Hemoglobinopathy

* This article describes the laboratory investigation of an unusual hemoglobinopathy involving hemoglobin (Hb) S, HbSG^sub Philadelphia^, and alpha-thalassemia-2 in a patient whose phenotype was HbSC by alkaline electrophoresis. Findings of a mean corpuscular volume of 62 fL and microcytes on the blood smear were inconsistent with HbSC disease. The patient's clinical course over several years had been mildly symptomatic. Testing in our hospital laboratory using isoelectric focusing and cation-exchange high-performance liquid chromatography to separate hemoglobins showed an unknown variant. Additional studies, including globin chain electrophoresis, reverse-phase high-performance liquid chromatography, and polymerase chain reaction-based DNA analysis were performed at reference laboratories, which reported the following findings: HbG^sub Philadelphia^ associated with alpha-thalassemia-2, HbS and HbG^sub Philadelphia^, and the alpha-globin deletions defining the -alpha^sup 3.7^/-alpha^sup 3.7^ genotype. The hemoglobin molecular defects, alpha-thalassemia-2, and the pattern of inheritance are discussed. (Arch Pathol Lab Med. 1999;123:963-966)

The discovery of an abnormal hemoglobin (Hb) in sickle cell disease and description of the electrophoretic property of the sickle cell Hb (HbS) by Pauling in 1949 were benchmarks in the study of human hemoglobins. Since then, approximately 700 structural Hb variants, most of which are benign, have been described.' These variants represent globin chains with 1 or 2 amino acid substitutions, deletions, insertions, cross-over products, or fusion products. The mutations may occur on the alpha chain, the beta chain, or on both. When both the alpha and beta chains are affected, double heterozygote Hbs may form. These variants, sometimes referred to as hybrid Hbs, produce their own unique electrophoretic pattern, which is different from that of the individual variant produced by the alpha- or beta-chain mutation. Hemoglobin S is the most common abnormal beta-chain variant in the United States. The abnormal beta-chain gene is expressed with normal adult HbA as sickle cell trait (HbAS) in about 8% of the African American population; fewer than 1% have sickle cell disease (HbSS).2 The second most common beta-chain variant, HbC, is found in about 2% of the African American population. Other beta-chain variants, such as HbD^sub Los Angeles^, HbE, HbO^sub Arab^, and the alpha-chain variant, HbG^sub Philadelphia^, are encountered in less than 1% of the African American population.2 These variants are frequently found in the trait condition with HbA and, less commonly, in the double heterozygous condition with HbS. A recent study of the distribution of hemoglobin variants in the multiethnic population of California reported a low prevalence (

Methods available for the detection of abnormal hemoglobins include the solubility test, zone electrophoresis at alkaline and acid pH, isoelectric focusing electrophoresis (IEF), globin chain electrophoresis at alkaline and acid pH, and cation-exchange high-performance liquid chromatography (HPLC). The principles and applications of these and other laboratory techniques have been published elsewhere.1,2,4 These techniques have different advantages and limitations for the separation of hemoglobins. Zone electrophoresis is widely used because it is simple and rapid, but a major disadvantage of this technique is the incomplete separation of different variants that have similar isoelectric points. For example, Hbs S, D, and G^sub Philadelphia^ migrate in the same zone at alkaline pH. Similarly, the hybrid HbSG^sub Philadelphia^ migrates into the same zone on alkaline electrophoresis as hemoglobins C, O, E, and Az. We describe a case that illustrates this problem and discuss how various laboratory methods were used to reveal the patients unusual hemoglobinopathy. The genetic and clinical aspects of the case are discussed.

