CASE REPORT

Hemoglobin Bethesda Causing Polycythemia in a Japanese Family Issei Kawashima, Koichiro Arima, Tetsuya Hanada, Teruo Harano*, Keiichi Harada, Yasuo Matsuoka and Shoichiro Irimajiri

A family with Hemoglobin Bethesda is reported. A 23-year-old man was hospitalized for the evaluation of polycythemia. Analysis of hemoglobin using high pressure liquid chromatography showed the presence of hemoglobinopathy. Separation of globin into a and B chains revealed approximately 50%of the B chain to be abnormal. Analysis of the DNAsequence of the B chain gene identified Hb Bethesda. The family study disclosed that his father and sister also had the same hemoglobinopathy. This case is the first report of Hb Bethesda in Japan. (Internal Medicine 33: 242-247, 1994) Key words: polycythemia, hemoglobinopathy

Introduction Table 1. Laboratory Values on Admission H e m ato lo g y Hemoglobin Bethesda is a variant of hemoglobin which R B C 6 07 x 1 0 7 mm J presents with polycythemia. Although there are several reports H b 19 .1 g /d l on this type of variant with development of polycythemia (1), H c t 5 7 . 7 % no case of Hb Bethesda in Japan has been. W B C 3 ,8 00 /m m3 N . b an d 4 % Here, we present the first documented case of Hb Bethesda N . se g me nt 5 2 % in Japan, and the results ofamino acid analysis, DNAsequencing M o n o 3 % analysis, and the hemoglobinoxygenequilibrium curve. L y m p h 4 1 % R e tic 1 5 % Case Report P L T s 6.7x 1 04 /mm3 C o ag u la tio n In 1993, a 23-year-old man visited our hospital for evalua- Pr ot hr om bi n t im e 7 2 % tion of general fatigue. He was hospitalized for analysis of A P T T 32 .7 sec (30. 4 sec) abnormal results in liver function tests and erythrocytosis. F ib rin o ge n 1 50 mg /d l Physical findings on admission revealed no abnormality. The results of diagnostic blood tests performed at the time of Ci rc ula tin g blo od vo lum e Re d ce ll vo lu me 3,3 81 m l (l, 61 6ｱ 30 7 m l) admission are presented in Table 1. In evaluation of the in- To ta l bl oo d vol um e 5 ,5 07 m l (3 ,72 5ｱ 59 6 ml ) creased red cell count, the circulating blood volume as meas- To ta l pl as m a vo lu me 2 ,1 26 m l (2 ,10 4ｱ 40 0 ml ) ured by sodium chromates (2), was found to be increased. These findings established the diagnosis of polycythemia. Serum E ryth ro p o ietin 2 1 .7 ml U/ m l ( 9 -4 0 m l U/ m l) 2 , 3 - D P G 4. 18 fim ol/ ml (1.8 -2. 2 L imol /ml RB C) erythropoietin level and arterial blood gas analysis were within V it am in B i 551 L ig/d l ( 210-8 40 ug/d l) normal limits. Neutrophil alkaline phosphatase score, serum Se ru m i on 18 3 ug/ dl (6 0-180 LLg/ dl) vitamin B12, total iron binding capacity and serum iron were also normal. However, an elevated value of4. 1 8 jimol/ml RBC B o ne m ar r ow bi o ps y was measured for 2,3-diphosphoglyceric acid (2,3-DPG) (3). n o rm o c ellu lar This patient had a normal myelogram. His serum was positive HBs and for HBe antigen. Fromthe Department of Internal Medicine, Kawasaki Municipal Hospital, Kawasaki and *the Department of Biochemistry, Kawasaki Medical School, Kurashiki Received for publication September 13, 1993; Accepted for publication February 17, 1994 Reprint requests should be addressed to Dr. Issei Kawashima, the Department of Internal Medicine, Kawasaki Municipal Hospital, 1 2-1 , Shinkawa, Kawasaki- ku,Kanagawa210

