AmJHum Genet 29:256-271, 1977
Inheritance of F Cell Frequency in Heterocellular Hereditary Persistence of Fetal Hemoglobin: An Example of Allelic Exclusion SAMUEL H. BOYER,1 LOUISE MARGOLET,' MARIAN L. BOYER,1 TITUS H. J. HUISMAN,2 WALTER A. SCHROEDER,3 W. G. WOOD,4 D. J. WEATHERALL,4 J. B. CLEGG,4 AND R. CARTNER5
Varying quantities of immunologically verified fetal hemoglobin (HbF) persist throughout adult life [1-2]. The amount is usually small (e.g., less than 1% in 90% of white Americans) and confined to a few erythrocytes (F cells) [3-8] where it coexists [9] with adult hemoglobins. The quantity of HbF per F cell (6%-40% of cell hemoglobin) when multiplied by the proportion of F cells (0. 1%-4% of all red cells) was sufficient in healthy individuals to account for the HbF found in unfractionated hemolysate [10]. Thus varying proportions of two discrete populations of erythrocytes, F cells and non-F cells, are naturally present in adults. This report describes F cell analysis of two previously reported families (kindred G [I 1] and kindred A [12]) with hereditary persistence of HbF (HPFH). In kindred G, HbF levels (2%-7%) were lower than usual for HPFH but were thought to be uniformly distributed in at least one individual (designated D. G. in reference [11 ] and III-4 in this report). In kindred A, HbF levels were higher (6%-23% and remarkable for their variability among family members and their uneven distribution among cells. The two families were opposite with respect to the type of HbF-y chains present: most chains were Gy in kindred G [11 ] and Ay in kindred A [12]. In both families, prior assay of intracellular distribution relied on the Kleihauer technique (i.e., resistance of cellular HbF to elution with acidic buffers [13]). The acid elution method is useful for detection only with cells having large amounts of HbF; its detection threshold for HbF is about 20 times higher [7] than the single cell Received July 7, 1976; revised January 19, 1977. This investigation was supported in part by grants GM-19489, HL-1-2403, HL-05168, HL-15158, and HL-02558 from the U.S. Public Health Service, National Institutes of Health; and by grants from the Wellcome Trust and the Medical Research Council of the United Kingdom. 1 Howard Hughes Medical Institute Laboratory of Biochemical Genetics and Division of Medical Genetics, Department of Medicine, The Johns Hopkins University School of Medicine and Hospital, 720 Rutland Avenue, Baltimore, Maryland 21205. Address reprint requests to S. H. Boyer. 2 Laboratory of Protein Chemistry and Sickle Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30902. 3Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. 4 Nuffield Department of Clinical Medicine, The Radcliffe Infirmary, Oxford, England OX2 6HE. 5Central Laboratory, Sunderland Group of Hospitals, England. © 1977 by the American Society of Human Genetics. All rights reserved.
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immunological assays with anti-HbF devised by Katsura [3] and Hosoi [4] and used by us [6-8] for F cell analysis. Consequently, in families where HbF levels are only moderately elevated above normal [11,12,14-17], the acid-elution technique cannot distinguish between variable concentrations of HbF in all red cells and constant amounts localized to a few. This paper shows that erythrocyte patterns in kindred G [11 ] and kindred A [12] take
the form of F cells which constitute nearly 50% of all erythrocytes in heterozygotes and close to 100% in homozygotes. We thus demonstrate what in heterozygotes appears to be inherited allelic exclusion of an HPFH gene in approximately half the erythrocytes. However this arises, it leads to a condition which is phenotypically different from pancellular HPFH [11, 17-20] where HbF is present in all erythrocytes from
heterozygotes. MATERIALS AND METHODS
Samples Blood from members of kindred A in England and kindred G in Georgia was collected in 0.2 vol of acid-citrate-dextrose. Refrigerated samples intended for immunodiffusion analysis were received in Baltimore within 36 hr after sampling. F cell assays using Hosoi's method [4, 8] were performed in Oxford. The identity and kinship of individuals in family G were unknown to those who performed immunological assays. Blood samples collected in heparin from 41 healthy white adults were used as controls.
