Sickle Cell Syndromes. I. Hemoglobin SC-A-Thalassemia
HEMOGLOBIN SC-a-THALASSEMIA 613
3. Belle, A.. and Ktm. Y S: Rat \mall ~nte\llnallilucln Is~ilat~onand ch;trilcterl~.~- 5x5 (1967) tlon of ;I water-soluble mucln fract~on.Arch. Blochem. H~oph!s. 150- 079 14. Jakow\ka. S Cystic F~hrostaand Rclitted Human and Animal I)~\ea\c\, p I62 (1972). Gordon and Breach Scientific Publ~sh~ngCo.. New York) 4. Bettelheim. F. A,: [Light-sc:tttcr~ng studlea of hov~ne suhm;~rlllar! mucln. IS. Joe\. R. W.. and Carr. C W. The b~ndlngof ci~lctum in mlrture\ of Biochim. Biophys eta, 236- 702 (1971) pho\phol~pids.Proc. Soc 'xp Blol. Med.. 1.74 1268 ( 1967). 5. Boat.T. F., W~esrnan.U. N.. and Pilllaviclni. .I C.: I'urtf~cat~oni~nd propcrtiea of 16. Negua. V.. Mucus Proc KO!. Soc. Med.. 60. 75 (1967). the c:tlc~um-prec~p~tnbleprotcln In \uhmar~llar! \:t11\:1 of normal .tnd c!\t~c 17 P~gman. W.. and tlerp. ,A: Aspects blochlmlquea et ph!\~olog~qucs des fihros~ssubjects. I'cdiat. Re\. 8. 531 (1974). gl)coproteldc\ des ?ecretlon\ muqucuaea. !Ann. Anat. Pitthol . 17 227 (1972). 6 Buddccke. I:.: In: A. Ciottachalk: M~scellnneoo\gl)coprotetns In gl)coprote~ns. IX. Roherts. (i. P. Isolation and ch:~r;~cter~/ittton~~fglycopr~lle~ns from sputu~n. bur. thetr cornporit~on.structure and function. p 558 (l:lse\~c.r Puhl~sh~ngC'O., J. Blochem . 30 265 ( 1974). Amsterdam. 1966) 14 Kou\\el. P.. I amhl~n.(; , and Degand. P.: lleterogene~t)of the c;~rhoh)drntr 7. di Sant'Agnesc. P. A,. and 'T:il;~mo, K. C : P:~thogcne\~\and ph!stop;~tholog) of chain\ of sull';tted hronch~algl!coproteins ~solatedfrom ;I patlent \ul'fer~ng cyat~cfihrow of the p;tncreas h' I-ngl J. Med.. 277 12x7 ( 1967) from c!\tic fthroz~\..I. Biol Chcm.. 2.51) 21 I4 (1975). X Foratncr. I. 1,- .. and kor\tncr. (i. (i.: C'i~lciu~iihlnd~ng to ~nte\ttn;tl ohlet ccll 20. Ta!lor. hl. The ongin\ and function\ of naul mucu\ I.ar!ngorcopc. 34 612 mucln Rlochtm. Hloph!\. act:^. 386 2x3 (1975) (1974). 9. Fcir\tner. .I F . Jahhitl. I.. itnd t.or\lner. Ci. (i: <;ohlet ccll mucln of r,lt smitll 21. \h arner. R R.. and Coleman. J. K . tlectron prohe anal!\^\ olcalc~urntran\port Inte\tlnc. Chcm~c;~litnd ph!s~c;~lch:~r:tcterl/;~tlon Can J. H~,ichcm..51. 1154 by \mCill tntc\tlnc. J Cell Blol., 64 54 (1975). (1973). 22 W:~rton. K. L.. and Blo~nficld.J H!droxydp:ttttc In the p:ithogcnez~\ tllc!\tlc 10. For\tner. .I. k . and Milncr!. 1. 1. Ci~lc~ulnb~nd~ng h) human er!throc!tc fthro\~a.Br~t. Med. .I . J: 570 ( I97 l ) rncrnhranca. Hlochcm J.. 124 563 ( 1971). 23. The i~uthora;ire ~ndehtedto the Medlc;tl Rc\carch Counc~lof C.tnad.~ and the I I Forstncr. I. k.. Tn~chtnnn.N.. haln~n\,V.. and For\tncr. (i. (i. Inre\tln;tl gohlet C;ln:ldl;ln C)sr~ct~hroslh l.oundat~on f11r fin:tncl;~l aupport. to Dr Ditv~dKelh cell mucu\ l\olatl<~nand ~dcnt~flcntlonh) ~~nmun~~lluore~cenccof it gohlut ccll of the 0ep;lrtmcnt of B~ochcm~str!.Iln~vcr\~t! of Torontl~.I)r perfornm~ng gl)coprolc~n..I. Cell Scl . 12 585 (1973) anitlyt~cultr:~centr~lugat~on \rudlc\. and 10 Mr\. Cec~l~akcng lor technical I?. (iihson. L. t.. Mi~tthe~s.W. .I . Mlnlh;ln. I' T . and I'att~, J. A - Kclat~ng 3b\l\l:lIlCt. muc~us,cnlc~um and sue;lt In a ncu concept of c)\tlc f~hro\~\Pcdiatrlcs. 