242 Internal Medicine Vol. 33, No. 4 (April 1994) A Family with Hemoglobin Bethesda Isolation of the abnormal hemoglobin peptide Methods and Results The B chain was aminoethylated, digested with trypsin, and the obtained peptide fragments were separated by reversed phase on HPLC(SG120 column, 4.6x250 mm) (6) (Fig. 4). The Cation exchange highpressure liquid chromatography (HPLC) peak for the B T-15 peptide was absent, but no abnormal peak was observed. Aminoacid analysis of each peak disclosed that of hemoglobin fraction histidineB T-15 hadhademergedbeenwithsubstitutedB T-6,7,8 forpeptidetyrosine(Tablein 2),the andB T-15that HPLC(Fig. 1) revealed an abnormal variant peak (abn.Hb), (Table 2). This substitution, B145 Tyr -> His, has already been which emerged after HbAand HbA2using cation exchange reported in Hb Bethesda. HPLC(Micropeal BPG- 17, 7.5x150 mmcolumn). Absorbance Analysis of the DNAsequence infi-chain globulin gene To further clarify the results of the amino acid analysis, DNA (4 1 5 nm) was measured to determine the elutes of the individual sequencing was done. The nucleotide sequence in the B-chain hemoglobin bands (4). globulin gene was analyzed using DNAobtained from the patient's peripheral blood (7). Two amplification primer sets were used for the PCRmethod (8). The amplification of DNA was carried out by the dideoxy method for sequencing. B Hemoglobinfractionation by isoelectric electrophoresis Bethesda and B normal clone were obtained. Then the reaction Using a polyacrylamide gel plate (1), isoelectric electro- gel.sampleswere analyzed by electrophoresis on a polyaerylamide phoresis resolved two major components, HbAand abn.Hb Both T and C were observed for the first base of the B 145 (Fig. 2), which were demonstrated as abnormal slow-moving codon, indicating that the patient globulin was a heterozygote bands.

Separation of hemoglobin a-chain andfi-chain by carboxyl- methyl (CM) cellulose column Hemoglobin was biosynthesized with incorporation of tritium-labeled leucine (5), and then subjected to uread CM- cellulose column chromatography. The abnormal B peak was eluted after the peak of the normal B chain (Fig. 3).

'.i g at

*-HbA -HbA

_abn.Hb

|S s g 1A S A-HbA2 ..Mr In ^7 1/- ^IJ\JW 1^ ,^^

cd ro 1^ S ®s ^r 22is S3s « en og c^ Control Patient Fig. 1. Cation exchange HPLCofhemolysate. An abnormal Hb (abn.Hb) was eluted after HbAand HbA2.

243 Internal Medicine Vol. 33, No. 4 (April 1994) Kawashimaet al

HbA HJA2 abirHb-j J j-HbA.c Patient Control

Fig. 2. Isoelectrofocusing of hemolysates on a polyacrylamide gel plate.

Norm /?-chain (2,930 dpm) Norm *-chain t5'060 dPm> Abn /?-chain (2,980 dpm)

-1 «

20 30 40 50

60 70 Fraction No. Fig. 3. Urea CM-cellulose column chromatography of the 3H-leucine-labeled globin. -: absorbance, : radioactivity.

LO

* 7" M 2 ^^* 8 .S H co HI H H C J w r\ II I^ O | 1 II I SWI

I å , T , r- 0 20 40 60 80 (Minutes) Fig. 4. Separation of the tryptic digest of the aminoacetylated B-Bethesda chain by HPLCon a Capcell Pak 5C,8 SGI20 column (4.6 mmI.D. x 250 mm)with a lineargradientofacetonitrile (from 0 to 50%) in 9 mMtrimethylamine-acetic acid buffer (pH 6.0) at a flow rate of 0.7 ml/min for 100 minutes.

