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From www.bloodjournal.org by guest on June 12, 2017. For personal use only. Increased HbF in Sickle Linked By Paul F. Milner, Members of sickle cell 7 large anemia hemoglobin F cells amounts of cell trait count (AS) than marked HbF and had were also range. had only HbF It seems Ford, with HbF and in the remaining values in both F-cell a factor small and siblings whereas a parents. the F cells, fetal hemoglobin (a2”y2, HbF) persists normally in a small proportion of erythrocytes called F cells.’3 Synthesis of “y-chains in these cells is probably completed very early in their maturation,4 and HbF constitutes only about lO%-30% of the hemoglobin, or a mean of about 4.4 ± 0.3 pg HbF/F cell. Population data indicate that, among normal individuals, F-cell frequency has a skewed distribution, a range of about 0.5%-7.5% and 2.5%.’ About 3% of Europeans cells,”7 and family studies have inherited as a heterozygous trait There is also good evidence that the ‘y#{244}f3-genecluster and that a mode of about have more than 8% F indicated that this is for a dominant gene.7 the gene is linked to there is linkage disequi- librium with the i9S gene,9”#{176}so that, among subjects with sickle cell trait, there is a higher prevalence of increased F cells than among subjects with a normal hemoglobin genotype.8”0 Among proportion than patients of HbF 1% to over 20%, and (F retics) vary from constant over time.6 subjects have normal majority it is increased cell,’1 and From lar in these and gia. Augusta. containing the less HbF subjects, there ofPathology. the Sickle Cell is preferential Medicine, Center. sur- Cell and Molecu- Medical College of Geor- GA. Supported Lung reticulocytes (SS), from 2% to 55%,6I The levels remain Although about a quarter of SS amounts of HbF per F cell, in the to about a mean of 8 pg HbF/F the Departments Biology. anemia varies in part and Blood by Grant Institute. HL/ilS8from National the National Institutes ofllealth, Address Center, January reprint Medical 0 1 984 6. 1983; requests College by Grune to Dr. ofGeorgia. & Stratton, 0006-497//84/630/-0008$0/.00/0 64 accepted Inc. July Paul Augusta, GA 309/2. the same the 55 offspring it their “increased of it greatly HbF per as that the result of to of AA marrow similarity in Swiss it sickle may cell type, anemia to the ultimate level if few F cells survival will amount, and this in most black SS only of HbF were increase alone in any by the can patients. HbF account by F cells contain for further increases in HbF found, increased number of F cells have this will provide, but a lessening increase in red have these et al.’2 similar patients have not only that high limit that HbF frequently F reticulocytes in of F cells in their et al.’3 found that 10% HbF had one than 0.4% I-IbF, their upper workers’4 have also observed might and if a property, rate and an total hemoglo- 55 siblings HbF levels and that correlate with numbers parents. Serjeant with more than very level more survival of the hemolytic cell count and noted modest HbF their survival preferential patient. a for the If the improved modulaI-IbF/F contrib- preferential HbF, levels, overall bin. its of the of 55 produced, account greatly their the infancy. additions of HbF. Thus, each or all of these lions-increased numbers of F cells, increased cell, and preferential survival of F cells-may ute is control vival of F cells in the peripheral od6’ because well-known inhibitory effect on HbS polymerization Obviously, of hereditary (HPFH). HbF subjects, hyperplasia The that parents increased, heterocellular suggested HbF in AS greatly anemia. hemoglobin synthesis F cell was in these increased of normal it gene” is in early is segregating the hemolytic fetal and persistent was F-cell persistence 14/16 parent 55 patients with more of normal, and these the rate of decline of HbF in SS infants over the first 6 yr of life is related to the maximum HbF level in either parent. The data we present here indicate that, in families where 55 patients have more than about 10% of HbF, this tendency teristic appears to be inherited as a dominant linked to the f3 gene of one parent. charac- Heart, Bethesda, MATERIALS 