polyclonal hypergammaglobulinaemia with hyperviscosity syndrome

Correspondence

TIME FOR A REDEFINITION OF TYPE 2M VON WILLEBRAND DISEASE

The recent paper by Riddell et al (2002) was interestingand timely. However, the results were entirely predictablewithout any experimentation because of preselection ofpatients and the currently used definition of type 2M vonWillebrand disease (VWD). Both the ristoctin cofactor(VWF:RCo) assay and the collagen-binding (VWF:CB)assay will be sensitive to loss of von Willebrand factor(VWF) protein and especially to the loss of the higher-molecular-weight (HMW) multimers. The current defini-tion of type 2M VWD is �decreased platelet-dependentfunction that is not caused by the absence of HMW-VWFmultimers�. Defects in the VWF glycoprotein Ib bindingsite will lead to loss of platelet-dependent function, causetype 2M VWD by this definition, and be detected by theVWF:RCo (but not the VWF:CB). Defects in the VWFcollagen binding site will lead to loss of collagen-depen-dent function, cause a currently unclassified form ofVWD, and be detected by the VWF:CB (but not theVWF:RCo).

I suggest that �platelet-dependent� is removed from thedefinition of type 2M VWD. Type 2M can then be subtypedas type 2Mp – decreased platelet-dependent function (theold 2M), or as type 2Mc – decreased collagen-dependentfunction. The VWF:RCo detects only the former and theVWF:CB only the latter. That type 2Mc exists is clear (Ribba

et al, 2001). We will only know the relative importance oftype 2Mp and type 2Mc when the VWF:CB is used as widelyas the VWF:RCo.

David KeelingOxford Haemophilia Centre andThrombosis Unit, ChurchillHospital, Headington, Oxford, UK.E-mail: [email protected]

REFERENCES

Ribba, A.S., Loisel, I., Lavergne, J.M., Juhan-Vague, I., Obert, B.,

Cherel, G., Meyer, D. & Girma, J.P. (2001) Ser968Thr mutation

within the A3 domain of von Willebrand factor (VWF) in tworelated patients leads to a defective binding of VWF to collagen.

Thrombosis and Haemostasis, 86, 848–854.

Riddell, A.F., Jenkins, P.V., Nitu-Whalley, I.C., McCraw, A.H., Lee,

C.A. & Brown, S.A. (2002) Use of the collagen-binding assay forvon Willebrand factor in the analysis of type 2M von Willebrand

disease: a comparison with the ristocetin cofactor assay. British

Journal of Haematology, 116, 187–192.

Keywords: type 2M von Willebrand disease, VWF:RCo,VWF:CB.

POLYCLONAL HYPERGAMMAGLOBULINAEMIA WITH HYPERVISCOSITY SYNDROME

Amati et al (2002) describe a case of polyclonal hypervis-cosity in a human immunodeficiency virus (HIV)-infectedman. This is not the first report, as suggested. A case wasreported by Martin et al (1989) and we reported two casesfrom Zimbabwe (Paul et al, 1990). As the majority of HIVcases are in subsaharan Africa, it is likely that this is not asrare as reported but reflects a lack of diagnostic facilities inmany of these countries. Both our cases died rapidly afterthe diagnosis was made.

Beverley PaulDepartment of Haematology &Blood Transfusion, BassetlawDistrict General Hospital, Worksop,Nottinghamshire, UK.E-mail: [email protected] [email protected]

REFERENCES

Amati, F., Canellini, G. & Beris, P. (2002) Polyclonal hypergam-

maglobulinaemia with hyperviscosity syndrome. British Journal

of Haematology, 116, 2.

Martin, C.M., Matlow, A.G., Chew, E., Sutton, D. & Pruzanski, W.(1989) Hyperviscosity syndrome in a patient with acquired

immunodeficiency syndrome. Archives of Internal Medicine, 149,

1435–1436.

Paul, B., Houston, S. & Latif, A. (1990) HIV infection associatedwith the plasma hyperviscosity syndrome: a report of two fatal

cases. AIDS, 4, 1302–1304.

Keywords: hyperviscosity, HIV, polyclonal gammopathy.

