Studies congenital dyserythropoietic type DNA, RNA ... · genital dyserythropoietic anaemia, type `.1-4 The peripheral blood film showed severe poikilocytosis andanisocytosis with

J Clin Pathol 1986;39:881-890

Studies of erythroblast function in congenitaldyserythropoietic anaemia, type I: evidence ofimpaired DNA, RNA, and protein synthesis andunbalanced globin chain synthesis in ultrastructurallyabnormal cellsSN WICKRAMASINGHE,* MJ PIPPARDt

From the *Department ofHaematology, St Mary's Hospital Medical School, University ofLondon, andfSection ofHaematology, Northwick Park Hospital and Medical Research Council, Clinical Research Centre,Harrow, Middlesex

SUMMARY Two patients with congenital dyserythropoietic anaemia, type I (CDA) were studied.Their blood reticulocytes showed unbalanced globin chain synthesis with increased a:,B globin chainsynthesis ratios. A high proportion of the erythroblasts displayed the characteristic "Swiss cheese"abnormality of the nuclear chromatin and some also showed cytoplasmic intrusions lined withnuclear membrane within the nucleus. Occasional erythroblast profiles contained intracytoplasmicinclusions that were ultrastructurally indistinguishable from precipitated a chains. The technique ofcombined Feulgen microspectrophotometry and 3H-thymidine autoradiography showed grossabnormalities of proliferation in the early polychromatic erythroblasts. The proliferative abnor-malities included an arrest ofDNA synthesis after the progress of cells through part of the S phaseand the formation of several mononucleate and binucleate cells with hypertetraploid total DNAcontents. The bone marrow cells gave a normal deoxyuridine suppressed value, indicating that therewas no impairment of the methylation of deoxyuridylate. Electron microscope autoradiographicstudies showed that a high proportion of the erythroblasts with the "Swiss cheese" nuclear abnor-mality suffered from a severe impairment, or arrest of DNA, RNA, and protein synthesis.

Congenital dyserythropoietic anaemia, type I (CDAtype I) is a rare disorder of unknown aetiology char-acterised by a congenital macrocytic anaemia, mega-loblastoid erythroid hyperplasia, the occurrence ofintererythroblastic nuclear chromatin bridges, thedevelopment of secondary haemochromatosis in laterlife, and, probably, an autosomal recessive inher-itance.' 2 Multinucleate erythroblasts, althoughpresent in increased numbers, are not a prominentfeature of the marrow and the acidified serum lysistest is negative. Ultrastructural studies have shownthe presence of nuclei with a "Swiss cheese" appear-ance in a high proportion of the mononucleate earlyand late polychromatic erythroblasts; these nucleihave multiple rounded electron lucent areas withinabnormally electron dense heterochromatin. In thispaper we report the results of studies into erythro-

Accepted for publication 5 March 1986

blast function in two new patients with CDA type I.The functions investigated included the efficiency ofthe methylation of deoxyuridylate to thymidylate inbone marrowcells; proliferative behaviour as indi-cated by the cell cycle distribution of the erythro-blasts; and the capacity of the ultrastructurally abnor-mal erythroblasts to synthesise macromolecules.

Material and methods

CASES STUDIEDTable 1 summarises the essential clinical and labora-tory data. In both patients the haematologicalfindings were unexpected. Case 1 was first found to beanaemic at the age of 32 years when he had infectiousmononucleosis. In case 2 persistent mild jaundice andsplenomegaly led to a diagnosis of unexplained "hae-molytic anaemia" at the age of 24 years (haemoglobin

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Table 1 Essential clinical and laboratory data in both male patients studied

Case 1 (34 years) Case 2 (59 years) Normal values

(a) (b)Splenomegaly 4 cm 2 cmHepatomegaly 4 cm 4 cmHaemoglobin (g/dl) 10 1 11 1 134-170Red bloods cells ( x 10"2/1) 2 91 3-41 44-5 8Mean cell volume (fl) 101 9 97-8 80-92Retics ( x 109/1) 92 75 18-158White cells (x 109/l) 3.5 5 3 4-11Platelets ( x 109/l) 189 178 140-400Red cell folate (ug/l) 340 2353 145-450HbA2(%) 31 29 1 5-34HbF (%) 3 7 16 <2.0oe:f globin chain synthesis ratio (blood retics; one hour

incubation)Radioactivity ratio 1 8 1 5 0-9-1 2Specific activity ratio 1 6 1-3 09-1 2

