Am J Hum Genet 27:765-777, 1975
A GY Type of the Hereditary Persistence of Fetal Hemoglobin with P Chain Production in Cis T. H. J. HUISMAN,' A. MILLER,1 AND W. A. SCHROEDER2
The hereditary persistence of fetal hemoglobin (HPFH) is a well known and extensively characterized condition. Not long after its discovery, heterogeneity in expression of this benign hemoglobinopathy was observed: the percentage of hemoglobin F (Hb F) is higher in most affected blacks than in their Greek counterparts [1]. The investigations of Hb F in HPFH conducted since the detection of heterogeneity in the y chains of Hb F [2] have demonstrated the very great heterogeneity of the genetic bases of HPFH despite virtually identical clinical expression (reviewed in [3-5]). The hemoglobin of the homozygote for HPFH consists solely of Hb F: Hb A and Hb A2 are not detectable [6-9]. Individuals who have a combination of HPFH and a /3 chain variant such as Hb S, C, or E or a 8 chain variant such as Hb A2' also produce no Hb A or Hb A2, respectively. The conclusion has been, therefore, that Hb A and Hb A2-more specifically /8 and 8 chains-are not produced in cis to the HPFH determinant. Deletion of the /8 and 8 genes in cis to the HPFH determinant is the probable cause of failure to produce /3 and 8 chains. The present report concerns a family with a 0y type of HPFH (i.e., one whose Hb F has only Gy chains) in which /3A chains are produced in cis to the HPFH determinant. The genetic aspects of this information are discussed. MATERIALS AND METHODS Blood samples were collected in Vacutainers with EDTA as anticoagulant and refrigerated during transport to Augusta, Georgia. Hematological Examination Hemoglobin, packed cell volume, red cell count, and red cell indices were determined by conventional techniques [10]. The distribution of Hb F in the cells was studied with the acid elution method of Kleihauer [11] and with density gradient fractionation [12]. In the latter, red cells were fractionated by centrifugation in a discontinuous density gradient of Dextran 40, and the quantity of Hb F in each layer of cells was determined Received January 16, 1975; revised April 4, 1975. Contribution no. 5039 from the Division of Chemistry and Chemical Engineering, California Institute of Technology. This investigation was supported in part by grants HL-05168, HL15158, and HL-02558 from the U.S. Public Health Service, National Institutes of Health. 1 Laboratory of Protein Chemistry and Sickle Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30902. 2 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. o 1975
by the American Society of Human Genetics. All rights reserved.
765
766
HUISMAN ET AL.
by ion exchange chromatography. Red cell indices were also determined on all isolated fractions. Hemoglobin Analyses The method of starch gel electrophoresis of hemoglobins has been described [13]. The quantities of the several hemoglobins were determined by analytical chromatography on 0.9 X 55-cm columns of DEAE-Sephadex [14, 15]. Larger columns of DEAESephadex were used for preparative isolation of hemoglobins. Hb F was determined not only by this method (percentage FCh) but also by amino acid analysis of the chromatographically isolated Hb F (percentage F11) [16] and by alkali denaturation (percentage FAD) [17]. A microchromatographic method for the determination of Hb A2 [18] was applied in some instances. Globins from chromatographically isolated Hb F were examined by previously described methods [2] to ascertain the type of y chain. Biosynthetic Studies The incorporation in vitro of 14C-leucine by the method of Friedman et al. [19] was used to determine the relative rates of synthesis of the a, 13, and y chains. Saline-washed red cells were incubated with 14C-leucine for 2 hr. and the total radioactivity of each of the chains was used for the calculation of the ratio. The average value for 24 normal adults was 1.07 with a range of 0.90-1.30. For nine Hb S heterozygotes with normal hematological characteristics, the value was 1.20 with a range of 0.95-1.40. RESULTS