British Journal of Haematology, 2002, 118, 922–928

922 � 2002 Blackwell Science Ltd

POSSIBILITY OF SOMATIC MOSAICISM OF ELA2 MUTATION OVERLOOKED

IN AN ASYMPTOMATIC FATHER TRANSMITTING SEVERE CONGENITAL NEUTROPENIA

TO TWO OFFSPRING

The article by Germeshausen et al (2001) describes apedigree in which severe congenital neutropenia (CN) hasbeen transmitted by a father to two children. Sequenceanalysis of the neutrophil elastase gene (ELA2) identifiedheterozygous changes at amino acid 122 in both theaffected children as well as the father. The authors interpretthe lack of neutropenia in the father to be evidence that themutation in ELA2 is not sufficient to cause CN. CN is knownto be primarily a sporadic disease and as such the authorshave overlooked the most likely interpretation of theirresults which is that the father is mosaic for the C122Ymutation. Somatic and germline mosaicism is a commonoccurrence in severe genetic diseases in which reducedgenetic fitness mandates that de novo mutations account fora substantial proportion of new cases. These includeDuchenne’s muscular dystrophy, achondroplasia, tuberoussclerosis, Hunter’s disease, osteogenesis imperfecta, retino-blastoma, neurofibromatosis type 1 and haemophilia A(Leuer et al, 2001 and references within). Most recently,Ancliff et al (2001a) have demonstrated somatic mosaicismof a C42R ELA2 mutation in the father of a girl presentingwith CN. Further analysis determined that, while about halfthe father’s lymphocytes and other cells contained the C42RELA2 mutation found in his daughter, < 5% of hisneutrophils contained a mutant allele, unequivocallydemonstrating the pathogenic nature of ELA2 mutationon neutrophil production and CN genesis.

The authors go on to suggest that their one case provesthat mutations in the ELA2 gene are not the single causeof CN; however, it has never been stated that there is asingle cause of CN. An elegant study by Ancliff et al(2001b) has shown that mutations in ELA2 account forapproximately 75% of sporadic cases of CN, a figuresimilar to that obtained by Dale et al (2000). ELA2mutations are less common in familial cases of the diseaseand linkage analysis and the exclusion of the 19p13.3region in one pedigree with autosomal dominant CNsuggests that at least one other gene is involved (Ancliffet al, 2001b). The possibility that this other gene or genesmay function in a common pathway or actually directlyregulate neutrophil elastase expression is supported by theauthors themselves who have pointed out that CN patientswith undetectable mutations in the ELA2 gene nonethelessdemonstrate reduced elastase activity (Germeshausen et al,2000).

In closing, the demonstration of somatic mosaicism in thecase of CN presented by Germeshausen et al (2001), whichwe believe to be a likely scenario that has not beenaddressed, would lead to the opposite conclusion as thatproffered by the authors: mutations in ELA2 are causative ofCN and sufficient.

Kathleen F. Benson

Marshall Horwitz

Markey Molecular MedicineCenter, Division of MedicalGenetics, Department of Medicine,School of Medicine, Universityof Washington, Seattle,WA, USA. E-mail:[email protected]

REFERENCES

Ancliff, P.J., Gale, R.E., Hann, I.M., Strobel, S. & Lynch, D.C.(2001a) Paternal mosaicism proves the pathogenic nature of

mutations in neutrophil elastase in severe congenital neutrope-

nia. Blood, 98, 439a.

Ancliff, P.J., Gale, R.E., Liesner, R., Hann, I.M. & Lynch, D.C.(2001b) Mutations in the ELA2 gene encoding neutrophil elas-

tase are present in most patients with sporadic severe congenital

neutropenia but only in some patients with the familial form of

the disease. Blood, 98, 2645–2650.Dale, D.C., Person, R.E., Bolyard, A.A., Aprikyan, A.G., Bos, C.,

Bonilla, M.A., Boxer, L.A., Kannourakis, G., Zeidler, C., Welte, K.,

Benson, K.F. & Horwitz, M. (2000) Mutations in the geneencoding neutrophil elastase in congenital and cyclic

neutropenia. Blood, 96, 2317–2322.

Germeshausen, M., Schulze, H., Ballmaier, M., Lang, M., Schaper,

Y., Zeidler, C. & Welte, K. (2000) Decreased neutrophil elastaseactivity in patients with severe congenital neutropenia is not

correlated with mutations in ELA2. Blood, 96, 610a.

