with essential thrombocythaemia (ET) and persistent intractable night sweats despite good disease control. Following the identification of late-onset hypogonadism (LOH) and the initiation of topical testosterone gel, the night sweats completely resolved with improved sense of well-being. The development of night sweats in a haemato-oncology patient may trigger the hunt for an underlying sinister cause, such as an atypical infection, lymphoma or disease progression. An increased awareness and earlier recognition of LOH in the older male haematology patient may reduce unnecessary investigation and anxiety. Case 1 presented at 78 years of age with an unexplained thrombocytosis following a bleeding duodenal ulcer, complicated by post-operative pneumonia. The platelet count remained elevated at 732 · 109/l eight months following his initial presentation, prompting further investigation. He reported a history of night sweats preceding the duodenal ulcer by six months with no other symptomatology. Past medical history included osteoporosis for which he was on calcium and vitamin D supplementation. A bone marrow biopsy confirmed the diagnosis of ET. Mutational analysis of the JAK2 gene was normal. He commenced cytoreductive ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 145, 424–437 435