244 Internal Medicine Vol. 33, No. 4 (April 1994) A Family with Hemoglobin Bethesda

Table 2. Amino Acid Composition of a Peptides Mixture Containing the The oxygen equilibrium curves of the patient's red cell Abnormal B T-15 suspension did not show the typical sigmoid shape especially at Found Theoretical molecular ratio low O2 saturation (Fig. 6). (nmol) T-6 T-7 T-8 Comparison of the P50 values indicated that the Hb Bethesda red blood cells had an oxygen affinity 2.7 to 2.9 times greater 7.33 1 7.93 1 than the normal red blood cells. The low saturation region of the 6.18 1 oxygen equilibrium curve for this patient's red blood cells 0.00 showed the loss of cooperative intersubunit interaction. 16.85 1 1 19.91 1 Family study The data on this family are presented in Table 3. His father and his younger sister carried the same abnormal hemoglobin and had similar polycythemia. ofHb Bethesda. Furthermore, clones of the B globin genes were separated, and the results assigned either TAT(tyrosine) or Discussion CAT (histidine) for codon 145 (Fig. 5). There have been six previously reported cases of Hb The oxygen equilibrium curve Bethesda [B145(HC2)Tyr -> His], including North European The oxygen equilibrium curves were measured on whole Caucasians (5, 10, 1 1), a French Canadian (12), and a Chinese blood that had been diluted 150 times with isotonic phosphate living in America ( 13). This is the first report ofHb Bethesda in buffer solution (0.15M K2HPO4+0. 15M NaH2PO4), pH of 6.9 Japan. All WoBethesda patients had secondary polycythemia or 7.45, at 37°C. The final concentration of hemoglobin was because of inefficient O2 dissociation from the abnormal adjusted to 60 jiM on hemo basis (9). hemoglobin.

GTAC GTAC 3' ***' 9Bá"""* 3' I **-.- fpT*^ I

^1 , "*"** ' «*>» ******* mtm IL/ G1 -- .. ''à"*,.à" /G A\ ^.....-à"à" -^««- /A TermA\ .:^ S^,^ ATerm T\ -"å ' "---^ -- T

14b \_--,__ à". -_ ;. J * 146 His A -* »-' *"--.««* A His

:|145i !-H§l r- "^^S :**fe8*i^5 ^n rr" ll45tt jJjiS^'SXsassJ*! jl /\ t*t»"»! I #&£*»«& 3w8ssM?& ^^^^- «w«www å ,*.j r\ ;A..v....AV..J;tff;.

3-^"**"-^^?I i»3 /^^&J#XI ^^'W s ^ ^^" 1 vT~^ a -^- y -^- ft"5 144G - r-- ^144 Lys a/ ^' lA Lys Hlsr å à"" "-r AHis I ^ - *-*,, _ ^ I 57 ^r ._^ å * -_ 5; /?- Bethesda /?- N ormal

Fig. 5. DNAsequence ofB-Bethesda gene. B145 codon was changed from TAT(tyrosine) to CAT(histidine).

Internal Medicine Vol. 33, No. 4 (April 1994) 245 Kawashimaet al

1 00 1 .»-»-*-t-fc84

cm Mr/r^ /

0 1.0 10 50 100 PO2 (mmHg)

Fig. 6. Oxygen equilibrium curves oferythrocytes from aheterozygote ofHbBethesda (Beth./A) and a normal control (A/A). The curves were obtained using the automated method ofImai et al (9). - à" -: Beth./A (pH 7.45), - O -: Beth./A (pH 6.9), -x-: A/A (pH 7.45), - å¡-: A/A (pH 6.9).