Germeshausen, M., Schulze, H., Ballmaier, M., Zeidler, C. &

Welte, K. (2001) Mutations in the gene encoding neutrophilelastase (ELA2) are not sufficient to cause the phenotype of

congenital neutropenia. British Journal of Haematology, 115,

222–224.Leuer, M., Oldenburg, J., Lavergne, J.-M., Ludwig, M., Fregin, A.,

Eigel, A., Ljung, R., Goodeve, A., Peake, I. & Olek, K. (2001)

Somatic mosaicism in hemophilia A: a fairly common event.

American Journal of Human Genetics, 69, 75–87.

Keywords: congenital neutropenia, somatic mosaicism,ELA2 mutation.

REPLY TO BENSON AND HORWITZ

In their comment regarding our article (Germeshausenet al, 2001), Kathleen F. Benson and Marshall Horwitzrefer to the possibility of a germline mosaic for an ELA2

mutation in the father of two children suffering fromcongenital neutropenia (CN). Indeed, a somatic mosaicismof the ELA2 mutation C42R in the father of a girl

� 2002 Blackwell Science Ltd, British Journal of Haematology 118: 922–928

Correspondence 923

presenting with CN was demonstrated by Ancliff et al(2001). We presented our data at the same meeting(Germeshausen et al, 2001), demonstrating a mosaicismfor an ELA2 mutation in the father of the siblings sufferingfrom CN described in our paper. Comparable to the casedescribed by Ancliff et al (2001), we found approximately50% mutated cells in CD3+ cells and in haematopoieticprogenitors (CFU-GM). However, in contrast to the resultsof Ancliff et al (2001), we also detected the ELA2 mutationin 31% of the mature neutrophils of the father, excludingan effective depletion of neutrophil precursors bearing theELA2 mutation C122Y. In our opinion, these results alsoargue against a simple causal relationship between ELA2mutations and the neutropenia seen in CN.

The conclusions made in our manuscript are notimpaired by these new data: we argued that ELA2 muta-tions are not sufficient for causing CN, although �they mightbe a necessary prerequisite�. We, like Drs Benson andHorwitz, think that the increased occurrence of ELA2mutations in CN argues for the pathophysiological signi-ficance of this protein, however, we propose a more complexpathomechanism for CN.

In the father of a CN patient demonstrating the mosaicismdescribed by Ancliff et al (2001), < 10% of the neutrophils(< 5% mutant alleles), about half of the CD3+ cells, but 20%of the CD3– cells (most likely B lymphocytes and monocytes)contained a mutant allele. The latter fact raises the questionon the fate of the monocytes: the monocytosis seen in CN(up to 10 · 109 monocytes ⁄ l) also argues against a deple-tion of cells bearing an ELA2 mutation.

To recapitulate the arguments against a single mono-causal correlation between ELA2 mutations and CN: todate, no one has demonstrated a correlation between theclinical phenotype in CN (granulocyte colony-stimulatingfactor requirement, leukaemic development, infections) andthe presence of ELA2 mutations. There is an overlap of themutations found in cyclic neutropenia (CyN) and CN (e.g.C26Y, S97L, P110L, cluster of mutations at the intron 4splice donor site), which makes it difficult to explain thevariation in phenotype and clinical severity, in particularthe different risks for malignant transformation (CyNpatients do not develop leukaemia). Until now, there hasbeen no pathophysiological concept to explain the effects ofsuch a variety of different ELA2 mutations. The extensivework of Li & Horwitz (2001) has shown no hint for a toxicgain of function and ⁄ or an apoptotic mechanism for anumber of different ELA2 mutations. However, they foundthat mutant-elastase forms inhibited the wild-type enzymewhen both were co-expressed within the same cell, leading

to a decreased elastase activity for some mutants. Indeed,we found a decreased activity of neutrophil elastase ingranulocytes of both CN and CyN patients, but independentof the occurrence and type of ELA2 mutations (Germes-hausen et al, 2000). Decreased elastase activity alone seemsunlikely to be the pathomechanism for CN, as mice lackingneutrophil elastase (Belaaouaj et al, 1998), as well as miceengineered to express a mutated neutrophil elastase (V72M,detected in two unrelated CN patients), did not show a CNphenotype (Grenda et al, 2001).

We hope, that the combined efforts of all groupsworking in this field and a lively unbiased discussion willelucidate the role of ELA2 mutations in CN and CyN in thenear future.