17, 1983. F. Milner, was that MD. Submitted subjects. with presence for 55 associated and Fred A. Garver parent, in the in Dover with sickle cell in a hemolysate of one suggesting this E. Grenett, is responsible discussed, N ADULTS, with gene and by a Factor Parent Hernan to the sickle family, above P. Barton, F cells had increased Betty One and hemoglobin counts for From families F-cell parents is Determined linked of fetal of the I of 8 normal and that Janet levels both F cells; but Gene patients one higher family, at borderline siblings, 9S to measure a much and Anemia immunofluorescence families, in one in HbF siblings, “normal” using of these other; F cells 20 by high a radloimmunoassay parents the of 14 AS (AA) studied In five Increase Seven I and containing characterized were HbF. to the D#{246}bler Leibfarth, families, (SS) (HbF). to count Johanna Cell Sickle Cell Hematologic using determined tified investigations a Coulter Model were S cell by cellulose by DEAE-cellulose AND acetate METHODS carried counter. out by standard Hemoglobin electrophoresis, methods phenotypes and fractions were quan- chromatography. Blood, Vol. 63, No. 1 (January), 1984: pp. 64-72 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. INCREASED HbF Family IN SICKLE CELL 65 ANEMIA Studies Blood from multiple 12 patients, blood CDE, group Jk’Jk’, drogenase (G6PD) TX). red was cell surface determined to antigens which (Helena there AB, II S acetate 10 Laboratories, was doubt about full from this analysis. Measurements HbF was alkali 8 measured in parents denaturation munoassay method (RIA).” methods were values measured 1% low the than by alkali and (AD) At similar, below S a dehy- by cellulose reagents in subjected P. Glucose-6-phosphate were commercial Families siblings and isoenzymes were excluded HbF for MNSs, using Beaumont, and analysis FyFyb, electrophoresis sibship parents, of Betke levels of HbF, denaturation’5 55 and patients more by S 7 the the S two reproducible patients, S by the by a radioim- results much In HbFAD. of 55 et al.’5 and being HbFRIA the siblings at HbF ‘6 S was S by a microchromatographic S technique lab Inc., (MC)’7 Akron, patients, the but were HbF using commercially OH). At the high gave HbFMC considered measured values more by other obtained by elution liquid chromatography I prepared microcolumns (Isolevels of HbF found in some 55 about 20% accurate higher because chromatographic from cellulose (unpublished than they the and S y S S. with and acetate strips observations). S HbFAD, correlated procedures” S S S S S by values S S 5*5 high-pressure S 2 S S #{149}1 .5 F-Cell S Measurements The technique modified used as follows. absorbed with divided HbA, as into small across frosted I hr. and smears has the excess, fixed to dry, for 30 mm were stained glycerol. goat Diego, 30 washing mm at anti-rabbit smears and, following examined Fig. 1 . Appearance of F cells in blood smears visualized by indirect immunofluorescence using antiserum to HbF (see Materials and Methods). (A) Wi I-i ; (B) Wi 11-3; (C) E 11-9; (D) D 1-2; (E) D 11-3; (F) D 11-6 (see Fig. 3). 7.2, wiped thoroughly M away. PBS, Molu for The washed phosphate), to the smeared with the (Calbiochem-Behring under wash, were phase-contrast Females Prenont Females Fig. 2. Percentage of F cells in immunofluorescent-stainea blood smears from 60 unrelated individuals with sickle cell trait (AS). The pregnant women were in the late part of the first or early part of the second trimester. areas smears ultraviolet fluorescein-isothiocyanate- a final S. A made to dry for 2 mm, 0.01 applied y was diluted, were allowed circles, 37#{176}Cwith globulin were slides, pH batch smears (9: 1 , v/v) serum and and exhaustively was Blood glass (PBS, the anti-HbF CA), The frozen. the frosted saline and for described,’6 and outside described3’9#{176} in rabbits in acetone:methanol at 37#{176}C. After conjugated San previously on immunologic in phosphate-buffered allowed previously prepared aliquots, circles were been Anti-HbF, Corp., mounted and light using a Leitz equipped with a Ploem’s mercury vapor lamp. in eyepiece was by counted. When used A to define the Dialux incident fluorescent 20-EB Miller the