Hematological Examinations Red cell indices, hemoglobin analyses, and biosynthetic assays were obtained for kindred G using methods described previously [21-27] and noted in footnotes to tables 1-4. A partial set of such analyses were reported earlier [11]; equivalent analyses [12] and methods [28] for kindred A have already been described. Immunological Assays Except where indicated, specific antisera were used in our studies (i.e., anti-HbF was unreactive with HbA, and anti-HbA was unreactive with HbF). F cells were enumerated after reaction with anti-HbF using both microscopic immunodiffusion analysis [3, 5-7] and binding of fluorescein isothiocyanate (FITC)-labeled antibody [4, 8]. Diameters of microscopic immunodiffusion reactions were used for semiquantitative estimation of HbF per F cell [5, 10] and in several individuals from kindred A, for analysis of the size distribution of HbA reactions. Estimates of HbF per F cell were standardized [10] through measurement of the mean immunodiffusion reaction diameters surrounding 100 red cells from each of three different individuals with HPFH. Hemolysate HbF in these three persons represented 100% [17], 69% [17], and 20% [29] of all hemoglobin; corresponding mean corpuscular hemoglobin (MCH) values in the three subjects were 23.1, 14.2, and 23.4 pg, respectively. A plot [5] of the mean reaction diameters vs. log (% hemolysate HbF x MCH/100) formed a straight line through three
points. Measures of hemolysate HbF levels by alkali denaturation [26, 28] and/or isoleucine [27] analysis in kindreds A and G were supplemented by macroscopic immunodiffusion assay [1-2, 10] with anti-HbF. Areas of macroscopic immunodiffusion rings associated with HbF reactions were nonspecifically augmented 1.1 to 1.4-fold by the HbA present in samples from our subjects. To circumvent this effect, samples were diluted with a solution of purified HbA; the HbF standards used for construction of calibration lines contained added HbA levels (24 mg/ml) approximating that in the samples analyzed. Without such precautions, the quantity of HbF in sample hemolysates would be slightly overestimated. Correlation coefficients for the regression of immunodiffusion ring area upon concentration of HbF usually exceeded .996.
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Inheritance of F Cell Frequency in Heterocellular Hereditary Persistence of Fetal Hemoglobin: An Example of Allelic Exclusion SAMUEL H. BOYER,1 LOUISE MARGOLET,' MARIAN L. BOYER,1 TITUS H. J. HUISMAN,2 WALTER A. SCHROEDER,3 W. G. WOOD,4 D. J. WEATHERALL,4 J. B. CLEGG,4 AND R. CARTNER5
Varying quantities of immunologically verified fetal hemoglobin (HbF) persist throughout adult life [1-2]. The amount is usually small (e.g., less than 1% in 90% of white Americans) and confined to a few erythrocytes (F cells) [3-8] where it coexists [9] with adult hemoglobins. The quantity of HbF per F cell (6%-40% of cell hemoglobin) when multiplied by the proportion of F cells (0. 1%-4% of all red cells) was sufficient in healthy individuals to account for the HbF found in unfractionated hemolysate [10]. Thus varying proportions of two discrete populations of erythrocytes, F cells and non-F cells, are naturally present in adults. This report describes F cell analysis of two previously reported families (kindred G [I 1] and kindred A [12]) with hereditary persistence of HbF (HPFH). In kindred G, HbF levels (2%-7%) were lower than usual for HPFH but were thought to be uniformly distributed in at least one individual (designated D. G. in reference [11 ] and III-4 in this report). In kindred A, HbF levels were higher (6%-23% and remarkable for their variability among family members and their uneven distribution among cells. The two families were opposite with respect to the type of HbF-y chains present: most chains were Gy in kindred G [11 ] and Ay in kindred A [12]. In both families, prior assay of intracellular distribution relied on the Kleihauer technique (i.e., resistance of cellular HbF to elution with acidic buffers [13]). The acid elution method is useful for detection only with cells having large amounts of HbF; its detection threshold for HbF is about 20 times higher [7] than the single cell Received July 7, 1976; revised January 19, 1977. This investigation was supported in part by grants GM-19489, HL-1-2403, HL-05168, HL-15158, and HL-02558 from the U.S. Public Health Service, National Institutes of Health; and by grants from the Wellcome Trust and the Medical Research Council of the United Kingdom. 1 Howard Hughes Medical Institute Laboratory of Biochemical Genetics and Division of Medical Genetics, Department of Medicine, The Johns Hopkins University School of Medicine and Hospital, 720 Rutland Avenue, Baltimore, Maryland 21205. Address reprint requests to S. H. Boyer. 2 Laboratory of Protein Chemistry and Sickle Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30902. 3Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. 4 Nuffield Department of Clinical Medicine, The Radcliffe Infirmary, Oxford, England OX2 6HE. 5Central Laboratory, Sunderland Group of Hospitals, England. © 1977 by the American Society of Human Genetics. All rights reserved.