Manuela Germeshausen

Matthias Ballmaier

Karl Welte

Department of PaediatricHaematology and Oncology,Medizinische Hochschule,Hannover, Germany.E-mail: [email protected]

REFERENCES

Ancliff, P.J., Gale, R.E., Hann, I.M., Strobel, S. & Lynch, D.C. (2001)

Paternal mosaicism proves the pathogenic nature of mutations inneutrophil elastase in severe congenital neturopenia. Blood, 98,

439a.

Belaaouaj, A., McCarthy, R., Baumann, M., Gao, Z., Ley, T.J.,

Abraham, S.N. & Shapiro, S.D. (1998) Mice lacking neutrophilelastase reveal impaired host defense against gram negative

bacterial sepsis. Nature Medicine, 4, 615–618.

Germeshausen, M., Schulze, H., Ballmaier, M., Lang, S., Schaper, Y.,

Zeidler, C. & Welte, K. (2000) Decreased neutrophil elastaseactivity in patients with severe congenital neutropenia is not

correlated to mutations in ELA2. Blood, 96, 610a.

Germeshausen, M., Ballmaier, M., Zeidler, C., Schulze, H. & Welte,K. (2001) Mutations in the gene encoding neutrophil elastase

(ELA2) are not sufficient to cause congenital neutropenia. Blood,

98, 440a.

Grenda, D.S., McLemore, M.L., Johnson, S.E., Horwitz, M. & Link,D.C. (2001) Mice expressing neutrophil elastase bearing the

V72M mutation found in patients with Severe Congenital

Neutropenia do not display an SCN phenotype. Blood, 98,

440a.Li, F.Q. & Horwitz, M. (2001) Characterization of mutant neutrophil

elastase in severe congenital neutropenia. Journal of Biological

Chemistry, 276, 14230–14241.

Keywords: congenital neutropenia, ELA2 mutations,neutrophil elastase.

THE USE OF VITAMIN K FOR REVERSAL OF OVER-WARFARINIZATION IN CHILDREN

We have followed the discussion about reversal of warfarintherapy with interest (Pendry et al, 2001; Watson et al,2001). An increasing number of young children are nowreceiving long-term warfarin, following surgery for majorheart defects. Control of therapy is more difficult than in

adults (Streif et al, 1999). Few data are available aboutwarfarin reversal in children; fresh-frozen plasma is notappropriate for non-bleeding patients and may not produceadequate correction in bleeding patients (Makris et al,1997). Blood products should be avoided in small children


924 Correspondence

because of anxieties concerning possible transmission ofinfectious agents.

Because low-dose vitamin K (0Æ5–1 mg) produces alowering of a high International Normalized Ratio (INR)to near or within the therapeutic range in adults (Hunget al, 2000), we decided to extrapolate this to children withhigh INRs (venous INR > 8). The INR was successfullylowered in six out of seven cases (Fig 1) with a single dose of30 lg ⁄ kg (1 ⁄10th of the full dose) given intravenously(doses ranging from 350 to 1000 lg). These six childrenwere well and treated as outpatients. One other sickinpatient with deranged liver function tests required threedoses to bring the INR into the therapeutic range. Ourweight-adjusted regimen is safer than a universal dosage(0Æ5–2 mg subcutaneously). Even 0Æ5 mg (sufficient formany adults) is likely to be too high for most young children(too high for five of our seven children). Seriously illchildren with liver dysfunction may require more than asingle dose; this is one reason why it is important to confirmadequate correction at a chosen time interval: 4–6 h inthose at highest risk or 24 h in milder cases.

Oral or subcutaneous vitamin K is effective in adults (Rajet al, 1999), but the INR fall is slower than i.v. injection.Our preferred route is therefore i.v., but in a child with poorvenous access, the subcutaneous or oral route may be used,particularly if the INR is 6–10. A paediatric protocol should

include specific advice that vitamin K should not be givenintramuscularly to children on anticoagulants because ofthe risk of intramuscular haemorrhage. Paediatricians aremore used to giving Vitamin K i.m., as this is standardpractice for neonatal prophylaxis.

Paula Bolton-Maggs

Lynda Brook

Royal LiverpoolChildren’s Hospital,Liverpool, UK.E-mail: [email protected]

REFERENCES

Hung, A., Singh, S. & Tait, R.C. (2000) A prospective randomized

study to determine the optimal dose of intravenous vitamin K in

reversal of over-warfarinization. British Journal of Haematology,

109, 537–539.Makris, M., Greaves, M., Phillips, W.S., Kitchen, S., Rosendaal, F.R.

