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Clinical Chemistry 43: 1850-1856, 1997;
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(Clinical Chemistry. 1997;43:1850-1856.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Combinations of ß chain abnormal hemoglobins with each other or with ß-thalassemia determinants with known mutations: influence on phenotype

Titus Hendrik and Jan Huisman

Department of Biochemistry and Molecular Biology, Research and Education Bldg., Room CB-2208, Medical College of Georgia, Augusta, GA 30912-2114. Fax 706-721-3092; e-mail research.acarver{at}mail.mcg.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hematological and hemoglobin (Hb) data are presented for numerous patients with compound heterozygosities for different ß chain variants and for a ß chain variant with different ß-thalassemia (ß-thal) alleles. Considerable variations, which result from the type of ß chain variant and ß-thal mutation, can be noted. The comparison again emphasizes the importance of determining the diagnoses at the molecular level to aid the physician in the management of patients with different combinations of abnormalities. Simplification and commercialization of modern technology may make the introduction of this approach in some clinical chemistry laboratories possible.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recent advances in the identification procedures have made it possible to rapidly characterize the amino acid replacement in an abnormal hemoglobin (Hb) and the nucleotide mutation in the ß-globin gene that results in a ß-thalassemia (ß-thal). In particular, the PCR amplification procedure with (automated) sequencing, dot-blot analysis, or allele-specific amplification is most useful for this purpose. The importance of this approach is demonstrated by comparing hematological and Hb composition data for numerous patients with compound heterozygosities for different ß chain variants or for a ß chain variant and one of the many ß-thal alleles that have been identified during the past 15 years.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
subjects
Nearly 200 patients (ages 2 months to 60 years) were involved in this study; most were members of local families, but several lived in other states or in different foreign countries. Blood samples were collected in EDTA and transported by car to the laboratory or shipped by air or fast courier service from the country of origin to Augusta, GA. Informed consent was obtained. None of the patients had been transfused during the 6 months before blood collection.

methods
As a rule, each sample was analyzed within 48 h after its arrival in the laboratory. These initial studies included routine hematology with an automated cell counter, examination of blood smears, and reticulocyte counts. Erythrocyte lysates were analyzed by starch gel- or cellulose acetate electrophoresis (1) or by isoelectrofocusing (2) with commercial agar plates (Isolab). The lysates were also applied to cation-exchange HPLC (3)(4) and reversed-phase HPLC (5)(6) columns to quantitate Hb A2, Hb F, and Hb A. When a slowly moving variant like Hb D was present, the Hb A2 zone was often contaminated with small amounts of minor (modified) Hb D, resulting in Hb A2 values that were too high. Furthermore, Hb E and Hb A2 often do not separate on a cation-exchange HPLC column; the concentration of Hb A2 in such samples was determined as %{delta} chain (100 · {delta}/[ß + {gamma} + {delta}]) by reversed-phase HPLC.

Identification of the Hb variant was by protein analysis as described before (7) or by sequence analysis of an amplified segment of DNA that included the ß-globin gene (8)(9). Excluded from these analyses were the samples from patients with Hb S, Hb E, or Hb C, which were evaluated by electrophoretic and chromatographic procedures only. Many patients were studied repeatedly, and the data presented here are those obtained for the first samples that were collected. The repeat samples were primarily used for confirmation of the diagnosis and for identification of the ß-thal alleles. Methodology included sequence analysis of amplified DNA and dot-blot analysis with 32P-labeled, mutation-specific probes (8)(9) and allele-specific amplification (10). The possible presence of an {alpha}-thal or an iron deficiency was not evaluated.