fluorescence illumination square microscope system graticule fields in which cells were and fluorescent greater a lOO-W placed than in cells about one were 75%, From www.bloodjournal.org by guest on June 12, 2017. For personal use only. MILNER ET AL. 66 Li) I- LACLA e’NcO COO)COCO NNWO) COCO LAN 1’)NO CDC)COCO IC’) ,-,-o- NC’4 +1 + +1 LA CO N O)N’ CO 0) CO LOC’) ‘_C4 +1 41 +1 +1 COOCO C000 tACO NCO 0’- ‘-N +1 +1 +1 c’4CON C’4LA NCI +1 +1 +1 LAO (0’- COLA (00 NO)C’) ‘-0) C0NC’ (0000) ‘ NC,C’) NCI4 C44 C’NC’) C1C1 N 0C’)C’) NC’ C1C’)CS1 C’44 LA N CO LA +1 +1 NCO 0(0 0)N ‘- 4; 4; +1 +1 00’-’- ONN’- CO (‘40) 0)N0) CO 0) N COO)’- (‘4(0 + +1 ,.: +1 +1 LACO LAN +3 NC0 CO( NC’) NN tACO CON (00)0) (0(0 0)(0 CO 0)C1 C,)’+1 ‘- NLAN’ +1 COLA C’)COCO CI0) +1 C’4C0 0000) ON ‘-0 C’(0 +1 +1 +1 CON CO ON LAO) ,.: CO +1 +1 0C’)c’) +1 ‘4’ ‘4’ LA 4; +1 CO ON LC +1 +1 N +1 qo Lii +1 C’)C’) C’)C’)C’)C’) +1 u C)C’) LA (‘4 ‘*C’) C’)C’) C’) C’)C’) ‘4’ 0)0) c’)O)O) C’)LALA C’)C’)C’) +1 +1 +1 +1 +1 NN C’)C’) C’)C’) C C OON e C +1 Cl) LA ‘N’-’- 00(0 I-. a. +1 C’)CiC) CO NC0 e I- O LA LA C) LA C’)C’)C’)C’) NC’) ,+1 4; 4; 4; +1 c’-q coo N COCO 00 +; LA 00 +1 0 C’)O) +i +1 +1 +1 NO CO LAO) LAO) Cf)C’) C’)C’) C’4 C’)N C’)N (“Jo) COCA C’)e4 01’) 4; LA,(0 (0’- C’)C’) .C .v COC’)O ‘-‘-(0(0 0 C C C >-. C’)LANN + 0 +1 0 C’) LA ‘4’ NC’)CO +1 Ci0Lt CO(’40 000 C +1 4; +1 0 +1 +1 +1 O +1 LAO NO) NC’) +1 +1 g0N, (‘10) 0)0) O’-OC) O)N (‘4 +1 +1 +1 +1 +1 COLA C’)LA OCO N CO 00) NC’) OCO OCO 0)0) C a. Cl) Cl) OCON CO CO 0 CO Lc? 0 NN C’4 +1 +1 C C’) C + 0 +1 0) 4; 4; (‘4 +1 +1 CO CO NNN 0)LA(’4 C +1 +1 NCO 0)0 NC’) C’JN COOn CC ‘- 0’- 4-I N0(’4 NN C’)NNN +1 +1 #1 +1 OC’ C’)’- 4; 4; NOC’) 0(0 NO) ‘COCO OLA CO C’)NN C’)N NN LA’N NO) O NO C’)N OOa 00 +i e CO ‘‘-‘-NC) OCOC”I 0 0000) +1 0 0 0 C N x E CO OaOo +1 C’) ‘4’ +1 N CO N +1 N C)N 00 N C’) 00 +1 +1 +1 CO +141 +1 CO +1 +1 N0(0 C’)CO C)N OCOLACO (‘4NC’) 0 C) NC’) +1 +1 +1 dO +1 +1 CON’LOLAN C’4LA 0)’- O)O O,O C’)NC’) NC’) C’)C’) 00 00 +1 I LAC’)C’) LACOCO e .a +1 C CO I- a V +1 N 4; ‘: +1 +1 +1 C) LA LA CO +1 N CO LO’-C’) do dd 000 -H-H +I+1 LALA O)(0 +1 N +1 +1 +1 C’) +1 +1 O) C’) N’- 00)0) 0O)LA ON 0)0) O)CO C’)N NNN C’)’-’- N’- U. U. U.LL ULL. LA. C?C9 CL) C9 L)C (‘4C’) 0)CO’CONC’) ‘-0CC ‘4NO 0)0) C’)N NN’-’- C’)C’)C’)’- LLLu. .5 C’)LA 00 C C’) Ci)- U 0.0 N ‘-LA OCO L( ‘-Ce COLC) NLA N NN NN NC’) COCO NC’4 d c’iL(i C’)O NC) ‘- ‘4’O) ‘-C’) 00) 0- 00 od ON C’)(’ -JO)? VC’) 0 . 0 O,4’ LA; . DC’) .0 DC’). 0.0 DC’) 0.0 +1 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. INCREASED fading fluorescence cent cells were were the the smears from was using in each counts were smear. is shown were were used F-cell for the count, sources. same I . The between smears light by the in Fig. 60 AS subjects during both Reproducibility done 2. Blood not a problem counted scanned All 67 HbF IN SICKLE CELL ANEMIA was observer. range the ages of 15 and also examined after 2,000 better 30 are of in smears shown elution, 2), ages in Fig. but these enumeration. RESULTS Seven HbFMC could group families containing above 10% were studied be reasonably antigens and 20 55 patients in detail because certain, on the G6PD isoenzyme evidence patterns, with we of blood that the offspring were all full siblings. These families intact at the time of the study, and the 20 patients been followed as outpatients hematologic details are given Hematologic values blood smear phoresis results, were available family counts, for several in Table 1. obtained appearances, and the for every pedigrees, and percent years. F-cell in any AA or AS comparable to the siblings subject the other 2.2%, ± whereas among 1 .8% subjects only although fractionally those above carriers arbitrary family Their the mean was that, given F cells, while all the 55 offspring more I Kindred (L)s HbF% F.cslls III HbF % ‘Ia .0 12.5 0.6 4.5 an SC child (111-4), Kindred HbF% F.c.IIs JJL- 1.0 9.5 0.4 1.5 18.7 84.0 HbF % 6.5 F.cslls % 23.5 14.3 65.0 AS AA 0.5 0.6 5.0 1.7 0.3 2.6 % iitJLI 1.3 8.8 1.1 8.4 1.2 12.5 14.0 65.0 0.5 4.7 15.4 64.0 3.6 3.6 23.0 12.3 I E 2 A llE HbF % 0.2 0.9 F.cSIIs % 2.0 7.8 HbF ‘6 F.c.IIs % ho l II 14.7 72.0 0.3 1.5 14.1 62.0 3.8 12.4 0.5 4.4 - 0.4 5.4 3.5 13.8 112 - 23.4 92.0 - _____ HbF % 0.8 F.cslls % 6.1 K 20.6 88.0 cI I #{243} 4 0.6 6.9 Pedigrees 22.6 95.0 25.7 96.0 of 7 kindreds DISd 595 13 0.5 6.7 showing HbF% F.cslls % segregation 2 _____ Wa 0.9 8.6 HbF% F.cslls % - - - - 1.1 7.8 12.8 58.0 of the “high 12 13 0.2 3.5 - 2.3 13.5 14 0.3 4.9 0.4 3.0 Is 18.6 78.0 I DIsdIn Accldsnt gene” L_i F.cSlIs ‘,‘ 3.7 /o ll#{243} 0.4 3.5 F-cell Ii 0.1 0.75 Kindred l[ Kindred ILj Kindred % 113 0.6 6.5 I 2 HbF% F.cslls - HbF% 1 2 Kindred 1 Fig. 3. have i I Ii ‘#{149}l HbF % 0.7 F.cslls % 8.0 also 12 lli 0.3 1.2 who I 1.0 7.5 19.8 92.0 Kindred HbF % 16.0 F.c&Is % 74.0 and A 16.5 85.0 - #{176}/o F.cslls 0.3 1.8 i.