& Preston, E.F. (1997) Emergency oral anticoagulant reversal:

the relative efficacy of infusions of fresh frozen plasma and clot-ting factor concentrate on correction of the coagulopathy.

Thrombosis and Haemostasis, 77, 477–480.

Pendry, K., Bhavnani, M. & Shwe, K. (2001) The use of oral

vitamin K for reversal of over-warfarinization. British Journal ofHaematology, 113, 839–840.

Raj, G., Kumar, R. & McKinney, W.P. (1999) Time course of

reversal of anticoagulant effect of warfarin by intravenous and

subcutaneous phytonadione. Archives of Internal Medicine, 159,2721–2724.

Streif, W., Andrew, M., Marzinotto, V., Massicotte, P., Chan, A.K.,

Julian, J.A. & Mitchell, L. (1999) Analysis of warfarin therapy inpediatric patients: a prospective cohort study of 319 patients.

Blood, 94, 3007–3014.

Watson, H.G., Baglin, T., Laidlaw, S.L., Makris, M. & Preston, F.E.

(2001) A comparison of the efficacy and rate of response to oraland intravenous Vitamin K in reversal of over-anticoagulation

with warfarin. British Journal of Haematology, 115, 145–149.

Keywords: over-warfarinization, warfarin reversal, vitaminK, children.

REVERSAL OF COUMARIN-INDUCED OVER-ANTICOAGULATION

The recent article in this journal on the management ofcoumarin-induced over-anticoagulation (Makris & Watson,2001) provides a general review on oral anticoagulants andthe risk of bleeding. Also, important management recom-mendations are given for the reversal of oral anticoagula-tion. I agree with their recommendations on the treatmentof non-emergency situations with stopping the anticoagu-lant and ⁄ or giving vitamin K. On the other hand, for thetreatment of life-threatening bleeds where complete andrapid reversal of anticoagulation is needed, the authorsrecommend withholding the anticoagulant, administrationof vitamin K and the use of fresh-frozen plasma (FFP) orprothrombin complex concentrates (PCCs). The use of FFPor PCCs have multiple disadvantages as described in the

article and both are plasma-derived products. A newproduct, currently on the market, is recombinant factorVIIa (NovoSeven, Denmark). In my opinion, this is a betteralternative and efficacious treatment for fast reversal of oralanticoagulants.

Recombinant factor VIIa (FVIIa) is an activated coagu-lation factor that is approved in Europe and the USA forthe treatment of patients with congenital haemophilia withinhibitors. The effect of recombinant FVIIa in coagulationis through the formation of a complex with tissue factor atthe site of injury, generating sufficient amounts of throm-bin to activate platelets and to form a clot. The reason forits use in the setting of vitamin K deficiency from oralanticoagulants is to replace factor VII given its short

Fig 1. Low-dose i.v. vitamin K for warfarin reversal in children.


Correspondence 925

half-life and low levels. Recombinant FVIIa has beenshown to completely normalize the prothrombin time (PT)in animals and humans with acquired deficiencies of thevitamin K-dependent factors (Diness et al, 1990; Bernsteinet al, 1997; Erhardtsen et al, 1998; Muleo et al, 1999).Erhardtsen et al (1998) evaluated the use of different doses(5–320 lg ⁄ kg) of intravenous recombinant FVIIa tohealthy volunteers that had an international normalizedratio (INR) greater than 2 while on acenocoumarol. Afterone dose of 5 lg ⁄ kg of recombinant FVIIa, the INRnormalized for 12 h, and doses between 120 and320 lg ⁄ kg normalized the PT for 24 h. Muleo et al(1999) treated four patients with deficiencies of vitaminK with small doses of recombinant FVIIa, in preparationfor surgery without hemorrhagic complications.

NovoSeven is an alternative treatment for rapid reversalof oral anticoagulation. Advantages for its use are minimaltransmission of blood-borne agents, good safety profile,immediate effect and ease of use.