Marrow erythroid:myeloid ratio 4-1 3 2 3-0 0 12-0 5Serum iron (pmol/l) 42 48 14 12-27Serum total iron binding capacity (pmol/l) 48 49 69 40-75Plasma iron turnover (pmol/l whole blood/24 hours) 1103 996 104-152Red cell 59Fe utilisation (% at 14d) 19 74-865tCr-labelled red cell survival-T'/2 5Cr (d) 18 21 25-33Mean cell life-span (d) 52 50 110Serum bilirubin (ymol/l) 28 99 75 1-15Serum ferritin (pg/I) 3869 3695 212 17-230Liver iron (% of dry weight) 3-0 2 6 <0 1%

(a) Values obtained at age 56 years before treatment for iron overload; (b) values obtained at the time of this study.Conversion: SI to traditional units: serum iron, total iron binding capacity and plasma iron turnover I pmol/l 5 59 ug/I 00 ml; serumbilirubin I pmol/l- 0058 mg/100 ml.

1 I g/dl, mean cell volume 106 fl). Severe iron overloaddeveloped in both patients (Table 1), providing evi-dence for lifelong erythroid hyperplasia associatedwith excessive iron absorption: neither had everreceived any blood transfusions. On reinvestigation atthe age of 56 years, case 2 had hepatic cirrhosis andhis iron overload (about 20 g) was subsequently com-pletely removed with a combination of regular phle-botomy and desferrioxamine infusions before thepresent studies were conducted. Neither patient hadany family history of anaemia or jaundice.

In both cases the light microscopic appearances ofthe blood and bone marrow were typical of con-genital dyserythropoietic anaemia, type `.1-4 Theperipheral blood film showed severe poikilocytosisand anisocytosis with obvious macrocytes, as well assome poorly haemoglobinised cells, microcytes, andred cells with basophilic stippling. The marrowsmears showed severe erythroid hyperplasia. Poly-chromatic erythroblasts, many showing a megalo-blastic chromatin pattern, dominated the marrow(56% and 53% of nucleated cells in cases 1 and 2,respectively). Internuclear fine chromatin bridgeswere seen in 1-0% (case 1) and 0 3% (case 2) of poly-chromatic and orthochromatic erythroblasts. In addi-tion, 4-2% (case 1) and 5 8% (case 2) of these cellswere binucleate; the two nuclei were often of differentsize and chromatin structure and were sometimesincompletely separated. Marrow smears stained for

iron showed moderately increased numbers of eryth-roblasts with abnormally coarse iron containing gran-ules (abnormal sideroblasts) but no ringed side-roblasts: in case 2 an earlier marrow had shown asmall proportion of ringed sideroblasts before treat-ment for iron overload. The presence of a grossdegree of expansion of the erythroid marrow wasindicated not only by the erythroid:myeloid ratios butalso by the increased plasma iron turnover (Table 1). 5That this was related to ineffective erythropoiesisrather than haemolysis was confirmed by the normalabsolute reticulocyte counts and only moderatelyreduced autologous red cell survival,6 as well as thesevere reduction in the use of 59Fe by red cells (Tablel).6

GLOBIN CHAIN SYNTHESISGlobin chain synthesis was measured by incubating10 ml of washed reticulocyte rich red cells with100 1uCi of [3HI leucine for 60 minutes at 37°C.7 Afterincubation the cells were washed, lysed, and con-verted to globin by acid-acetone precipitation. Theglobin chains were separated by chromatography onCM cellulose in 8M urea/0-05M mercaptoethanol,8and the radioactivity and optical density of the frac-tions containing each chain were measured.