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immunological assays with anti-HbF devised by Katsura [3] and Hosoi [4] and used by us [6-8] for F cell analysis. Consequently, in families where HbF levels are only moderately elevated above normal [11,12,14-17], the acid-elution technique cannot distinguish between variable concentrations of HbF in all red cells and constant amounts localized to a few. This paper shows that erythrocyte patterns in kindred G [11 ] and kindred A [12] take
the form of F cells which constitute nearly 50% of all erythrocytes in heterozygotes and close to 100% in homozygotes. We thus demonstrate what in heterozygotes appears to be inherited allelic exclusion of an HPFH gene in approximately half the erythrocytes. However this arises, it leads to a condition which is phenotypically different from pancellular HPFH [11, 17-20] where HbF is present in all erythrocytes from
heterozygotes. MATERIALS AND METHODS
Samples Blood from members of kindred A in England and kindred G in Georgia was collected in 0.2 vol of acid-citrate-dextrose. Refrigerated samples intended for immunodiffusion analysis were received in Baltimore within 36 hr after sampling. F cell assays using Hosoi's method [4, 8] were performed in Oxford. The identity and kinship of individuals in family G were unknown to those who performed immunological assays. Blood samples collected in heparin from 41 healthy white adults were used as controls.
Hematological Examinations Red cell indices, hemoglobin analyses, and biosynthetic assays were obtained for kindred G using methods described previously [21-27] and noted in footnotes to tables 1-4. A partial set of such analyses were reported earlier [11]; equivalent analyses [12] and methods [28] for kindred A have already been described. Immunological Assays Except where indicated, specific antisera were used in our studies (i.e., anti-HbF was unreactive with HbA, and anti-HbA was unreactive with HbF). F cells were enumerated after reaction with anti-HbF using both microscopic immunodiffusion analysis [3, 5-7] and binding of fluorescein isothiocyanate (FITC)-labeled antibody [4, 8]. Diameters of microscopic immunodiffusion reactions were used for semiquantitative estimation of HbF per F cell [5, 10] and in several individuals from kindred A, for analysis of the size distribution of HbA reactions. Estimates of HbF per F cell were standardized [10] through measurement of the mean immunodiffusion reaction diameters surrounding 100 red cells from each of three different individuals with HPFH. Hemolysate HbF in these three persons represented 100% [17], 69% [17], and 20% [29] of all hemoglobin; corresponding mean corpuscular hemoglobin (MCH) values in the three subjects were 23.1, 14.2, and 23.4 pg, respectively. A plot [5] of the mean reaction diameters vs. log (% hemolysate HbF x MCH/100) formed a straight line through three
points. Measures of hemolysate HbF levels by alkali denaturation [26, 28] and/or isoleucine [27] analysis in kindreds A and G were supplemented by macroscopic immunodiffusion assay [1-2, 10] with anti-HbF. Areas of macroscopic immunodiffusion rings associated with HbF reactions were nonspecifically augmented 1.1 to 1.4-fold by the HbA present in samples from our subjects. To circumvent this effect, samples were diluted with a solution of purified HbA; the HbF standards used for construction of calibration lines contained added HbA levels (24 mg/ml) approximating that in the samples analyzed. Without such precautions, the quantity of HbF in sample hemolysates would be slightly overestimated. Correlation coefficients for the regression of immunodiffusion ring area upon concentration of HbF usually exceeded .996.
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