-kI li to a II, two and one an AS high 12 HbF#{176}/, F.csIIs % one than of the families (Fig. 3) illustrate this point. In kindred Wi, 9 of the 1 1 offspring were available and none have died. The four 55 subjects all have F-cell 3. It was decision rather (with had greatly increased F cells and The following brief descriptions (111-3) The F-cell our F, generation AS parent. value of the increased apparent that, where F cells and HbF were increased, the increases were often greater in AS offspring in the in either in the cut-off value of 7%, one AS parent (in one both parents) and half the AS offspring had exception) 10% HbF. F-cell counts, Hb electropercentage of HbF in lysates family member studied. The siblings (Table 2). In the had F-cell counts ± 7%, considered to have a count was level of HbF. Of the 4 AS in generation have HbF and F cells clearly above “normal,” of these subjects (11-3) has three children: than as F, generation 10.6% F, generation, it was 4.7% parental generation, several gene was 10.8% ± 3.2%. The most striking finding were have in the considered mean F-cell among Counter, phenotypes, shown in Fig. count of the families. Among subjects gene for increased F cells, the increased by Coulter hemoglobin HbF are an abnormally high was based on the levels reported in the literature,3’7”0 and on our own results in randomly selected subjects between the ages of 1 5 and 30 yr (Fig. I 0%. ± appearance counted acid cells than The of F cells regard so nonfluores- At least 1.0 8.9 17.5 78.0 (hatched) HbF% F-cells linked % 0.4 5.2 13.5 62.0 to the f 12.8 58.0 locus in one parent. le 20.1 88.0 From www.bloodjournal.org by guest on June 12, 2017. For personal use only. MILNER 68 Table 2. Segregation of “High Total Examined Kin&ed F cells Increased F-Cell Gene” Among Siblings ss AA AS >50% in one parent F cells >7% F cells WI 9 4/4 2/4 0/1 E 10 4/4 2/4 0/2 Wa 3 2/2 1/1 L 6 3/3 0/1 ?1/2 D 6 2/3 2/2 0/1 G 5 2/2 0/1 0/2 K 3 2/2 0/1 Total 19/20 42 MeanFcells(% Low F cells SD) SD) ± MeanHbF (% in both ± 26 (% 17.2±7.2 4.9 ± SD) ± HbF measured by microcolumn increased HbF who does not. F-cell tendency sumably and it increased gene the tendency gene from AA F-cell is clear HbF the from the borderline to increased mother SS and two of four In kindred E, an AA subjects child 2.2/4.7 ± 8.0/3.8 1.8 ± 0.65/0.44 ± ± 2.0 ± 0.73/0.36 0.14 0.16 ± has F cells. It appears and has is linked been passed similar 3.6 0.10 0.28 subjects. and her, the or F-cell possibly gene, which her F cells appears do to have not survive HbF. that to the ± 1.6 preferentially. This family is extended; the father has an 55 brother with 3.0% HbF, who has four AS daughters, all with normal HbF and F cells. One of the mother’s sisters has an 55 granddaughter with I 7% a slightly In kindred flS to all four situation. 0.36 in AS and AA missed grand- mother 1.8 the fS gene high pre- 0/2 0/10 3.8± by RIA siblings in generaIn considering the F cells AS offspring. there is a and (111-2) her who her maternal and AS counts. that and and y in 55 11-2 has not passed AS daughter (111-1), her father. The remaining tion II have normal parents, chromatograph F cells, Patient to her inherits 10.6 1.68 0/14 MeanFcells(%±SD) HbF - 71/8 parents lifamilies Mean - 7/14 76 ± 14/23.5 17.4 ± 3.8/6.5 ET AL. G, the cells, and her two 16.6% and 20.1%, AS offspring In kindred The mother has 55 daughters respectively, increased HbF have a mean but two AA have not inherited K, both parents this have and F HbF of and one characteristic. an increased HbF mother’s blood was studied on several occasions, and among