REPORT OF A CASE

A 31-year-old African American man who had been monitored in the sickle cell clinic for many years underwent hip surgery because of avascular necrosis secondary to sickle cell disease. His past medical history included infrequent, uncomplicated, painful episodes that required about 3 hospitalizations per year. He had required few blood transfusions throughout his life and did not manifest splenomegaly, ulcers, or other serious symptoms frequently seen in patients with sickle cell disease and related sickling syndromes. His hemoglobin concentration remained stable at approximately 90 g/L, and the phenotype HbSC had been observed repeatedly over many years on cellulose acetate electrophoresis at alkaline pH. Laboratory findings in a peripheral blood sample following a routine clinic visit in 1995 included the following data (reference ranges in parentheses): hemoglobin concentration, 91 g/L (140-180 g/L); hematocrit, 0.289 (0.420.52); mean corpuscular volume (MCV), 61.8 fL (80-94 fL); and reticulocyte count, 0.043 (0.005-0.015). A peripheral blood smear was examined for cell morphology. At this time, the patient's blood sample was examined for hemoglobin variants by HPLC and IEF and, in addition, was sent to a reference laboratory for globin chain electrophoresis. Subsequently in 1998, a blood sample collected during a clinic visit was referred for DNA analysis. The patient's physician and his medical record indicated that family studies had not been done.

METHODS AND MATERIALS

Hematology

The routine hematologic studies were performed with a Coulter STKS electronic cell counter (Coulter Corporation, Miami, Fla). The reticulocyte count was determined by flow cytometry using the Coulter EPICS-SL-MCL System II. Cell morphology was examined on a Wright-stained peripheral blood smear.

Biochemical Analyses

Hemoglobin electrophoresis in our laboratory at this time was performed only on agarose gels at pH 6.0 using the Beckman Paragon Electrophoresis System (Beckman Instruments, Diagnostics Group, Brea, Calif); the hemoglobin electrophoresis method on cellulose acetate strips at pH 8.4 had been discontinued the previous year. Isoelectric focusing electrophoresis was performed on Resolve-Hb agarose gels at pH 6-8 (Isolab, Inc, Akron, Ohio). The electrophoresis procedures were performed according to the kit instructions provided by the respective manufacturers. Hemoglobins A, S, C, F and A^sub 2^ were separated by cation-exchange HPLC on the Bio-Rad Variant Automated Hemoglobin Testing System using an adaptation of the Bio-Rad Beta-Thalassemia Short Program (Bio-Rad Laboratories, Diagnostics Group, Hercules, Calif), as previously reported.4

Further hemoglobin studies by IEF alkaline electrophoresis, acid electrophoresis, and globin chain electrophoresis were referred to the Mayo Medical Laboratories (Rochester, Minn). Additional analyses in 1998 were performed at the Hemoglobin Laboratory, Sickle Cell Center, Department of Medicine at the Medical College of Georgia (Augusta, Ga) using IEF, cation-exchange HPLC, reverse-phase HPLC, and DNA analysis based on automated sequencing of polymerase chain reaction-amplified betaand alpha-globin genes.

RESULTS

The peripheral blood smear revealed sickle cells, target cells, microcytes, and polychromatophilia (Figure 1). The low MCV (61.8 fL) and microcytes on the blood smear were inconsistent with HbSC, the phenotype assigned previously by alkaline electrophoresis (Figure 2). The findings in our laboratory by acid electrophoresis and IEF (Figure 2, middle and bottom, respectively) and by HPLC (Figure 3) further disputed the presence of HbC. The unusual features of the HPLC analysis of the patient's sample (Table and Figure 3) include the absence of the normal HbA peak and presence of 2 major peaks representing abnormal hemoglobins. Thus, the results obtained by 3 different separation methods in our laboratory at this time confirmed the presence of HbS and demonstrated that the unknown variant was not HbC. The variants were presumptively identified as HbS and Hb "unknown."

The alkaline electrophoresis results reported by the Mayo Medical Laboratories were consistent with our results by cation-exchange HPLC (Table). Globin chain electrophoresis revealed 2 hemoglobin variants identified as HbS and HbSG^sub Philadelphia^. Based on the proportion of HbSG^sub Philadelphia^ detected (0.45), the reference laboratory also diagnosed the patient as having homozygous alpha-thalassemia-2.