SPECIAL STUDIES ON MARROW CELLSFreshly aspirated marrow was mixed with 2 ml

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Studies of erythroblast function in congenital dyserythropoietic anaemia

Hanks's solution containing 50 units of preservativefree heparin. An aliquot of the mixture was used toprepare a red cell deficient single cell suspension ofmarrow cells by forcing it through 21 and 25 gaugeneedles, as previously described.9

DEOXYURIDINE SUPPRESSION TESTThis test was performed on the single cell suspension,using the method of Wickramasinghe.'0

CELL CYCLE DISTRIBUTIONOne ml of the single cell suspension was incubatedwith 20 pCi [methyl-3H] thymidine (specific activity50 Ci/mmol; Amersham International Ltd) for 30minutes and the labelled cells smeared on glass slides.The radioactive smears were used to determine thedistribution of various classes of erythroblasts andneutrophil precursors in the three stages of inter-phase, GI, S, and G2 by a combination of Feulgenmicrospectrophotometry and 3H-thymidine auto-radiography.9 "

ELECTRON MICROSCOPYMarrow fragments were removed from the aliquot ofundispersed heparinised marrow, fixed in 2 5%glutaraldehyde in 0-1 M phosphate buffer (pH 7T3) forone and a quarter hours at room temperature, andprocessed for transmission electron microscopy asdescribed previously.12

ELECTRON MICROSCOPE AUTORADIOGRAPHYAliquots of the single cell suspension were mixed with25% (v/v) group AB serum and separately incubatedwith 40 pCi [methyl-3H] thymidine (specific activity50 Ci/mmol), 50 pCi [5-3H] uridine (specific activity29 Ci/mmol), and 50 pCi L-[4,5-3H] leucine (specificactivity 56 Ci/mmol) per ml for one hour; the radio-labelled chemicals were obtained from AmershamInternational Ltd. The radioactive cells were used forthe preparation of electron microscope auto-radiographs, as described previously,9 except that thecells were stained en bloc with 2% aqueous uranylacetate. One set of autoradiographs was exposed forone and another for six weeks.

Results

GLOBIN CHAIN SYNTHESISAlpha:beta globin chain synthesis ratios wereincreased in both patients. Table 1 gives the radio-activity and specific activity ratios.

DEOXYURIDINE (du) SUPPRESSION TESTThe dU suppressed value given by the bone marrowcells was 3-4% in case 1 and 4-8% in case 2 (normalrange 144-8.6%).1o

CELL CYCLE DISTRIBUTIONTable 2 gives the cell cycle distribution of the baso-philic erythropoietic cells (proerythroblasts and baso-philic erythroblasts) in both cases studied. There wasan increased percentage of cells in the S phase with acorresponding decrease in the percentage in the G,phase. There was also a slight increase in the propor-tion in the G2 phase and some reduction in the ratioof the number of cells in S to that in G2 (S:G2 ratio).

Figs. I and 2 show the distribution of the totalFeulgen absorbance values (relative DNA contents)given by individual mononucleate and binucleatepolychromatic or orthochromatic erythroblasts ofcases 1 and 2, respectively: most of the mononucleatecells had DNA contents around and between the 2and 4c values and most of the remainder had DNAcontents between the 4 and 8c values. Most of thebinucleate cells had total DNA contents between the4 and 8c values. The percentages of DNA syn-thesising mononucleate polychromatic and ortho-chromatic erythroblasts in cases I and 2 were 28 and24, respectively.

In both cases studied the distribution of pro-myelocytes plus myelocytes in the different stages ofinterphase was essentially normal, the average valuesfor the percentages of cells in GI, S, G2, and U(Table 2) being 58-9, 34.7, 5 7 and 1-0 (normal ranges59-70, 20-34, 3-5, and 0-1 -2, respectively).