a random group of people, her HbF and F cells would fall in the upper part of the normal distribution. and F cells. Two 55 offspring have only moderately increased HbF. If the F-cell gene is linked to the flA locus of the father, this could explain its absence in the The AS contrast with her husband, however, is striking, offspring who has inherited the flA gene from the with parents low of and all four of her 55, and two of three AS, offspring available have increased F cells. In kindred Wa, the two SS and one AS siblings mother and the flS gene from Data from large families HbF are limited, but we have examined from the 14 such patients and some of their siblings. The results are summarized in Table 2, where they are compared with the families in which a “high F-cell gene” is segregating. have increased father, although F cells, presumably his HbF and inherited F cells are barely above the normal range. In kindred L, both parents have F-cell counts HbF levels bordering on the upper limit of normal. eldest group son is probably is not compatible illegitimate, with that and The because his Jk blood of his father. There are three SS siblings with HbF levels among the highest encountered in black subjects, and they could be homozygous for an F-cell gene inherited from each parent. But, if this is so, 11-7 has to be a heterozygote, and 11-1 and 11-3 are examples of crossovers of the F-cell In gene during parental meiosis. kindred D, the mother clearly has increased cells and has passed this to two of three SS and both AS offspring, but not to the AA offspring. The eldest daughter may be an example of a crossover between the father. of 55 subjects examined both The calculated HbF per F cell in 55 patients and their AS relatives is compared in Table 3. F cells from nonanemic AS subjects have a normal content of HbF, no matter whether this is calculated from HbF measured by alkali denaturation or by RIA. The 55 patients have a considerably higher content of HbF per Table 3. Calculated Parents 40) 4.19 ± 1.84pg MCHx HbF per F Cell AS Siblings (n - 14) (n F Mean 3.76 HbF F cells % ± 1.2pg (± SD) AA Siblings (n - 8) 3.85 ± 1.68pg SS Patients (n - 20) 7.80 ± 0.97 pg From www.bloodjournal.org by guest on June 12, 2017. For personal use only. INCREASED HbF IN SICKLE CELL 69 ANEMIA F cell. This could be an artifact because of the method used for measuring HbF (microcolumn chromatography), but, on the other hand, the calculated mean HbF per F cell is not very different from that reported by other workers4’6 Katsura method.2’ when measured directly using the at the extreme mendelian autosomal dominant inheritance of a their ods? parents by biochemical The recent investigations that there factor for increased HbF and F cells linked to the f3 locus, which segregates in the families described here, is probably the same as that described in several families reported in the literature92224 where small increases in HbF and F cells in one AS parent have pearance age, and resulted over in much thalassemia patient with large ally larger offspring, homozygous amount increases or of HbF thesis is beneficial gene is probably lular hereditary (HPFH), which it may in in other /3-thalassemia has 55 be different from that F cells, and HPFH described Weatherall et F cells al.,28’29 in the absence type heterocellular Polish woman who 25% associated anemia.3#{176} An there similar obvious 7.8% 20% homozygotes of hemolytic have anemia. Swiss HPFH has been documented in a had a transient increase in HbF to with acquired difficulty autoimmune in defining this hemolytic gene is that may be an age-related diminution in its effect, to that found in baboons,3’ making it difficult to assess al.7 had gation shown with with from the nondeletion in an English kindred where whether the same a parent experience of Swiss type that, in normal has the gene or not. Zago et when studying the segre. HPFH, people, and recently it has HbF does decrease been with age.32 It is not clear whether the high levels of HbF and F cells reported for the majority of SS patients among the Shiite Arabs3335 process as described guinity and the can be accounted here. The high increased F cells for by the same level of