The electrophoretic analyses by the Hemoglobin Laboratory at the Medical College of Georgia showed unique patterns specific for HbS and HbG^sub Philadelphia^, which were confirmed by cation-exchange and reverse-phase HPLC (Abdullah Kutlar, MD, oral communication, January 1999). Polymerase chain reaction-based DNA analysis identified the mutant beta- and alpha-chain genes and revealed the homozygous -alpha^sup 3.7^ gene deletion. The diagnosis given was HbSS with HbG-alpha^sub Philadelphia^

COMMENT

Hemoglobin G^sub Philadelphia^ is the only alpha-chain variant commonly encountered in the United States. Because it is an alpha-chain variant, it is able to form heterozygous hemoglobins with normal adult A hemoglobin (AG^sub Philadelphia^) as well as with the beta-chain variants, such as S (SG^sub Philadelphia^) and C (CG^sub Philadelphia^). The hemoglobinopathy involving homozygous S and the hybrid SG^sub Philadelphia^ has been described only rarely.2,5,6 On zone electrophoresis at alkaline pH, HbSG^sub Philadelphia^ migrates as a band at a cathodal position where Hbs C, E, O, and others are detected.2 Thus, each time this patient's sample was examined by the alkaline electrophoresis method previously used at our laboratory, the cathodal band was misidentified as HbC. The error was suspected only after the IEF and cation-exchange HPLC studies were performed.

The association between G^sub Philadelphia^ and alpha-thalassemia was suggested in 1971 by French and Lehmann.7 Since that time, various molecular studies have been performed to establish that alpha-thalassemia-2 is linked in cis with HbG.8 Other investigators have shown that the quantity of HbG^sub Philadelphia^ depends on the number of alpha-globin genes deleted or affected by a mutation, and the expression has been recognized in 3 different proportions. Individuals with approximately 0.25 HbG^sub Philadelphia^ have 4 globin genes (alpha^sup G^alpha/ alphaalpha) due to a base mutation in 1 of the alpha-globin genes; those with approximately 0.35 HbG^sub Philadelphia^ have 3 alpha-globin chains (alpha^sup G^- / alphaalpha) with a 3.7kilobase deletion that affects both alpha-globin genes; and those with approximately 0.45 HbG^sub Philadelphia^ have 2 globin chains (alpha^ sup G^- /alpha -).9 The last genotype occurs because the alpha-thalassemia-2 haplotype (alpha- /) is present in approximately 27% of the African American population.10

The genetic interaction of HbG^sub Philadelphia^ (alpha^sup G^), HbS (Beta^sup s^), and alpha-thalassemia-2 (heterozygous, alpha-/alphaalpha or homozygous, alpha- / alpha-) can be explained by the pattern of inheritance. One beta chain and 2 alpha chains are inherited from each parent, producing 4 hemoglobins. The genes for HbS trait and HbG^sub Philadelphia^ with 1 alpha gene deletion are represented by the following: alpha^sup G^- / Beta^sup A^, alpha^sup G^- / Beta^sup s^, alpha alpha / Beta^sup A^, and alphaalpha / Beta^sup s^. These combinations produce normal HbA (alphaalpha / Beta^sup s^) and 3 abnormal hemoglobins, namely, G^sub Philadelphia^ (alpha^sup G^ / Beta^sup A^), S (alphaalpha/ Beta^sup s^), and SG^sub Philadelphia^ (alpha^sup G^- / Beta^sup s^). On alkaline electrophoresis, SG^sub Philadelphia^ represents 0.15 of the hemoglobins, about half of the expected amount.2,5 This is because the remaining HbG (0.15 to 0.2) migrates with S and cannot be determined using this method alone.

The genes for HbSS and HbG^sub Philadelphia^ with 2 alpha gene deletions, as in the case presented, are represented by the following: alpha^sup G^- / Beta^sup s^; alpha^sup G^- / Beta^sup s^; alpha- / Beta^sup s^; alpha- / Beta^sup s^. The possible combinations produce 2 hemoglobins: S (alpha- / Beta^sup s^) and SG^sub Philadelphia^ (alpha^sup G^- / Beta^sup s^ ). As expected, SG^sub Philadelphia^ comprises approximately 0.45 of the total hemoglobin separated by alkaline electrophoresis. Although the patient's parents were not studied for hemoglobin variants, it may be assumed that each parent carried at least 1 of these genes (alpha- / Beta^sup s^ or alpha^sup G^- / Beta^sup s^) and passed them on to the propositus.