   Results
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Discussion
 References
 
hb s (or hb c) in combination with another ß chain variant
As many as 22 different SX compound heterozygotes have been detected; in addition to the 13 listed in Table 1 , others are S-D-Iran [ß22(B4)Glu->Gln] (11), S-Caribbean [ß91(F7)Leu->Arg] (12), S-Mobile [ß73(E17)Asp->Val] (13), S-Maputo [ß47(CD6)Asp->Tyr] (14), S-Siriraj [ß7(A4)Glu->Lys] (15), S-I-Toulouse [ß66(E10)Lys->Glu] (16), S-San Diego [ß109(G11)Val->Met] (17), S-Osler [ß145(HC2)Tyr->Asp] (18), and S-Shelby [ß131(H9)Gln ->Lys] (19). Table 1 also lists five combinations of Hb C with a second ß chain variant. The listing in Table 1 is restricted to the patients whose samples were analyzed by HPLC methodology so that quantitative data can be compared. Comparable hematological values were obtained for most patients, showing a mild anemia to normal values. Exceptions are the patients with the S-D-Los Angeles, S-O-Arab, and S-Hofu compound heterozygosities, who have a more severe anemia. Hb C, Hb D-Los Angeles, and possibly Hb O-Arab and Hb Hofu affect the sickling process directly or indirectly; the low production of Hb Hofu in the patient with the Hb S-Hofu combination results in a high relative concentration of Hb S and in sickling. A considerable variation in the percentages of Hb S (or Hb C) and Hb X was observed; although the Hb X/Hb S (or Hb C) ratio for many combinations averaged ~1, low ratios were seen for the SE, CE, and S-Hofu combinations, and high ratios were seen for the S-N-Baltimore, C-P-Galveston, and the S-Hope compound heterozygotes. The concentrations of Hb F were, in most instances, <5% except in the three babies, 2–3 months old, with SE and SD disease. Some of the previously published data (20)(21)(22)(23)(24) are referenced in Table 1 .


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  		Table 1. Hematological and hemoglobin composition data for patients with a compound heterozygosity involving Hb S or Hb C and another ß chain variant.

hb x in combination with a ß-thal with a known mutation
These combinations concerned 10 different ß chain variants and numerous ß-thal alleles (Table 2 ). Some of these cases have been reported before (25)(26)(27)(28)(29)(30)(31)(32)(33). Great variations in hematological value can be noted; many patients with Hb E-ß-thal, some with Hb S-ß-thal, and the adult patient with Hb Lulu Island-ß-thal, have a moderate-to-severe anemia. Patients with Hb X and a ß+-thal [the alleles -88 (C->T), -19 (A->G), IVS-I-5 (G->T or G->C), poly(A) (T->C or A->G), IVS-I-110 (G->A), IVS-I-6 (T->C)] had variable concentrations of Hb A (6.2–41.6%), the quantity being directly related to the type of ß-thal mutation. The concentration of Hb F was also most variable; although age was a factor (a few patients were ages 1–5), more important was the type of ß chain variant and the ß-thal alleles, as is evidenced by the high concentration of Hb F (7–65%) in many patients with E-ß0-thal or E-ß+-thal. The Hb F concentration was <15% in all other patients with the different compound heterozygosities. Microcytosis and hypochromia were present in all subjects, although some high mean corpuscular volumes were observed, mainly in blood samples shipped from abroad. The concentrations of Hb A2 were increased (4.1–8.0%); the numbers are somewhat higher than expected because of contamination with minor abnormal Hbs, whereas values calculated from the %{delta} chain obtained by reversed-phase HPLC are always 10–20% higher than those determined by cation-exchange HPLC. The concentration of Hb A2 in the one patient with the Hb E-Lepore combination fell into the normal range, as expected because of the presence of only one functional {delta}-globin gene.