consan- in many outside We is a relationship of HbF the level AS subjects population of 120 15 yr of age distribution, rate of disap- HbF at whether (Fig. and I 54 female 4). These we converted among 55 results 1 yr of this be a patients are based on for each patient over total Hb level has on each percent HbF to an result on HbF (g/dl) for each patient. The shown in Fig. 4B. While curve accentuates resulting distribution this manipulation the fact that most of these patients HbF, it does not divide the population nor can the distribution mode, as would be heterocellular expression There is have very little into two groups, be explained by a three-group expected if a recessive gene for HPFH was segregating, in heterozygotes. is a lot of evidence from with studies partial in both baboons3”338 and humans39”#{176} that erythropoietic stress produced by anemia induces increased “y-chain synthesis in early erythrocyte precursors. In both baboons37 and humans,”#{176} some subjects respond with much higher levels of HbF than others, and this appears to be genetically determined. These studies have been done iron-deficiency from iron on animals anemia,37 with adult aplastic commences resultant induced humans deficiency39”#{176} or marrow by cyclophosphamide,39 and recovering from transient anemia.5 In sickle hemolytic or recovering repression caused on children over 6 mo old erythroblastopenia39’#{176} or cell anemia, hemolysis first month of life, and the stress would be expected to increase 7-chain synthesis to some extent. In fact, in the majority of 55 infants, this response to increasing hemolysis is negligible, and it is mainly because of the relatively within the erythropoietic longer S/f3#{176}-thalassemia than those reported compartment postnatal life. higher among the infants, their in one parent, male an in absolute HbF (g/dl), using the hemoglobin that blood sample, and recalculated mean that the increased. “y-chain precursors are, on the whole, and our patients are 55 also have detectable or within the normal range. looked at the distribution of HbFMC in that population8 would suggest that an F-cell gene with a high frequency, dominant in all SS offspring, would result in a high frequency of 55 with increased HbF. However, the levels of HbF in Shiite SS and patients here, between in 55 of HbF this by Stama- for black or morphological methof Mason et al.’4 suggest to have a of HbF Do they and not the mean of several estimations several years. To see what effect In both SS HbF syn- described FAD and heterocellular 100% 5//3#{176}- in prolonging red cell survival. The similar to the Swiss type of heterocelpersistence of fetal hemoglobin is found in about 2% of Europeans,”7 et al.,’#{176} where an AS father F cells had an 55 daughter by range where a an unusu- found F cells.9’2527 increased toyannopoulos HbF and 30% 100% or families and been parent with increased HbF and and homozygous /3-thalassemia, but upper patients. Lastly, there remains the question of those 55 patients with intermediate levels of F retics and F cells, who are not entirely explained by preferential survival of normal numbers of F cells. F-cell gene, less well expressed DISCUSSION The those survival of the few F cells produced F-cell count and HbF in peripheral blood is The possession of a factor for persistence of synthesis in a greater number of red cell presumably allows for augmentation of this when hemolytic anemia Persistent reticulocytosis begins in our early in patients From www.bloodjournal.org by guest on June 12, 2017. For personal use only. MILNER ET AL. 70 A 0 Male $5 PatIents Female SS PatIents D U) C 0 U) 0. 0 HbF % E Male SS Patients % Female SS PatIents % U) C 0 U) 0. 0 <0.2 0.2- 0.4- 0.6- 0.8- 1.2- 1.0- HbF is evidence because, of even when continued F-cell some cells is insufficient ture senescence. These are 1.6- 1.8- presumably high, the HbF in to prevent sickling and premapatients are also distinguished by an increased average level of HbF per F cell, which may be the result of an expanded, stressed erythron. Baboons made anemic tamed at a hematocrit and subjected to further baric conditions, by hemolysis of about 20% erythropoietic maintain high and then mainby phlebotomy, stress by hypo- F-cell counts and acquire a higher HbF per F cell.4’ To maintain this state, however, they must be kept in a severely anemic or hypoxic condition, with reticulocyte counts of greater than 50%. The mean reticulocyte count in our patients was 9.1% (± SE 4.5%), yet they maintain a mean (± SE in the HbF of 16.9% (± SE 4.0%). This is a very experimental baboons. 