ELECTRON MICROSCOPYNo ultrastructural abnormalities were found in thebasophilic erythropoietic cells of either of the cases

Table 2 Distribution ofbasophilic erythropoietic cells in different stages ofinterphase

Case No Percentages S/G2 Total No ofcells studied

Gi 5 G. U*

Case 1 11-7 76 7 11-7 0 6-57 120Case2 14-2 74-3 115 0 6-46 113Normal ranget 19-36 58-72 3-9 0-09 8-2-25-5

*Cells that were not synthesising DNA, as judged by their failure to incorporate =H-thymidine but which had DNA contents between the GIand G2 values.tIncludes both normal and haematologically normal adults.11

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Wickramasinghe, Pippard

25-, Case 1

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Fig. 1 Distribution ofrelative DNA contents ofmononucleate (upper histogram) and binucleate (lowerhistogram) polychromatic and orthochromatic erythroblasts ofcase 1. Open bars represent cells that wereunlabelled with 3H-TdR and stippled bars cells that were labelled with 3H-TdR. Hatched and solid bars showpairs oferythroblasts joined by internuclear chromatin bridges in which nuclei were unlabelled and labelledwith 3H-TdR, respectively. In the case ofbinucleate erythroblasts and cells with internuclear chromatinbridges DNA contents shown are total DNA contents ofboth nuclei.

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DNA content (arbitrary units)Fig. 2 Distribution ofrelative DNA contents ofmononucleate (upper histogram) and binucleate (lowerhistogram) polychromatic and orthochromatic erythroblasts ofcase 2. Open bars represent cells that were

unlabelled with 3H-TdR and stippled bars cells that were labelled with 3H-TdR. Hatched and solid bars showpairs oferythroblasts joined by internuclear chromatin bridges in which nuclei were unlabelled and labelledwith 3H-TdR, respectively. In the case ofbinucleate erythroblasts and cells with internuclear chromatinbridges DNA contents shown are total DNA contents ofboth nuclei.

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Fig. 3 Part ofa late polychromatic erythroblast with spongy heterochromatin showing multipleintracytoplasmic inclusions resembling precipitated a globin chains. Case 1. x 46500.

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Fig. 4 Electron microscopic autoradiograph ofmarrow cells that had been incubated with 3H-thymidineforone hour. Two erythroblasts in centre with normal looking nuclei are heavily labelled. Cells on either sideshow multiple electron lucent areas within heterochromatin and are either unlabelled (left) or associated withveryfew autoradiographic grains (right). Autoradiograph was exposedfor 3d. Case 1. x 6575.

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0Fig. 5 (a) and (b) High resolution autoradiographs oftwo 3H-thymidine-labelled early polychromatic erythroblasts fromcase 2. Both cells show multiple electron lucent areas within heterochromatin. They also show some increase in electrondensity ofheterochromatin. More heavily labelled cell in (a) shows lesser degree ofabnormality than more weakly labelledcell in (b). Autoradiograph was exposedfor 3d. x 9850.

studied. Qualitatively similar abnormalities werefound in the polychromatic erythroblasts of bothcases. In cases 1 and 2 62% and 79%, respectively, ofthe profiles of the mononucleate early and latepolychromatic erythroblasts contained abnormalnuclei. These were characterised by the presence ofmultiple electron lucent areas within abnormally elec-tron dense heterochromatin that gave the hetero-chromatin a spongy or moth eaten appearance (Swisscheese like nuclei). Some of the affected nuclei alsocontained nuclear membrane lined masses of cyto-plasm that had invaginated through small or largedefects in the nuclear membrane associated hetero-chromatin. In some cells these cytoplasmic intrusionsfilled the areas between adjacent masses of hetero-chromatin so that the invaginating nuclear membranewas closely apposed to the surfaces of the hetero-chromatin. In occasional cells cytoplasmic organelleswere seen within nuclear territory. Cell profiles con-taining abnormal nuclei sometimes displayed variouscytoplasmic abnormalities such as autophagic vacu-oles, myelin figures, and iron laden mitochrondria.Such profiles occasionally contained many small,rounded, or irregular masses of amorphous electrondense material in the cytoplasmic matrix (Fig. 3), oraround the centrioles. These masses tended to becomeconfluent and were indistinguishable from the a chainprecipitates found in the f thalassaemia syn-dromes. 13 14

Some large erythroblast profiles contained twonuclear masses, both of which showed the Swisscheese appearance. In such profiles the two nucleiwere either partially fused together over a wide areaor linked together by a bridge of chromatin; occa-sionally, one of the nuclear masses was ultra-structurally considerably more abnormal than theother.