Figure 1 depicts the results of starch gel electrophoretic analyses at pH 9 of some members of family P whose relationships are shown in figure 2. An elevated level of Hb F had first been detected in the proposita (D. P.) in a routine testing program, and a presumptive diagnosis of HPFH was made. Only Gy chains were detected in her Hb F (table 1).* Further study of the family revealed the presence of both Hb A and Hb S in two of the six additional members with an increased amount of Hb F and of sickle cell anemia in one young child (Ch. P.). The DEAE-Sephadex chromatogram of hemoglobin from subject W. P., for example, showed the presence of Hb A, Hb S, and an elevated Hb F (fig. 3). The Hb A and Hb S were characterized further by amino acid analysis, N-terminal determinations, solubility tests, and sickle cell preparations. The results were as anticipated and excluded the presence of crossover products between /3, y, and 8 loci.t In all seven subjects with elevated levels of Hb F, the Hb F had only 0y chains (table 1). * The amino acid analyses for glycine and alanine in yCB-3 are the basis for this statement. Interpretation of these data has been presented [2, 3]. Briefly, if glycine in yCB-3 is integral at 1.0 as it is here, only Gy chains with glycine in residue 136 are present. If glycine is 0.0, then only Ay chains (alanine is then 3.0 instead of 2.0) are present. If glycine is nonintegral, a mixture is present. tThat an amino acid analysis can be definitive in distinguishing Hb A and Hb S from crossover products with the y chain can be seen from the following considerations. The absence of isoleucine eliminates y sequences between residues 11 and 116. If the distinctive methionine of y133 and/or arginine of y144 were present, either would be obvious from the analysis. Residues 145 and 146 are Tyr-His in both chains. Hence, in the absence of these differences, 11-146 are /3 sequence. Any crossover between residues 5 and 10 would change charge because of glutamic acid in y5. Residue yl is glycine and /81 is valine; y3 is phenylalanine and 183 is leucine. Although the amino acid analysis accords with valine and leucine (i.e., /3), the data are less definitive because of the larger numbers of those residues. However, the N-terminal determination supports even this conclusion from the analyses.
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
S..
767
..
FIG. -.-Starch gel electrophoresis at pH 9 of some members of family P
When the cells of individuals with increased percentage of Hb F were examined by the acid elution technique [11], the Hb F was present in each cell although in varying amounts as is common in the HPFH condition (fig. 4). The distribution of Hb F, A2, and S in red cell fractions which were obtained by density gradient centrifugation from subjects W. P., S. P., and D. P. is presented in figure 5 together with similar data on a /3+ thalassemia homozygote, one heterozygous subject with the GyAy type of HPFH, and two persons with sickle cell anemia. Uniform distribution of these hemoglobins within the red cells of these members of family P was also demonstrated by this method. The percentage of Hb A2 in these persons is in the low normal or below normal range. The biosynthetic studies reveal the presence of a thalassemia in the family. The mother (C. P.) not only has sickle cell trait but is also heterozygous for a thalassemia. This has been deduced from the non-a/a chain ratio of 1.63 and from the low amount of Hb S (29%). The same condition appears in four of the children of whom W. P. also has the HPFH condition and J. P. the sickle cell trait with 29%o Hb S. The presence of a thalassemia in conjunction with HPFH does not
768
HUISMAN ET AL. t
BB. 62
T.P Jr 27
W. R 23
D.P 21
J.P 16
R.R 13
Ea Dw.R 2
S.P 12
F. P 11
Eli)
a-Thal.