2.0- 2.2- 2.4- (g/dI) hemolysis, counts 1.4- Fig. 4. (A) Distribution of percent HbF, measured by microcolumn chromatography, in 1 24 male and 1 54 female patients over the age of 1 5 yr. (B) Distribution of absolute level of HbF (g/dl) in the same 55 subjects. 1.0%) and F cells different situation of 74% to that Among AS with lO%-l3% controlling those with subjects (Fig. F cells are increased 7% F cells F-cell might 2), it could homozygous be that those for a gene production, in which case be heterozygotes. Among the homozygotes, one of the genes would have to be associated with the chromosome carrying the /3” locus, and half of the AA offpsring of such subjects should have increased F cells, as well as half the AS offspring. The data presented here (Fig. 3), and the frequency of increased F cells among AA and AS subjects tested at random,3’7”#{176} do not support increased F-cell production this concept. If the gene for is in disequilibrium with the flS locus, however, homozygotes more common among 55 subjects, of such subjects would be expected for it would be but both AS parents to have increased F cells, as would do not support all their AS offspring. this, except possibly could inheritance be that of two The in genes present family for data L. It increased From www.bloodjournal.org by guest on June 12, 2017. For personal use only. INCREASED HbF IN F-cell production high HbF levels Arab 55 SICKLE CELL 71 ANEMIA in trans is responsible reported in some black subjects.8 The levels of HbF subjects reported here, however, heterozygosity for an increased the /3 locus of one parent. It has recently been found and for the very most Shiite in most that treatment is associated with an increase in F cells. In a sickle caused to phlebotomy to have higher cell anemia patient, F reticulocytes alone. resting 5-azacytidine to increase per F high from with ‘y-globin cells. demethylation of DNA genes recovered from bone Heterocellular HPFH of dif- methylation in postnatal /3S of these areas locus could first, because more reproductive gotes with survival have come the ability F cells would age, increased advantage life. about of the have in the Linkage by natural f3 homozygote favored to selecto survival to and second, because /3 heterozyF cells might also have a marginal in a malarious environment. ACKNOWLEDGMENT We thank Bank to 52%. 10% associated with erythroid be caused by mutations DNA, which inhibits animals levels.37 treatment been ferent degrees9”#{176}’28’29 may ‘y-globin gene associated make In the Such HbF has associated marrow the tion; f3 homozy5-azacytisynthesis, anemic baboons, there was an increase in HbF cell, and the response was greatest in genetically F-cell responders have been shown to to of anemic baboons42 and human /3-thalassemia43 and gotes with DNA methylation inhibitor, dine, causes a great increase in -y-chain which of the 55 appear to be due F-cell gene linked This Joyce M. Laboratory invaluable Larison of the and her Medical technologists College of at the Georgia Blood for their assistance. REFERENCES I . Marti HR: Normale und Abnormale bine. Gottingen, 1963, pp 8 1-89 Boyer 2. Belding SH, TK, Menschliche Hemoglo- Margolet L, Noyes AN: Fetal acquired elevations of HbF. Blood 46:671, 1975 4. 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Dover GJ, Boyer SH, Pembrey ME: F-cell production in sickle cell anemia: Regulation by genes linked to fl-hemoglobin locus.Science2ll:144l, 1981 I 3. Serjeant GR, Serjeant BE, Mason K: Heterocellular heredi- homozygous 17. Abraham EC, Carver J, D#{246}bler J, Milner PF, Huisman THJ: Microchromatographic quantitation of fetal hemoglobin in patients 22. Interaction cell M, Huisman cation 1978 JB: Y, Hayes Post-natal WG: Br J Haematol 20. Blood and I 5. Betke K, Marti HR. Schlicht I: Estimation of small ages of foetal haemoglobin. Nature 4702: 1877, 1959 O’Sullivan adults. KP, Grandison 2 1 . Katsura Arabia. Wood Wood Genetic haemoglobin 1977 5, Wood in homozygous levels. fetal I :795, Vaidya fluorescent 7. Zago of Lancet 14. Mason GR, Branch, 1981, pp 48-54 M, persistence hemo- globin restriction to a few erythrocytes (F cells) in normal human adults.Science 188:361, 1975 3. Wood WG, Stamatoyannopoulos G, Lim G, Nute PE: F-cells in the adult: Normal values and levels in individuals with hereditary and tary disease. of Martinez foetal synthesis? 