Miguel A. EscobarUniversity of North Carolina atChapel Hill, Department ofHematology ⁄Oncology, Chapel Hill,NC, USA. E-mail: [email protected]

REFERENCES

Bernstein, D.E., Jeffers, L., Erhardtsen, E., Reddy, K.R., Glazer, S.,

Squiban, P., Bech, R., Hedner, U. & Schiff, E.R. (1997)

Recombinant factor VIIa corrects prothrombin time in cirrhotic

patients: a preliminary study. Gastroenterology, 113, 1930–1937.

Diness, V., Lund-Hansen, T. & Hedner, U. (1990) Effect of

recombinant human FVIIa on warfarin-induced bleeding in rats.Thrombosis Research, 59, 921–929.

Erhardtsen, E., Nony, P., Dechavanne, M., Ffrench, P., Boissel, J.P. &

Hedner, U. (1998) The effect of recombinant factor VIIa (Novo-

Seven) in healthy volunteers receiving acenocoumarol to anInternational Normalized Ratio above 2.0. Blood Coagulation

and Fibrinolysis, 9, 741–748.

Makris, M. & Watson, H.G. (2001) The management of coumarin-

induced over-anticoagulation. British Journal of Haematology,114, 271–280.

Muleo, G., Santoro, R., Iannaccaro, P.G., Papaleo, P., Leo, F., Zap-

pala, D. & Elia, L. (1999) Small doses of recombinant factor VIIa

in acquired deficiencies of vitamin K dependent factors. BloodCoagulation and Fibrinolysis, 10, 521–522.

Keywords: anticoagulation, warfarin, recombinant factorVIIa, anticoagulation reversal.

REPLY TO ESCOBAR

We thank Dr Escobar for his comments but disagree thatrecombinant factor VIIa (rFVIIa) concentrate should be thetreatment of choice for warfarin-induced bleeding. rFVIIawas introduced for the treatment of haemophilic patientswith inhibitors and we are convinced of its efficacy in thissetting. More recently, it has been advocated as a universalhaemostatic agent but, in the setting of warfarin-inducedbleeding, we feel that at present it has no place asmonotherapy.

The major flaw in Dr Escobar’s conclusion relates to themisconception that correction of the prothrombin timeequates with complete correction of the underlying coag-ulopathy. We accept that rFVIIa will correct the prolongedprothrombin time of volunteers taking coumarins. Theprothrombin time is, however, an in vitro test and amarker for only part of the coagulopathy associated withcoumarin therapy as it is normal in patients withundetectable levels of factor IX. It is important that thelimitations of in vitro testing, especially using broad-basedtests of coagulation, are recognized. For example, while weaccept that rFVIIa may have a role in securing haemo-stasis in a variety of clinical situations such as hereditaryor acquired platelet function disorders, it is clear that thisphysiological function is not best measured by theshortening of the prothrombin time which universallyaccompanies its administration.

We are unaware of any publication in a major peer-reviewed journal documenting the efficacy of rFVIIa instopping the bleeding of patients anticoagulated withwarfarin. Although it may be that in future rFVIIa will beshown to be effective in this situation (clinical trials arebeing carried out by the manufacturer), we believe thatthere is insufficient evidence at present to recommend thisproduct for this indication. Until such evidence becomesavailable, if ever, best clinical practice dictates that theappropriate therapy for bleeding in a subject on warfarin isthe specific correction of the relevant coagulation factorlevels.

M. Makris1

H. G. Watson2

1Sheffield Haemophilia andThrombosis Centre, RoyalHallamshire Hospital, Sheffield, and2Department of Haematology,Aberdeen Royal Infirmary,Aberdeen, UK. E-mail: [email protected]

Keywords: warfarin-induced bleed, recombinant factorVIIa.


926 Correspondence

REGARDING THE LOSS OF CD20 AFTER RITUXIMAB THERAPY

We read with interest the recent short report by Foran et al(2001) regarding the apparent loss of CD20 expressionfollowing treatment with rituximab. We noticed, however,that the possibility of immunohistochemical staining inhi-bition (blocking) of the CD20 antibody used (L26) byrituximab, at least in recently treated patients, did not seemto be considered in the interpretation of the results. Thisproperty of antibodies could possibly explain some of thefindings reported. In fact, the �acute loss� of CD20 expressionin the one post-mortem case may be attributed to blockingof the L26 antibody binding by the recently infused(circulating) rituximab. Another issue apparently notaddressed by the authors is the possibility that bone marrowprecursor B cells (haematogones), which are CD79a+ but donot express CD20 in early maturational stages (McKennaet al, 2001), may have been incorrectly interpreted asresidual ⁄ recurrent disease. The interpretation of the datamay be more valid if these issues were addressed.