A potential benefit of the presence of homozygous alphathalassemia-2 is that it may lessen the severity of sickle cell disease. Patients tend to have higher hemoglobin levels and a milder course with fewer complications. There is indirect evidence that a-thalassemia inhibits in vivo sickling, thereby reducing manifestations such as acute chest syndrome and leg ulcers. However, other complications, such as avascular necrosis of the femoral head, still occur, presumably because of the higher hemoglobin present in these patients.11

In summary, the patient in this case was found to have homozygous HbS, the hybrid HbSG^sub Philadelphia^, and alpha-thalassemia-2. Results of a commonly used alkaline electrophoresis method were misleading; more advanced analyses were necessary to reveal the identity of the hemoglobinopathy. The investigation of this unusual hemoglobinopathy helped to explain some of the features of the patient's clinical course. Accurate identification of Hb variants is important not only to understand the clinical course, but also, in some cases, for proper clinical management, especially in those cases involving HbS. The determination of rare variants may also assist in family studies or genetic counseling.

The authors thank our colleague and the laboratory personnel at the Medical University of South Carolina for providing the following assistance: Chris Brunson, MD, Division of Hematology and Oncology, provided the patient history and Jiaann King, MT(ASCP), MS, and Cheryl Thomas, MT, of the Special Chemistry Laboratory and Richard Sillivant, MT(ASCP)SH, of the Hematology Laboratory, Department of Pathology and Laboratory Medicine, provided technical assistance.

References

1. Fairbanks VF, Klee GG. Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry 3rd ed. Philadelphia, Pa: WB Saunders: 1998:1642-1710.

2. Fairbanks VF. Hemoglobinopathies and Thalassemias: Laboratory Methods and Clinical Cases. New York, NY: Brian C Decker; 1980:8-32.

3. Lorey FW, Arnopp J, Cunningham GC. Distribution of hemoglobinopathy variants by ethnicity in a multiethnic state. Genet Epidemiol.1996;13:501-512.

4. Papadea C, Cate IC. Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin Chem. 1996;42:57-63.

5. Pugh RP Monical TV, Minnich V. Sickle cell anemia with two adult hemoglobins: hemoglobin S and hemoglobin G-Philadelphia/S. Blood. 1964;23: 206-215.

6. Charache S, Zinkham WH, Dickerman JD, et al. Hemoglobin SC, SS/GPhiladelphia and SO-Arab diseases: diagnostic importance of an integrative analysis of clinical, hematologic and electrophoretic findings. Am J Med. 1977;62: 439-446.

7. French EA, Lehmann H. Is haemoglobin Ga-Philadelphia linked to a-thalassemia? Acta Haematol.1971;46:149-155.

8. Felice AE, Ozdonmez R, Headlee ME, et al. Organization of a-chain genes among HbG-Philadelphia heterozygotes in association with HbS, Beta-thalassemia, and a-thalassemia-2. Biochem Genet. 1982;20:689-701.

9. Molchanova TP, Pobedimskaya DD, Ye Z, et al. Two different mutations in codon 68 are observed in HbG-Philadelphia heterozygotes. Am Hematol. 1994; 45:345-346.

10. Dozy AM, Kan YW, Embury SH, et al. Alpha-globin gene organisation in blacks precludes the severe form of a-thalassemia. Nature. 1979;280:605-607.

11. Higgs DR, Aldridge BE, Lamb I. et al. The interaction of alpha-thalassemia and homozygous sickle-cell disease. N Engl J Med. 1982;306:1441-1446.

Accepted for publication March 26, 1999. From the Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC.

Reprints: Christine N. Papadea, PhD, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Ave, Suite 309, PO Box 250908, Charleston, SC 29425.

Copyright College of American Pathologists Oct 1999
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