Erythroblasts with a spongy appearance of the het-erochromatin were recognisable at various stages ofdegradation within the cytoplasm of some of the bonemarrow macrophages.

ELECTRON MICROSCOPE AUTORADIOGRAPHYAfter exposure of the autoradiographs for three toseven days the percentages of polychromatic erythro-blasts with ultrastructurally normal nuclei whichincorporated 3H-thymidine in cases 1 and 2 were 55 6and 65 6, respectively. In case 1, however, only 12%of the mononucleate early and late polychromaticerythroblasts with the Swiss cheese nuclear abnormal-ity were either clearly labelled, or, more usually,weakly labelled with 3H-thymidine, as were a similarproportion of the binucleate cells showing thisultrastructural abnormality; the remainder wereunlabelled (Fig. 4). In case 2 only 5-4% of these cellswere labelled (Fig. 5). In both cases the majority ofthe mononucleate and binucleate erythroblasts dis-playing the Swiss cheese abnormality, including many

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Fig. 6 Electron microscopic autoradiograph ofmarrowhour. One erythroblast has a normal looking nucleus and"Swiss cheese" nuclear defect; two of these are unlabellezx 6575.

early polychromatic erythroblasts, remained totallyunlabelled after exposure of the autoradiographs foras long as six weeks.An average of 96-4% of mononucleate early and

late polychromatic erythroblasts with ultra-structurally normal nuclei incorporated 3H-uridine.By contrast, 25% of the mononucleate erythroblastswith Swiss cheese like nuclei in case 1 and 6-7% ofsuch cells in case 2 were labelled moderately well with3H-uridine. There was either a total absence of label-ling with 3H-uridine or very weak labelling (six auto-radiographic grains per cell or less) in most mono-nucleate and binucleate cells with this nuclearabnormality in the autoradiographs exposed for bothone and six weeks (Fig. 6).

In those exposed for one week 94 1% of poly-chromatic erythroblasts with ultrastructurally normalnuclei were labelled with 3H-leucine. In these auto-radiographs substantial labelling was observed inonly 25% of the mononucleate erythroblasts withSwiss cheese like nuclei in case 1 and 8-9% in case 2;the remaining cells were unlabelled or very weaklylabelled (Fig. 7). Many completely unlabelled eryth-roblasts with this ultrastructural abnormality persis-ted in the autoradiographs exposed for six weeks; insuch autoradiographs the ultrastructurally normalerythropoietic cells, including reticulocytes, wereextremely heavily labelled.

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cells that had been incubated with 'H-uridinefor oneis heavily labelled. All three remaining cells showd. Autoradiograph was exposedfor six weeks. Case 1.

Those cells with the Swiss cheese abnormality,which showed a moderate degree of labelling with3H-thymidine, 3H-uridine, or 'H-leucine, tended todisplay a lesser degree of nuclear abnormality thanthose that were completely unlabelled or very weaklylabelled (Fig. 4). There seemed to be no abnormahityin the distribution of autoradiographic grains oversuch labelled erythroblasts. Autoradiographic grainscaused by the incorporation of.H-thymidine intoDNA were more or less confined to the area over thenucleus and located mainly at or near the junctionbetween heterochromatin and euchromatin. Thosecaused by the incorporation of 3H-uridine into RNAwere located largely over the nucleus, mainly at ornear the junction between heterochromatin andeuchromatin, but also over the cytoplasm. Auto-radiographic grains caused by the incorporation of'H-leucine into protein were situated both over thecytoplasm and over the nucleus: most of those locatedover the nucleus were located close to the junctionbetween heterochromatin and euchromatin.

Discussion

Light and electron microscopic features characteristicof congenital dyserythropoietic anaemia, type I wereseen in the erythroblasts of both cases studied. Thesefeatures included the presence of occasional pairs of

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Fig. 7 High resolution autoradiograph ofmarrow cells that had been incubated with 3H-leucinefor one hour.Three erythroblasts are labelled; one shows well developed "Swiss cheese" abnormality ofheterochromatin andanother a mild degree of this abnormality. There are two unlabelled erythroblasts, both ofwhich show a severedegree ofabnormality. Autoradiograph was exposedfor 7d. Case 1. x 6575.

erythroblasts that were joined together by inter-nuclear chromatin bridges; Swiss cheese like nuclei inover 60% of the profiles of mononucleate early andlate polychromatic and orthochromatic erythro-blasts3 41 5 16; and large cytoplasmic intrusions linedby nuclear membrane in some of the abnormalnuclei."7 18 Like the cases described by Dell'orbo etall7 and Hiraoka et al, 8 the erythroblasts of our twocases did not show widening of nuclear pores or lossof parts of the nuclear membrane.

