Ajetunmobi, JF

Carella, M

Delaunay, J

Iolascon, A

Schettini Jr, F

Stewart, GW

English

UCL Discovery

Haematologica 1999; 84:862-864 scientiﬁc lettersgenes (genetic heterogeneity). Mapping of the DHStand FP genes7,9 now allows us to address this prob-lem by means of a linkage analysis. Genomic DNAwas obtained from each member of a pedigreedescribed elsewhere.9 Linkage analysis was per-formed using 8 microsatellite markers (D16S511,D16S3037, D16S520, D16S498, D16S3074,D16S3026, D16S3121) from the chromosome 16region where we have previously mapped both DHStand FP.7,8 The analysis reveals the absence of segre-gation between the above mentioned markers andthe disease locus, as shown in Table 1. These resultsindicate that these pseudohyperkalemic variants aregenetically heterogenous with a locus on 16q23 andat least one additional locus, which remains to bemapped. Work is in progress to map this new locusand to understand the molecular basis10 underlyingthis interesting form of stomatocytosis.Massimo Carella,* Gordon W. Stewart,° Joanne F. Ajetunmobi,°Federico Schettini Jr,# Jean Delaunay,@ Achille Iolascon#*Servizio di Genetica Medica, IRCCS-CSS, San Giovani Rotondo(FG), Italy; °Department of Medicine, Rayne Institute, London, UK;#Dipartimento di Biomedicina dell’Età Evolutiva, Università di Bari,Bari, Italy; @Service d’Hématologie, Hôpital de Bicêtre, AssistancePublique-Hôpitaux, INSERM U473, Le Kremlin-Bicêtre, FranceFundingThis work was supported by the Telethon Projects E783(to MC), the MURST, the Ministero Italiano delle Sanità(Italy), Action Research and The Wellcome Trust.Key wordsDehydrated hereditary stomatocytosis (DHSt), hemoly-sis, linkage analysis, pseudohyperkalemia CorrespondenceAchille Iolascon, M.D., Dipartimento di Biomedicina dell’EtàEvolutiva, piazza G. Cesare 11, 70124 Bari, Italy. Phone: inter-national +39-080-5478923 – Fax: international +39-080-5592290 – E-mail: a.iolascon@bioetaev.uniba.itReferences1.  Lock SP, Septhon Smith R, Hardistry RM. Stomato-cytosis: a hereditary red cell anomaly associated withhaemolytic anaemia. Br J Haematol 1961; 7:303-14.2.  Glader BE, Fortier N, Albala MM, Nathan DG. Con-genital hemolytic anemia associated with dehydratederythrocytes and increased potassium loss. N Engl JMed 1974; 291:491-6.3.  Entezami M, Becker R, Menssen HD, MarcinkowskiM, Versmold HT. Xerocytosis with concomitantintrauterine ascites: ﬁrst description and therapeuticapproach. Blood 1996; 87:5392-3.4.  Grootenboer S, Schischmanoff PO, Cynober T, et al.A genetic syndrome associating dehydrated heredi-tary stomatocytosis, pseudohyperkalaemia andperipheral oedema. Br J Haematol 1998; 103:383-6.5.  Miller G, Townes PL, Macwhinney JB. A new congen-ital hemolytic anaemia with deformed erythrocytes(?”stomatocytes”) and remarkable susceptibility oferythrocytes to cold hemolysis in vitro. PediatricsGenetic heterogeneity of hereditarystomatocytosis syndromes showingpseudohyperkalemiaSir,The hereditary stomatocytoses (HSt) are a groupof rare, dominantly inherited hemolytic anemiasshowing abnormal membrane permeability to theunivalent cations Na+ and K+. They are clinically andpathophysiologically heterogenoeous. The groupincludes  overhydrated HSt (OHSt), the original condi-tion,1 dehydrated HSt (DHSt; hereditary xerocyto-sis),2 the commonest form, now seen to be het-erogenous,3,4 cryohydrocytosis,5 in which red cellslyse markedly if stored at low temperatures, andfamilial pseudohyperkalemia,6a non-hemolytic con-dition in which the red cells show a net loss of K+ atroom