24. variant G, Colombo haemoglobin: Nature Makler MT. type a diffusible 252:735, of sickle-cell homogeneously B: A new Is of hereditary factor persistence regulating ‘y-chain 1974 Berthrong M, Locke disease distributed with high within red HR, Dawson levels cells. of foetal DL: A new haemoglobin Br J Haematol 26:5 19, I974 25. ME: 26. JH, Knox-Macaulay Thalassaemia Wood Barlow WH, AM: HHM, Weatherall in the British. Weatherall Heterocellular Di, Di, Clegg Br Med J 3:150, Clegg JB, hereditary JB, Pembrey TJ, Edwards 1973 Hamblin persistence of fetal From www.bloodjournal.org by guest on June 12, 2017. For personal use only. 72 MILNER haemoglobin (heterocellular thalassaemia. Br 27. Rutland in healthy Weatherall Mackenzie interaction fi with Davies T: The estimation by uneven production Haematol of Boyer 1977 30. 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Hall Young G: Br J Haema- fetal J, Anagnou NS, Nail 0, of sickle hemoglobin Acad baboons. Proc Natl GH, AW: in a patient Humphries 5-Azacytidine with fl thalasse- 1982 Smith cell K, Talbot anemia USA CC with production of DNA Sci Keller P, Nienhuis synthesis 307:1469, Dover NP, Heller hypomethylation Proc D: 5-Azacytidine in anemic 1982 ‘y-globin 5, Treatment L, Zwiers synthesis DeSimone PH, increased complex. expansion. Bid M, Heller P: Maintenance of fetal hemogloin the baboon by prolonged erythropoietic hemoglobin Charache nonrandom E, Stamatoyannopoulos erythroid 1982 increases 5: Vichinsky acute J, Heller mia. N EngI J Med J Haematol Genet Zinkahm Th, 60:519, Turner Boyer ME, Th, Chen hemoglobin during elevations fetal Sci RK, A comparison 65:224, production SH, fetal DeSimone stimulates of a mono- Perrine hemoglo- 1980 Blood unusu- 1981 anaemia: anemia Invest baboons Boyer . DeSimone 42. with GJ, Hb production bin (HbF) stressed siblings of fetal 1980 contain tol 44:535, relationship erythropoietically A didactic gene Genet synthesis cases anaemia. 1981 G: Two modifier J Med D: Genetic hemolytic J Clin in adult Papayannopoulou Fetal 1980 le-fl#{176} thalassemia 1980 WG, HbF Arab M, Zwiers $-thalassemia: Am persistence haemolytic cell disease 56: 187, 1981 I 979 41 J, Stamatoyannopoulos effect hereditary of acquired P. Bid levels homozygous type anemia Dover that to acute of F-cell 8:157, 39. cytes 1970 J, Heller between 32. F: Swiss 5: Sickle Dis Child P: Stimulation PE, Papayannopoulou induced 40. AL, M, Perrine Arch Heller factors. Acceleration Hematol of fetal J Hum SI, response by genetic 38. Nute J R: Inheritance Am P. Pembrey childhood. J, Bid hemoglobin controlled Schroeder persistence John in early DeSimone los G: Margolet frequency F cell JB, Wood RP, Arabs AL. bin synthesis in baboons by hemolysis and hypoxia. Proc Natl Acad Sci USA 75:2937, 1978 37. DeSimone J, Heller P, Amsel J, Usman M: Magnitude of the 1975 SH, Wood WA, cellular Perrine 36. of fetal Br J Haematol persistence haemoglobins 29:205, 29. R, Clegg of hereditary 35. in Saudi by radioimmunoassay. DJ, Cartner A: A form characterized of ME, adults its 1977 1983 28. the and 36:461, PC, Pembrey haemoglobin 53:673, HPFH) J Haematol ET around 80:4842, Jr., Moyer 5-azacytidine and the 1983 M, results is associated with ‘y-b-fl-globin gene From www.bloodjournal.org by guest on June 12, 2017. For personal use only. 1984 63: 64-72 Increased HbF in sickle cell anemia is determined by a factor linked to the beta S gene from one parent PF Milner, JD Leibfarth, J Ford, BP Barton, HE Grenett and FA Garver Updated information and services can be found at: http://www.bloodjournal.org/content/63/1/64.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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