In a recent study of two patients with CDA type Ithe blood reticulocytes of both patients displayedunbalanced globin chain synthesis with an increase inthe x:non-a chain radioactivity ratio.'9 Our findingthat the blood reticulocytes of both cases showed asimilar abnormality (Table 1) is therefore of interest.Furthermore, as we found an occasional erythroblastprofile which contained intracytoplasmic inclusionsthat were morphologically indistinguishable fromprecipitated a chains'31 in both cases it seems that atleast some of the erythroblasts also suffered -fromunbalanced globin chain synthesis. An increase in theblood reticulocyte a:# chain synthesis ratio has beenreported not only in the cases of CDA type I men-

tioned above but also in some cases of congenitaldyserythropoietic anaemia that could not be allocatedto one of the three classical types, and in somepatients whose erythroblasts showed the morpho-logical features of CDA type II but whose red cellsdid not express the HEMPAS antigen.20 -22

Previous studies of the cell cycle distribution of theerythroblasts in two cases ofCDA type I have shownthat DNA synthesis was virtually confined to thebasophilic erythropoietic cells.23 By contrast, in thetwo cases reported here a substantial proportion ofthe polychromatic and orthochromatic erythroblastsincorporated 3H-TdR. Many of the non-DNA syn-thesising mononucleate polychromatic and ortho-chromatic erythroblasts of our cases, however, hadDNA contents corresponding to and in between theG1 and G2 values (Figs. I and 2), suggesting that ahigh proportion of the mononucleate polychromaticand orthochromatic erythroblasts become arrestedduring their progress through the S phase of the cellcycle. Our cell cycle data also showed that somemononucleate erythroblasts, as well as the binucleateerythroblasts and the cells linked by internuclearchromatin bridges, had total DNA contents greater

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than 4c and usually between 4 and 8c. The normalresults with the dU suppression test indicate that nei-ther the arrest ofDNA synthesis nor the mild megalo-blastic changes in the erythroblasts could be attrib-uted to any abnormality of the methylation ofdeoxyuridylate.Our electron microscopic autoradiographic data

show that a high proportion of the erythroblasts withthe Swiss cheese nuclear abnormality fail to synthesiseDNA, RNA, or protein. As some of the erythroblastswith this nuclear abnormality, including some with amild degree of abnormality, were capable of syn-thesising these macromolecules, it seems that theSwiss cheese defect begins to develop before the fail-ure of synthesis of macromolecules.The primary biochemical defect affecting the eryth-

roblasts of patients with CDA type I remainsunknown. On the basis of the characteristic Swisscheese appearance of many of the erythroblast nucleiit has been suggested that the primary lesion may bean abnormally high degree of condensation of nuclearchromatin. X This possibility is corroborated byquantitative cytochemical studies of individual eryth-roblasts from a patient with CDA type I that haveshown an increase in the mean value for the fastgreen:Feulgen absorbance ratio-that is, histone:DNA ratio-due to an increase in the average fastgreen value, thus suggesting that there may be eitheran increase in the histone content per nucleus or achange of the staining quality of the nuclear his-tones.24 A change in the nuclear histones could possi-bly lead to an abnormal degree of chromatin con-densation. Our own finding that the failure ofmacromolecular synthesis follows rather than pre-cedes the development of the Swiss cheese nucleardefect also supports the view that the primary bio-chemical lesion may be in the nucleoproteins of thecell. We consider that the primary lesion is likely to beeither the production of a changed nucleoproteinmolecule (perhaps due to a mutation affecting a singleamino acid), or the production of reduced quantitiesof a specific nucleoprotein. The abnormality innucleoprotein synthesis may lead to various second-ary effects, including the impairment of erythroblastproliferation; the failure of DNA, RNA, and proteinsynthesis; and unbalanced globin chain synthesis withprecipitation of excess a chains. Although it has beensuggested that the primary defect may lie in thenuclear membrane,18 it is equally possible that theultrastructural abnormalities affecting this membranealso arise as a consequence of a nucleoprotein abnor-mality, which may lead to a change in the normalinteraction between nucleoproteins and the nuclearmembrane.