CM
A-HPFH Normal Not Studied
Th. P. 9
AS
E
Proposita
FIG. 2.-Pedigree of family P. Numbers under initials indicate
ages
to alter the expression of HPFH appreciably (compare W. P. with S. P. especially but also with T. P. Sr., T. P. Jr., D. P., and Th. P.). The balanced thalassemia synthesis in those individuals with increased Hb F but without identifies the HPFH condition in this family and excludes 8,8 thalassemia [5, 20]. appear
a
DISCUSSION
Diagnosis of the Condition The conclusion that the proposita has the HPFH condition is supported by hematological and biosynthetic data and by the rather uniform distribution of Hb F in the cells as observed with two methods. The glycine and alanine values pH 2.0
I
--
-
8.0
E
S
A
-(48.9%)
.2
F
(24.1%)
(24.8%)
z
cr
0.8-
~A
m~~~~~
A GY Type of the Hereditary Persistence of Fetal Hemoglobin with P Chain Production in Cis T. H. J. HUISMAN,' A. MILLER,1 AND W. A. SCHROEDER2
The hereditary persistence of fetal hemoglobin (HPFH) is a well known and extensively characterized condition. Not long after its discovery, heterogeneity in expression of this benign hemoglobinopathy was observed: the percentage of hemoglobin F (Hb F) is higher in most affected blacks than in their Greek counterparts [1]. The investigations of Hb F in HPFH conducted since the detection of heterogeneity in the y chains of Hb F [2] have demonstrated the very great heterogeneity of the genetic bases of HPFH despite virtually identical clinical expression (reviewed in [3-5]). The hemoglobin of the homozygote for HPFH consists solely of Hb F: Hb A and Hb A2 are not detectable [6-9]. Individuals who have a combination of HPFH and a /3 chain variant such as Hb S, C, or E or a 8 chain variant such as Hb A2' also produce no Hb A or Hb A2, respectively. The conclusion has been, therefore, that Hb A and Hb A2-more specifically /8 and 8 chains-are not produced in cis to the HPFH determinant. Deletion of the /8 and 8 genes in cis to the HPFH determinant is the probable cause of failure to produce /3 and 8 chains. The present report concerns a family with a 0y type of HPFH (i.e., one whose Hb F has only Gy chains) in which /3A chains are produced in cis to the HPFH determinant. The genetic aspects of this information are discussed. MATERIALS AND METHODS Blood samples were collected in Vacutainers with EDTA as anticoagulant and refrigerated during transport to Augusta, Georgia. Hematological Examination Hemoglobin, packed cell volume, red cell count, and red cell indices were determined by conventional techniques [10]. The distribution of Hb F in the cells was studied with the acid elution method of Kleihauer [11] and with density gradient fractionation [12]. In the latter, red cells were fractionated by centrifugation in a discontinuous density gradient of Dextran 40, and the quantity of Hb F in each layer of cells was determined Received January 16, 1975; revised April 4, 1975. Contribution no. 5039 from the Division of Chemistry and Chemical Engineering, California Institute of Technology. This investigation was supported in part by grants HL-05168, HL15158, and HL-02558 from the U.S. Public Health Service, National Institutes of Health. 1 Laboratory of Protein Chemistry and Sickle Cell Center, Medical College of Georgia, and Veterans Administration Hospital, Augusta, Georgia 30902. 2 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125. o 1975
by the American Society of Human Genetics. All rights reserved.
765
766
HUISMAN ET AL.
by ion exchange chromatography. Red cell indices were also determined on all isolated fractions. Hemoglobin Analyses The method of starch gel electrophoresis of hemoglobins has been described [13]. The quantities of the several hemoglobins were determined by analytical chromatography on 0.9 X 55-cm columns of DEAE-Sephadex [14, 15]. Larger columns of DEAESephadex were used for preparative isolation of hemoglobins. Hb F was determined not only by this method (percentage FCh) but also by amino acid analysis of the chromatographically isolated Hb F (percentage F11) [16] and by alkali denaturation (percentage FAD) [17]. A microchromatographic method for the determination of Hb A2 [18] was applied in some instances. Globins from chromatographically isolated Hb F were examined by previously described methods [2] to ascertain the type of y chain. Biosynthetic Studies The incorporation in vitro of 14C-leucine by the method of Friedman et al. [19] was used to determine the relative rates of synthesis of the a, 13, and y chains. Saline-washed red cells were incubated with 14C-leucine for 2 hr. and the total radioactivity of each of the chains was used for the calculation of the ratio. The average value for 24 normal adults was 1.07 with a range of 0.90-1.30. For nine Hb S heterozygotes with normal hematological characteristics, the value was 1.20 with a range of 0.95-1.40. RESULTS