Ronald Lee

Raul C. Braylan

Department of Pathology andLaboratory Medicine, University ofFlorida College of Medicine,Gainesville, FL, USA.E-mail: [email protected]

REFERENCES

Foran, J.M., Norton, A.J., Micallef, I.N., Taussig, D.C., Amess, J.A.,

Rohatiner, A.Z. & Lister, T.A. (2001) Loss of CD20 expression

following treatment with rituximab (chimaeric monoclonal anti-

CD20): a retrospective cohort analysis. British Journal of Haema-tology, 114, 881–883.

McKenna, R.W., Washington, L.T., Aquino, D.B., Picker, L.J. &

Kroft, S.H. (2001) Immunophenotypic analysis of hematogones(B-lymphocyte precursors) in 662 consecutive bone marrow

specimens by 4-color flow cytometry. Blood, 98, 2498–2507.

Keywords: rituximab, haematogones, CD20.

CONSEQUENCES AT mRNA LEVEL OF THE PKLR GENE SPLICING MUTATIONS IVS10(+1)G fi C

AND IVS8(+2)T fi G CAUSING PYRUVATE KINASE DEFICIENCY

Pyruvate kinase (PK; EC 2Æ7Æ1Æ40) is an important regulatorof glycolysis by the catalytic conversion of phosphoenol-pyruvate to pyruvate. Two different PK genes present inmammals encode four isoenzymes: PKM gene encodesisoenzymes M1 (skeletal muscle) and M2 (fetal and mostadult tissues) (Noguchi et al, 1986), and PKLR gene encodesisoenzymes L (liver) and R (erythrocyte) (Noguchi et al,1987).

In a previous report, we described two PKLR genemutations associated with PK deficiency, IVS10(+1)G fi Cand IVS8(+2)T fi G, that change the 5¢ splice donor site GTto CG on intron 10 and to GG on intron 8 (Manco et al,1999). One homozygous patient for mutation IVS10(+1)G fi C (patient 1) and two homozygous patients formutation IVS8(+2)T fi G (patients 2 and 3) presented atbirth with severe haemolytic anaemia and required regularblood transfusions until the spleen was removed at the age of4 years. Patient 1 also needed a cholecystectomy, due tomultiple gallstones. The three patients are now 15, 11 and10 years old, their growth and development are normal. Thehaemoglobin levels remain stable, around 8 g ⁄ dl, with highreticulocyte counts, 30–40%. Patient 1 has a more severeclinical phenotype, with 2–3 severe haemolytic crises everyyear, needing blood transfusion. The other two patients havesporadic haemolytic crises.

In this report, we present the consequences ofIVS10(+1)G fi C and IVS8(+2)T fi G mutations on reti-culocyte mRNA structure.

Reticulocyte poly A+ mRNAs of patients 1 and 2 and offour normal controls were reverse transcribed as previouslydescribed (Manco et al, 2000). cDNAs were amplified bypolymerase chain reaction (PCR) with oligonucleotides:8F-5¢-ATCGAGATCCCAGCAGAGAA-3¢ and 9R-5¢-AGTCT-CCCCTGACAGCATGA-3¢; 10F-5¢-GGCAGCTGTTTGAGGA-GCTA-3¢ and 11R-5¢-GGAGGTTCACGGTAAAGCAA-3¢;9F-5¢-AGCATGATTACCAAGCCCC-3¢ and 11R (Fig 1A).Reverse transcription (RT)-PCR fragments were sequencedon an ALFexpress II DNA Analysis System using theThermo Sequenase fluorescent-labelled primer cyclesequencing kit (Pharmacia Biotech).

Patient 1, homozygous for mutation IVS10(+1)G fi C.The semiquantitative RT-PCR procedure described in Mancoet al (2000) showed normal amounts of mRNA transcripts.RT-PCR products spanning exons 9–11 (Fig 1A) showed afaster moving band on agarose gel in relation to normalcontrols (not shown). Sequencing of cDNA between exons 9and 11 showed a normal nucleotide sequence of exons 9and 11, lacking exon 10 (Fig 1B). The IVS10(+1)G fi Cmutation, abolishing the intron 10 donor splice site, leads tothe skipping of exon 10 in the mRNA transcript. The


Correspondence 927

predicted translation of the abnormal mRNA terminatesprematurely in exon 11 (CCGTGA1556) resulting in atruncated R-PK polypeptide with 462 residues.