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  		Table 2. Hematological and hemoglobin composition data for patients with a combination of a ß chain variant and a ß-thalassemia with a known mutation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
hb s (hb c)–hb x compound heterozygotes
The data listed in Table 1Up concern combinations in which Hb X is a Hb D-like variant (D-Los Angeles, Richmond, P-Galveston, Osu Christiansborg, Korle Bu), a Hb C-like variant (Hb C, Hb O, Hb E), a fast-moving variant (Lufkin, N, Hofu, Hope), or a variant with a neutral amino acid replacement (Muscat, Iowa) that was accidentally discovered with an HPLC method. Thus, when studied by electrophoresis alone, these variants would likely be identified as Hb D-like, Hb C-like, and fast Hb, whereas the two with neutral amino acid replacements would not have been observed. One needs to go beyond this initial electrophoretic characterization and further define the SD, SC, and S-fast Hb diagnoses because of some special properties of the variant that can affect the health of the patient. The best example is Hb D-Los Angeles [ß121(GH4)Glu->Gln], which, like the other Hb D-like variants, is an innocent anomaly in the heterozygote, but will, in combination with Hb S, cause a hemolytic disease as severe as sickle cell anemia because Hb D interacts with Hb S to promote sickling (34)(35). Similarly, correct identification of the Hbs C, O-Arab, and E is important; compound heterozygotes for the Hbs SC, SO, or SE have their own characteristics and clinical expression (34).

An interesting aspect of the data listed in Table 1Up is the difference in the relative quantities of the various variants. This information is further detailed in Fig. 1 . The first group consists of six slow-moving variants, which in the simple heterozygote are present at 25–40% (34)(36)) and at 45–60% in compound heterozygotes with Hb S. The relative quantities of most ß chain variants in heterozygotes are influenced by the presence of an {alpha}-thal (34)(35)(36)(37), and the large spread in the data shown in Fig. 1 likely results from differences in the numbers of active {alpha}-globin genes. Unfortunately, no {alpha}-globin gene data are available; however, it can be assumed that the frequency of {alpha}-thal is the same in the groups with simple heterozygosities for specific ß chain variants as in those with corresponding compound heterozygosities, thus validating comparisons of percentages in both categories. The studies by Mrabet et al. (34) have detailed the electrostatic attraction as a major mechanism controlling the assembly of Hb dimers and tetramers. Their data suggest that the <50% concentration of these abnormal Hbs in simple heterozygotes results from the fact that {alpha}ßX dimer formation is impaired when the ß chain carries one (or more) extra positive charges (34)(35). When present with another such variant (e.g., Hb S) this disadvantage disappears, and the formation of both {alpha}ßS and {alpha}ßX proceeds at about the same rate. Similarly, one can expect at least no effect on the dimerization when the variant ßX chain carries one or more extra negative charges. Indeed, heterozygotes for Hb N and other fast-moving variants have about equal quantities of Hb X and Hb A, whereas Hb X is the major component in compound heterozygotes with Hb S or Hb C. Other important factors may influence the quantity of Hb X. Examples are Hb E, which is present in low quantities (20–30%) in heterozygotes because the GAG->AAG mutation at codon 26 activates another splicing site, thus reducing the production of the ßE chains (35), and Hb Hofu, which is known to be unstable (21). Notably the concentration of Hb E in SE or CE compound heterozygotes (30–40%) is ~10% higher than in the Hb E heterozygote; apparently the decrease in the rate of {alpha}ßS dimer formation has indirectly promoted the {alpha}ßE dimerization.



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  		Figure 1. The concentrations of the listed ß chain variants (Hb X) in simple heterozygotes (open circles) and in patients with compound heterozygosities for Hb X with Hb S (closed circles) or with Hb C (closed squares).

The percentages are derived from the data listed in Table 1Up .