Figure 1 depicts the results of starch gel electrophoretic analyses at pH 9 of some members of family P whose relationships are shown in figure 2. An elevated level of Hb F had first been detected in the proposita (D. P.) in a routine testing program, and a presumptive diagnosis of HPFH was made. Only Gy chains were detected in her Hb F (table 1).* Further study of the family revealed the presence of both Hb A and Hb S in two of the six additional members with an increased amount of Hb F and of sickle cell anemia in one young child (Ch. P.). The DEAE-Sephadex chromatogram of hemoglobin from subject W. P., for example, showed the presence of Hb A, Hb S, and an elevated Hb F (fig. 3). The Hb A and Hb S were characterized further by amino acid analysis, N-terminal determinations, solubility tests, and sickle cell preparations. The results were as anticipated and excluded the presence of crossover products between /3, y, and 8 loci.t In all seven subjects with elevated levels of Hb F, the Hb F had only 0y chains (table 1). * The amino acid analyses for glycine and alanine in yCB-3 are the basis for this statement. Interpretation of these data has been presented [2, 3]. Briefly, if glycine in yCB-3 is integral at 1.0 as it is here, only Gy chains with glycine in residue 136 are present. If glycine is 0.0, then only Ay chains (alanine is then 3.0 instead of 2.0) are present. If glycine is nonintegral, a mixture is present. tThat an amino acid analysis can be definitive in distinguishing Hb A and Hb S from crossover products with the y chain can be seen from the following considerations. The absence of isoleucine eliminates y sequences between residues 11 and 116. If the distinctive methionine of y133 and/or arginine of y144 were present, either would be obvious from the analysis. Residues 145 and 146 are Tyr-His in both chains. Hence, in the absence of these differences, 11-146 are /3 sequence. Any crossover between residues 5 and 10 would change charge because of glutamic acid in y5. Residue yl is glycine and /81 is valine; y3 is phenylalanine and 183 is leucine. Although the amino acid analysis accords with valine and leucine (i.e., /3), the data are less definitive because of the larger numbers of those residues. However, the N-terminal determination supports even this conclusion from the analyses.
HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN
S..
767
..
FIG. -.-Starch gel electrophoresis at pH 9 of some members of family P
When the cells of individuals with increased percentage of Hb F were examined by the acid elution technique [11], the Hb F was present in each cell although in varying amounts as is common in the HPFH condition (fig. 4). The distribution of Hb F, A2, and S in red cell fractions which were obtained by density gradient centrifugation from subjects W. P., S. P., and D. P. is presented in figure 5 together with similar data on a /3+ thalassemia homozygote, one heterozygous subject with the GyAy type of HPFH, and two persons with sickle cell anemia. Uniform distribution of these hemoglobins within the red cells of these members of family P was also demonstrated by this method. The percentage of Hb A2 in these persons is in the low normal or below normal range. The biosynthetic studies reveal the presence of a thalassemia in the family. The mother (C. P.) not only has sickle cell trait but is also heterozygous for a thalassemia. This has been deduced from the non-a/a chain ratio of 1.63 and from the low amount of Hb S (29%). The same condition appears in four of the children of whom W. P. also has the HPFH condition and J. P. the sickle cell trait with 29%o Hb S. The presence of a thalassemia in conjunction with HPFH does not
768
HUISMAN ET AL. t
BB. 62
T.P Jr 27
W. R 23
D.P 21
J.P 16
R.R 13
Ea Dw.R 2
S.P 12
F. P 11
Eli)
a-Thal.
CM
A-HPFH Normal Not Studied
Th. P. 9
AS
E
Proposita
FIG. 2.-Pedigree of family P. Numbers under initials indicate
ages
to alter the expression of HPFH appreciably (compare W. P. with S. P. especially but also with T. P. Sr., T. P. Jr., D. P., and Th. P.). The balanced thalassemia synthesis in those individuals with increased Hb F but without identifies the HPFH condition in this family and excludes 8,8 thalassemia [5, 20]. appear
a
DISCUSSION
Diagnosis of the Condition The conclusion that the proposita has the HPFH condition is supported by hematological and biosynthetic data and by the rather uniform distribution of Hb F in the cells as observed with two methods. The glycine and alanine values pH 2.0
I
--
-
8.0
E
S
A
-(48.9%)
.2
F
(24.1%)
(24.8%)
z
cr
0.8-
~A
m~~~~~