Patient 2, homozygous for mutation IVS8(+2)T fi G.The semiquantitative RT-PCR procedure showed normalamounts of mRNA transcripts. Agarose gel electrophoresisof RT-PCR products spanning exons 8–9 (Fig 1A) showeda slower moving band in relation to normal controls (notshown). cDNA sequence showed the insertion of the first20 nucleotides of intron 8 between exons 8 and 9 (Fig 1C).The splicing system recognized a cryptic splice site (aggt)19 nucleotides 3¢ to the IVS8(+2)T fi G substitution.Predicted translation of the abnormal mRNA transcript isinterrupted by a stop codon (GCATGA1132) in exon 9,resulting in a truncated R-PK subunit 383 amino acidslong.

Due to the polypeptide structural alterations, the R-PKenzyme activity should be absent in both cases. Theresidual PK activity (18% and 15% of normal in patients 1and 2 respectively) is probably associated with thepersistence of the PK-M2 isoenzyme in affected erythro-cytes. The more severe clinical phenotype in patient 1,may be related to the 79 residues longer polypeptidetranslated from the IVS10(+1)G fi C transcript. Wehypothesize that the degradation of the longer transla-tional product of 462 amino acids, overloads the proteo-lytic mechanism of the cell more than the shorter 383amino acids chain.

LICINIO MANCO1,2

CELESTE BENTO1

M. LETICIA RIBEIRO1

GABRIEL TAMAGNINI1

1Unidade de HematologiaMolecular, Centro Hospitalar deCoimbra, and 2Departamento deAntropologia, Faculdade de Cienciase Tecnologia, Universidade deCoimbra, Portugal. E-mail:[email protected]

REFERENCES

Manco, L., Ribeiro, M.L., Almeida, H., Freitas, O., Abade, A. &Tamagnini, G. (1999) PK-LR gene mutations in pyruvate kinase

deficient Portuguese patients. British Journal of Haematology,

105, 591–595.

Manco, L., Ribeiro, M.L., Maximo, V., Almeida, H., Costa, A.,Freitas, O., Barbot, J., Abade, A. & Tamagnini, G. (2000) A new

PKLR gene mutation in the R-type promoter region affects the

gene transcription causing pyruvate kinase deficiency. BritishJournal of Haematology, 110, 993–997.

Noguchi, T., Inoue, H. & Tanaka, T. (1986) The M1 and M2-type

enzymes of rat pyruvate kinase are produced from the same gene

by alternative RNA splicing. Journal of Biological Chemistry, 261,13807–13812.

Noguchi, T., Yamada, K., Inoue, H., Matsuda, T. & Tanaka, T.

(1987) The L- and R-Type isozymes of rat pyruvate kinase are

produced from a single gene by use of different promoters. Journalof Biological Chemistry, 262, 14366–14371.

Keywords: pyruvate kinase deficiency, PKLR gene splicingmutations, mRNA analysis, cryptic splice site, exonskipping.

Fig 1. Sequence of the R-PK cDNA amplified fragments of patients1 and 2. (A) Diagram of the PKLR gene structure and position of

primers used to amplify cDNAs. Agarose gel electrophoresis

revealed the same normal band pattern, in relation to normal

controls, for patient 1 cDNA amplified with primers 8F-9R, and forpatient 2 cDNA amplified with primers 9F-11R and 10F-11R (not

shown). (B) Sequence of RT-PCR products spanning exons 9–11 in

patient 1, homozygous for IVS10(+1)G fi C mutation. The frag-ment shows the normal nucleotide sequence of exon 9 3¢-region

and of exon 11 5¢-region, lacking exon 10. (C) Sequence of RT-PCR

products spanning exons 8–9 in patient 2, homozygous for

IVS8(+2)T fi G mutation. The fragment shows the normal nuc-leotide sequence of 3¢-region of exon 8 and 5¢-region of exon 9 and,

between them, the insertion of the first 20 nucleotides of intron 8.

A

B

C


928 Correspondence

polyclonal hypergammaglobulinaemia with hyperviscosity syndrome

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