In both cases studied erythroblasts containingSwiss cheese like nuclei were found within the cyto-

plasm of macrophages, providing direct mor-phological evidence of increased ineffective erythro-poiesis. Presumably, some secondary abnormality ofthe biochemical composition of the cell membrane ofthe ultrastructurally abnormal erythroblasts resultedin their interaction with and phagocytosis by macro-phages. As dimethylsulphoxide induced Friend leu-kaemia erythroblasts treated with puromycin orcycloheximide have been shown to interact withmouse peritoneal macrophages to a greater extentthan untreated erythroblasts,25 the arrest of proteinsynthesis in the erythroblasts of our patients mayhave been indirectly responsible for the ineffectivenessof erythropoiesis, perhaps by causing changes in thestructure or configuration of the cell membrane.

We are grateful to Dr JA Easton, ConsultantHaematologist, Wexham Park Hospital, Slough, andDr MSA Qureshi, Consultant Physician, RoyalHalifax Infirmary, for referring the patients for study.We thank Ms Madeleine Hughes, Department ofHaematology, St Mary's Hospital Medical School,for invaluable technical help with the electron micro-scopic studies and Mr D Waters, Department ofHaematology, Northwick Park Hospital, formeasurement of globin chain synthesis.

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7 Dell'orbo C, Marchi A, Monafo V, et al. Type I congenital dys-erythropoietic anemia (CDA I): ultrastructural findings. Hae-matologica 1983;68:30-7.

18Hiraoka A, Kanayama Y, Yonezawa T, Kitani T, Tarui S, Hash-imoto PH. Congenital dyserythropoietic anemia type I: a freeze-fracture and thin section electron microscopic study. Blut1983;46:329-38.

9Alloisio N, Jaccoud P, Dorleac E, et al. Alterations of globin chainsynthesis and of red cell membrane proteins in congenital dys-erythropoietic anemia I and II. Pediatr Res 1982;16:1016-21.


20Weatherall DJ, Clegg JB, Knox-Macaulay HM, Bunch C, Hop-kins CR, Temperley IJ. A genetically determined disorder withfeatures both of thalassaemia and congenital dyserythropoieticanaemia. Br J Haematol 1973;24:681-702.

21 Hruby MA, Mason RG, Honig GR. Unbalanced globin chainsynthesis in congenital dyserythropoietic anaemia. Blood1973;42:843-50.

22Eldor A, Matzner Y, Kahane I, Levene C, Polliack A. Aberrantcongenital dyserythropoietic anemia with negative acidifiedserum tests and features of thalassemia in a Kurdish family. IsrJ Med Sci 1978;14:1 138-43.

23Queisser W, Spiertz E, Jost E, Heimpel H. Proliferation dis-turbances of erythroblasts in congenital dyserythropoietic ane-mia type I and II. Acta Haematol 1971;45:65-76.

24Meuret G, Tschan P, Schluter G, Keyserlingk DG, Boll I. DNA-,histone-, RNA-, hemoglobin-content and DNA-synthesis inerythroblasts in a case of congenital dyserythropoietic anemiatype 1. Blut 1972;24:32-41.

25Wiener E, Wickramasinghe SN. Impaired protein synthesis inerythroblasts enhances their phagocytosis by macrophages. Br JHaematol 1983;53:1 17-24.

Requests for reprints to: Professor SN Wickramasinghe,Department of Haematology, St Mary's Hospital MedicalSchool, London W2 IPG, England.

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Studies congenital dyserythropoietic type DNA, RNA ... · genital dyserythropoietic anaemia, type `.1-4 The peripheral blood film showed severe poikilocytosis andanisocytosis with