temperature, manifesting as high plasma [K]levels. The gene responsible for both DHSt and famil-ial pseudohyperkalemia (FP) has been mapped to16q23-qter.7,8 In spite of the variability of the pre-sentation, all kindreds that we have mapped so farshow linkage to this locus.Recently, Coles9 described an unusual form ofhereditary stomacytosis associated with very markedpseudohyperkalemia. Temperature studies of thepassive leak ﬂuxes of K showed the same shallow slopeabnormality as seen in classical familial pseudohy-perkalemia, but in addition, the ﬂuxes at 37°C weresignificantly increased; and frank hemolysis, withabnormal intracellular [Na] and [K] levels, was pre-sent. Superﬁcial examination might imply that thisnew condition is simply a more severe, franklyhemolytic, version of ‘classical’ FP.We have sought to establish whether the clinicaland biochemical heterogeneity of these conditionsstems from different mutations of the same gene(allelic heterogeneity) or from mutations in differentHaematologica vol. 84(9):September 1999Table 1. Pairwise LOD scores between FP family and chro-mosome 16 markers.Recombinant frequencies 0.0 0.01 0.05 0.10 0.20 0.30 0.40 Zmax qmaxD16S402 - Ï -9.01 -4.90 -3.21 -1.64 -0.82 -0.32 -2.75 0.69D16S511 - Ï -9.30 -5.18 -3.46 -1.84 -0.97 -0.40 -5.64 0.78D16S3037 - Ï -4.18 -2.16 -1.33 -0.58 -0.22 -0.05 -9.05 0.5D16S520 - Ï -10.7 -5.88 -3.87 -1.96 -0.96 -0.36 -1.79 0.63D16S498 - Ï -4.26 -2.23 -1.40 -0.63 -0.27 -0.07 -4.44 0.53D16S3074 - Ï -7.90 -4.44 -2.98 -1.58 -0.81 -0.32 -2.88 0.71D16S413 - Ï -2.51 -1.18 -0.67 -0.25 -0.08 -0.01 -2.48 0.50D16S3026 - Ï -10.7 -5.88 -3.87 -1.96 -0.96 -0.36 -1.79 0.63D16S3121 - Ï -8.27 -4.79 -3.30 -1.84 -1.00 -0.43 -1.02 1.001965; 35:906-15.6.  Stewart GW, Corrall RJ, Fyffe JA, Stockdill G, StrongJA. Familial pseudohyperkalaemia. A new syndrome.Lancet 1979; 28:175-7.7.  Carella M, Stewart G, Ajetunmobi JF, et al. A map-ping of dehydrated hereditary stomatocytosis (hered-itary xerocytosis) locus to chromosome 16 (16q23-qter) by genome wide search. Am J Human Genet1998; 63:810-6.8.  Iolascon A, Stewart G, Ajentoumbi JF, et al. Hereditaryxerocytosis and pseudohyperkaliemia map to thesame locus. Blood 1999; 93:3120-3.9.  Coles S, Ho M, Chetty M, Nicolaou A, Stewart G. Avariant of Hereditary stomatocytosis with markedpseudohyperkalaemia. Br J Haematol 1999 (in press).10. Iolascon A, Miraglia del Giudice E, Perrotta S, AlloisioN, Morle L, Delaunay J. Hereditary spherocytosis: fromclinical to molecular defects. Haematologica 1998;83:240-57.In vitro drug-induced cytotoxicity predictsclinical response to high-dose chlorambucil inB-cell chronic lymphocytic leukemiaSir,Chlorambucil (CLB) can be considered the goldenstandard of  front-line therapy for B-cell chronic lym-phocytic leukemia (CLL).1 In spite of its long use, thebest schedule of CLB administration has not yet beenestablished. We have demonstrated the clinicaladvantage of a high-dose continuous CLB schedule(HD-CLB) over standard-dose intermittent CLB2andthe CHOP regimen.3 In addition, according to theresults of an interim analysis of the ongoing ran-domized EORTC study, the efficacy of HD-CLB isapparently not inferior to that of ﬂudarabine,4 thusconﬁrming that we can now two efﬁcient therapeu-tic options for front-line therapy of CLL.We recenlty demonstrated that in vitro drug-sensi-tivity to fludarabine is associated with in vivoresponse.5This study was aimed at analyzing whetherthe ability of CLB to induce in vitrocytotoxicity in freshisolated CLL cells might be related to the