ßX-thal compound heterozygotes
The combination of Hb S or Hb E with ß-thal is a well-defined anomaly that has been described in all hematological text books. Considerable variation in severity of these hemolytic diseases has been noted, and differentiation is made between the Hb X-ß0 and Hb X-ß+ types, i.e., that without Hb A production and that with a decreased concentration of Hb A ((35) and references therein). Only more recently have reports appeared (26)(27)(28)(29)(30)(31)(32)(33) in which the ß-thal allele has been defined at the molecular level. The comparison provided in Table 2Up is based partially on published data (see references listed in the table) and on new information from my laboratory. None of the patients were transfused within the 6 months before blood collection, and all analyses were made with the same HPLC procedure. Fig. 2 summarizes the most important data, namely, the total concentration of Hb as a measure of the severity of the disease and the concentration of Hb F. Furthermore, three groups of ß-thal are recognized: ß0-thal (severe), ß+-thal (severe), and ß+-thal (mild), as defined in the legend of Fig. 2 . Hb S-ß-thal and Hb E-ß-thal are considered separately, whereas the others are combined into a third group of Hb X-ß-thal (see Fig. 2 legend). A considerable anemia (79–107 g/L) is noticed for all patients with Hb S-ß0-thal or Hb S-ß+-thal (severe) with only a modest increase in Hb F response (6.0–11.9%). Patients with these conditions often suffer from the same complications associated with a high percentage of Hb S as do patients with sickle cell anemia, who often may have even higher concentrations of Hb F. The subjects with the milder form of Hb S-ß+-thal consistently have higher total Hb concentrations and perhaps also higher Hb F concentrations. As a result, some 60–70% of their Hb is Hb S, and sickling-associated symptoms are less common also because the Hb A (present for ~20%) is equally distributed over all cells together with Hb S. Probably the most serious type of Hb S-ß0-thal is that in which the thalassemia allele has been identified as the IVS-I-2 (T->C) mutation (27).



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  		Figure 2. Total Hb and Hb F concentrations in patients with Hb Xß-ß-thal.

HbX = Hb C (6 Glu->Lys), Hb D-LA (121 Glu->Gln), Hb Lulu Island (107 Gly->Asp), Hb Shelby (131 Gln->Lys), Hb O-Arab (121 Glu->Lys), Hb E-Saskatoon (22 Glu-> Lys), Hb J-Baltimore (16 Gly->Asp), Hb N-Baltimore (95 Lys->Glu), Hb Porto Alegre (9 Ser->Cys). ß0-Thal (severe), codons 8 and 9 (+G), codon 15 (TGG->TAG), codon 35 (C->A), codons 36/37 (-T), codon 39 (C->T), codons 41/42 (-TTCT), codons 71/72 (+A), codons 106/107 (+G), IVS-I-2 (T->C), IVS-II-1 (G->A), IVS-II-849 (A->C), IVS-II-849 (A->G), -1292-kb deletion, Hb Lepore. ß+-Thal (severe), IVS-I-5 (G->C), IVS-I-5 (G->T), IVS-I-110 (G->A). ß+-Thal (mild), -88 (C->T), -29 (A->G), codon 19 (A->G) or Hb Malay, AATAAA->AATAGA, IVS-I-6 (T->C).

Hb E-ß-thal, a well-recognized disorder among East Asian populations, is characterized as a severe hemolytic disorder comparable with thalassemia major, which is quite understandable because the GAG->AAG mutation at codon 26 results in a ß+ type of thalassemia as well as in the formation of a ß chain with an amino acid replacement. The anemia is severe for all patients with Hb E-ß0-thal and Hb E-ß+-thal (severe); the total Hb concentration varies between 58 and 81 g/L (Fig. 2Up ). Hb F synthesis is markedly increased, as is often seen in patients with ß-thal major, and concentrations between 20% and 65% have been recorded in our subjects. The patients with Hb E-ß+-thal (mild) with the codon 19 (A->G) mutation, which involves the formation of Hb Malay (25), or with the poly(A) (A->G) mutation have indeed a much milder disease (Hb 95–100 g/L) but also a much lower Hb F response (7.0–7.5%).