Table 3. Hematologic Findings of the Family References P ro b a n d F ather S iste r Mori H. Relation between polycythemia and function of hemoglobin with amino acid substitution in al B2 contact, Hb Chesapeake [oc92 (FG4) R B C x l O 4 / m n r 6 07 6 5 3 6 7 5 Arg^Leu] and Hb J Cape Town [a92(FG4)Arg-^Gln]. Kawasaki Med Hb g/d l 1 9.1 1 9.1 14 .1 Jll:41, 1985. Hc t % 53.6 53.6 4 5.6 Read RC. Studies of red-cell volume and turnover using radiochromium. H b F ( 0. 1 -1 . 2% ) 0.32 0 .22 0 .1 5 NEngl JMed 250: 1021, 1954. H b A 2 (2 .2 - 3 .2 % ) 2.86 2 .28 2 .07 A b n o rm a l Hb ( % ) 49.5 4 8.6 48.3 Ericton A, Verdier CHD.Amodified method for the determination of2,3- diphosphoglycerate in erythrocytes. Scand J Clin Lab Invest 29: 85, 1 972. Trivelli LA, Ranny HM, Lai HT. Hemoglobin components in the patient with diabetes mellitus. N Engl J Med 284: 353, 1971. Hayashi A, Stamatoyannopoulos G, Yoshida A, Adamson J. Hemoglobin Rainier B145(HC2) Tyrosine --> Cysteine and Hemoglobin Bethesda: Oxygen equilibrium curve of the carriers is shifted to the left B145(HC2) Tyrosine -> Histidine. Nature New Biol 230: 264, 1971. and the normal sigmoid curve was replaced by a straight line at Sakai T. Determination of erythrocyte porphyrins by reversed phase the low saturation region. high performance liquid chromatography using capsule type silica gels coated with silicone polymer. J Chromatography 433: 73, 1989. An elevated value for 2,3-DPG was observed in our patient Poncz M, Solowiejezyk D, Harpel B, Mory Y, et al. Construction of in contrasttoprevious reports ofanormallevel (10, 12, 13). 2,3- humangene libraries from small amounts of peripheral blood: Analysis DPGfacilitates dissociation of oxygen from hemoglobin which of fi-like globin genes. Hemoglobin 6: 27, 1982. results in improved oxygenation of the tissues (14). Harano T, Harano K, Ueda S. Application of polymerase chain reaction (PCR) to molecular analysis of the B globin gene of Japanese B°- The neutrophil alkaline phosphatase score, serum vitamin Thalassemia-Hb San Diego [B109(Gl l)Val->Met]. Acta Hematol Jpn B 12 and erythropoietin levels were consistent with previously 53: 882, 1990. reported cases ofHb Bethesda by Schmidt (12) and Bunn (13). Imai K, Morimoto H, Jotani M, Watari H, Hitota W, Kuroda M. Studies The only clinical manifestation in Hb Bethesda is persistent on the function of abnormal hemoglobin. I. An improved method for cutaneous flush due to the polycythemia. Cardiac and cere- automatic measurementof oxygenequilibrium curve of hemoglobin. Biochim Biophys Acta 200: 189, 1970. brovascular complications have not been reported, and fetal Adamson JW, Hayashi A, Stamatoyannopoulos G, Burger WF. Erythro- oxygenation seemed undisturbed in a mother with Hb cyte function and marrow regulation in Hemoglobin Bethesda (B145 Bethesda (15). In this case, only the proband was found to Histidine). J Clin Invest 51: 2883, 1972. have cutaneous flush. Olsen JS , Gibson QH. The functional properties of Hemoglobin Bethesda. J Biol Chem 247: 3662, 1972. Acknowledgments:Authors appreciate Dr. Kiyohiro Imai, Department of Schmidt RM, Jue DL, Ali MM, Lyonnais J, Moo-Penn WF. Hemoglobin Physiology, Osaka University, School of Medicine, for his technical assistance Bethesda, B145(HC2)Tyr -> His, in a Canadian family. A J C P 66: 449, of oxygen saturation curve. 1976.

246 Internal Medicine Vol. 33, No. 4 (April 1994) A Family with Hemoglobin Bethesda

13) Bunn HF, Bradley TB, Davis WE, et al. Structural and functional studies the oxygen equilibrium of humanerythrocytes. Arch Biochem Biophys on Hemoglobin Bethesda (oc2B2145Hls), a variant associated with compen- 121: 96, 1967. satory erythrocytosis. J Clin Invest 51: 2299, 1972. 15) Charache S, Catalano P, Burns S, et al. Pregnancy in carriers of high 14) Chanutin A, Curnish RR. Effect of organic and inorganic phosphates on affinity hemoglobins. Blood 65: 713, 1985.

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