in vivo out-come of patients treated with the HD-CLB schedule. Twenty-nine previously untreated CLL patientsentered this study. Clinico-hematologic data, CLB-LD50 value and the in vivo clinical response to high-dose CLB are reported in Table 1. Criteria for treat-ment, high-dose CLB schedules and deﬁnitions ofresponse have been described elsewhere.2-4The drug-induced effect on cell viability was assessed by a non-clonogenic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay that has been repeatedlyused to test in vitro chemosensitivity of CLL cells toCLB.7, 8 Cells were continuously exposed to the drugfor 4 days. Median CLB-LD50value was 25, 16 and 58µg/mL in patients obtaining complete (CR), partial(PR) and no response (NR), respectively (p=ns forKruskall Wallis test).Rai and Binet stage, TTM score and age (Table 2)did not affect the response rate (CR+PR). However, asigniﬁcantly higher number of responses was observedin males and in those patients showing in vitro CLB-LD50 values below 26 µg/mL. In spite of the relativelysmall number of cases, the relevance of this parame-ter was conﬁrmed by multivariate analysis. The shortmedian follow-up time hampers a statistically reliableanalysis of survival. However, since response to thera-py is a parameter well known to be associated withlonger survival,9 it is reasonable to expect that in vitroresistance to CLB coulde be an early indicator of anoverall unfavorable outcome of patients treated withthis alkylating agent. In conclusion, in our experience the in vitro MTTassay is a useful tool for predicting in vivo treatmentfailure to HD-CLB in CLL. Thus, from the resultsobtained in cytotoxicity tests induced by differentScientiﬁc correspondence 863Haematologica vol. 84(9):September 1999Table 1. Main clinico-hematological data, CLB-LD50 valueand clinical response to high-dose CLB.Sex, male/female  13/16Age, mean ± sem  69.9 ± 2.5Rai stage 01I4II 9III 11IV 4Binet stageA7B1 1C1 1TTM score, mean ± sem 12.4 ± 0.72≤ 95> 9 24CLB-LD50 µg/mL (mean ± sem) 47.1 ± 7.2Clinical response  CR 16PR 6NR 7Table 2. Univariate and logistic multivariate regressionanalysis of prognostic variables for clinical response to HD-CLB.Variable CR+PR NR Uni-* Multi-°variateppRai stage (0-II v III-IV) 11 v 11 3 v 4 1.0 ---Binet stage (A-B v C) 15 v 7 3 v 4 0.3 ---TTM score (≤ 11.4 v > 11.4) 11 v 11 5 v 2 0.4 ---Age, years (≤ 74 v > 74) 11 v 11 4 v 3 1.0 ---Sex (male v female) 13 v 9 0 v 7 0.008 nsCLB-LD50, µg/mL (≤ 26 v > 26) 14 v 8 1 v 6 0.035 0.05*Fisher exact test (2-tail); °Logistic regression analysis.

A genetic syndrome associating dehydrated hereditary stomatocytosis, pseudohyperkalaemia and peripheral oedema. Br J Haematol

A mapping of dehydrated hereditary stomatocytosis (hereditary xerocytosis) locus to chromosome 16 (16q23-qter) by genome wide search.

A variant of Hereditary stomatocytosis with marked pseudohyperkalaemia.

Congenital hemolytic anemia associated with dehydrated erythrocytes and increased potassium loss.

Familial pseudohyperkalaemia. A new syndrome. Lancet

Hereditary xerocytosis and pseudohyperkaliemia map to the same locus. Blood

Stomatocytosis: a hereditary red cell anomaly associated with haemolytic anaemia.

Xerocytosis with concomitant intrauterine ascites: ﬁrst description and therapeutic approach. Blood

Genetic heterogeneity of hereditary stomatocytosis syndromes showing pseudohyperkalemia

Genetic heterogeneity of hereditary stomatocytosis syndromes showing pseudohyperkalemia

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