Nearly all subjects with Hb X-ß-thal (Hb X as defined in the legend of Fig. 2Up ) have a mild hemolytic anemia (Hb 91–145 g/L), which is comparable with that of a simple A-ß-thal condition. The Hb F response is also minor, and Hb F concentrations between 1% and 15% have been recorded. An obvious exception is the young adult with Hb Lulu Island-ß0-thal (Hb 85 g/L; Hb F 2.7%), who also suffers from a thalassemia intermedia because of the instability of the abnormal Hb (28). The differences in Hb E-Saskatoon [ß22(B4)Glu->Lys]-ß-thal and Hb E [ß26(B8)Glu->Lys]-ß-thal should be noted; the mildness of the first condition is striking, whereas differentiation between the two types requires advanced methodology.

In conclusion, the comparisons provided here for the multiple types of Hb S(C)–Hb X combinations and the Hb S(Hb E; Hb X)–ß-thal compound heterozygosities suggest that identification of these conditions should go beyond hematological evaluation and an electrophoretic examination. Because of the simplification (and commercialization) of procedures to isolate DNA from blood samples and the introduction of PCR techniques within the clinical laboratory, characterization of Hb types with substantial adverse properties and of ß-thal alleles with various pathologies [for a complete list see ref. 38] has come within the reach of routine hospital laboratories. The data presented here indeed show great variations in the clinical expression of the different Hb types and ß-thal alleles; a better defined diagnosis might assist clinicians greatly in the management of the individual patient.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Huisman THJ, Jonxis JHP. The hemoglobinopathies techniques of identification. Clinical and biochemical analysis, Vol. 6 1977 Marcel Dekker New York. .
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  5. Shelton JB, Shelton JR, Schroeder WA. High performance liquid chromatographic separation of globin chains on a large-pore C4 column. J Liquid Chromatogr 1984;7:1969-1977.
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  7. Wilson JB, Lam H, Pravatmuang P, Huisman THJ. Separation of tryptic peptides of normal and abnormal {alpha}, ß, {gamma}, and {delta} hemoglobin chains by high-performance liquid chromatography. J Chromatogr 1979;179:271-290. [Web of Science][Medline] [Order article via Infotrieve]
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  10. Postnikov YuV, Molchanova TP, Huisman THJ. Allele-specific amplification for the identification of several hemoglobin variants. Hemoglobin 1993;17:439-452. [Web of Science][Medline] [Order article via Infotrieve]
  11. Serjeant B, Myerscough E, Serjeant GR, Higgs DR, Moo-Penn WF. Sickle cell-Hemoglobin D Iran: a benign sickle cell syndrome. Hemoglobin 1982;6:57-59. [Web of Science][Medline] [Order article via Infotrieve]
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  14. Marinucci M, Boissel JP, Massa A, Wajcman H, Tentori L, Labie D. Hemoglobin Maputo: a new ß-chain variant ({alpha}2ß2 47 (CD6) Asp->Tyr) in combination with Hemoglobin S, identified by high performance liquid chromatography (HPLC). Hemoglobin 1983;7:423-433. [Web of Science][Medline] [Order article via Infotrieve]
  15. Rhoda MD, Arous N, Garel MC, Mazarin M, Monplaisir N, Braconnier F, et al. Interaction of Hemoglobin Siriraj with Hemoglobin S: a mild sickle cell syndrome. Hemoglobin 1986;10:21-31. [Web of Science][Medline] [Order article via Infotrieve]
  16. Tejuca M, Martinez G, Mendez J, Quintero I, Felicetti L, Colombo B. Association of Hb Toulouse with Hb S in a Nicaraguan girl. Hemoglobin 1987;11:43-46. [Web of Science][Medline] [Order article via Infotrieve]
  17. Williamson D, Perry DJ, Brown K, Langdown JV, De Silva C. Compound heterozygosity for two ß chain variants: Hb S [ß6(A3)Glu->Val] and the high affinity variant Hb San Diego [ß109(G11)Val->Met]. Hemoglobin 1995;19:27-32. [Web of Science][Medline] [Order article via Infotrieve]
  18. Hutt PJ, Donaldson MH, Khatri J, Fairbanks VF, Hoyer JD, Thibodeau SN, et al. Hemoglobin S/Hemoglobin Osler: a case with 3 ß globin chains. DNA sequence (AAT) proves that Hb Osler is ß145 Tyr->Asn. Am J Hematol 1996;52:305-309. [Web of Science][Medline] [Order article via Infotrieve]
  19. Adachi K, Surrey S, Tamary H, Kim J, Eck HS, Rappaport E, et al. Hb Shelby [ß131(H9)Gln->Lys] in association with Hb S [ß6(A3)Glu->Val]: characterization, stability, and effects on Hb S polymerization. Hemoglobin 1993;17:329-343. [Web of Science][Medline] [Order article via Infotrieve]
  20. Ramachandran M, Gu L-H, Wilson JB, Kitundu MN, Adekile AD, Liu J-C, et al. A new variant, Hb Muscat [{alpha}2ß232(B14)Leu->Val] observed in association with Hb S in an Arabian family. Hemoglobin 1992;16:259-266. [Web of Science][Medline] [Order article via Infotrieve]
  21. Brittenham G, Lozoff B, Harris JW, Nayudu NVS, Gravely M, Wilson JB, et al. Hemoglobin Hofu or {alpha}2ß2 [126 (H4) Val->Glu] found in combination with Hemoglobin S. Hemoglobin 1978;2:541-549. [Web of Science][Medline] [Order article via Infotrieve]
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  23. Gu L-H, Leonova JYe, Huisman THJ. Hb S-Hb Lufkin disease in a Black male infant. Hemoglobin 1995;19:291-294. [Web of Science][Medline] [Order article via Infotrieve]
  24. Duerr M, Gross S, Ramachandran M, Huisman THJ. Case report: Hb CE disease in a 5-year-old without clinical symptoms. Hematol Rev 1991;5:141-143.
  25. Yang KG, Kutlar F, George E, Wilson JB, Kutlar A, Stoming TA, et al. Molecular characterization of ß-globin gene mutations in Malay patients with Hb E-ß-thalassaemia and thalassaemia major. Br J Haematol 1989;72:73-80. [Web of Science][Medline] [Order article via Infotrieve]
  26. Gonzalez-Redondo JM, Kutlar A, Kutlar F, McKie VC, McKie KM, Baysal E, Huisman THJ. Molecular characterization of Hb S(C) ß-thalassemia in American Blacks. Am J Hematol 1991;38:9-14. [Web of Science][Medline] [Order article via Infotrieve]
  27. Gonzalez-Redondo JM, Stoming TA, Kutlar F, Kutlar A, McKie VC, McKie KM, Huisman THJ. Severe Hb S-ß0-thalassaemia with a T->C substitution in the donor splice site of the first intron of the ß-globin gene. Br J Haematol 1988;71:113-117.
  28. Gray GR, Manson HE, Gu L-H, Leonova JYe, Huisman THJ. Hb Lulu Island ({alpha}2ß2107[G9]Gly->Asp)-ß0-thalassemia (codon 15; TGG->TAG), a form of thalassemia intermedia. Am J Hematol 1995;50:26-29. [Web of Science][Medline] [Order article via Infotrieve]
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  31. Malcorra-Azpiazu JJ, Wilson JB, Molchanova TP, Pobedimskaya DD, Huisman THJ. Hb Porto Alegre or {alpha}2ß29(A6)Ser->Cys in unrelated families of the Canary Islands. Hemoglobin 1993;17:457-461. [Web of Science][Medline] [Order article via Infotrieve]
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  38. Huisman THJ, Carver MFH, Baysal E. A syllabus of thalassemia mutations. Atlanta: Sickle Cell Anemia Foundation, 1997:310 pp..



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Right arrow Molecular Diagnostics and Genetics
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