48 14 Kequc\t\ for reprint\ should he addre\\cd t ~ediat.Res. 10: 61 3 620 (1976) Erythrocytes sickle cell anemia hemoglobin a-thalassemia Sickle Cell Syndromes. I. Hemoglobin SC-a-Thalassemia (iEOK 111r\~c,r\rn.01 Illrr~or.\.Sr(,hIc, cc.11 (c31rrc~rcrrrcl L)c~prrrrr~rc~r~rof lJrdrrr/rrc,\. IIrc, Ahraha~~r1.irrc~olrr Sc,hool oJ Medrcitrr. L'rri\,er.rin. of 1lli1roi.r M~dicuICetrrer, C'hic,ago. Illirrois. L'S.4 Extract months typical findings of mild hemoglobin H disease appeared. with HbH making up 6.5% of the total hemoglobin. Hematologic and globin synthesis $tudie$ were performed in a hlack American family in which the genes for x-thalassemia and Speculation hemoglobins t Hb) S and C' were segregating. 'l'he following distribution of these abnormalities was found: father. sickle cell Ihe prewnce of tu-thalassemia in the proband of thi\ rtudy trait + n-thalassemia; mother, HbC trait + a-thalaswmia, proposi- appeared to modif, her HbSC direare ro as to reduce it$ clinical tus, HbSC + a-thalassemia; older sibling, n-thalasscmia trait; seterit!, ar nell ar its pathologic potential. The amelioratite effect of and younger sibling, hemoglobin H disease. tu-thalarremia would appear to he related to a reduction in the 'The child with HbSC'-e-thalasremia demonstrated more serere intracellular hemoglobin concentration in the patient'$ erythrocvter. anemia and a more hemolytic picture than is typical of HhS<' hut other factors may alro be responriblc for there change$. Further disease. Her erythrocytes exhibited decreased osmotic fragility in study of genetically modified sickle hemoglobinopathy ryndromer comparison with HbSC' er! throcytes, but had an indistinguishable may ultimatcl! aid in the detelopment of effectite mcanr for the oxygen equilibrium curre and 2.3-diphosphoglycerate (2,3-I)P(;) treatment of there di$orderr. letel. Erythrocyte sickling in the patient, honeter, was significantly reduced, with less than 35% sickle forms observed at nearly com- plete oxygen desaturation. The clinical expression of sickling disorders ma! var! widel?. The sibling with hemoglobin H disease exhibited 26'2 Bart's even aniong individuals having an apparently identical form of (y,) hemoglobin at birth, a level comparable with that seen in in- sickle cell disease. This variability has stimulated investigation of fants with HbH disease in Far Eastern populations. At age 5 the role of genetic as well as nongenetic factors in the hematologic 614 HONIG ETAL and clinical expression of the sickle hemoglobinopathies. In a samples also were drawn into syringes containing 5% formalin and number of atypical sickling disorders specific modifying factors 0.01 M Na,HPO, in isotonic saline. The formalin-fixed erythro- have been identified that seem to alter the ability of the er) throcyte cytes were examined in a hemocytometer counting chamber and to undergo sickle transformation. Other genetic traits appear to classified according to the criteria of Rampling and Sirs (31). For modify the clinical expression of sickling disorders by different. each sample. 1,000 cells were counted. but thus far unidentified mechanisms. All clinical studies were carried out with informed consent Abnormalities that have a genetic basis and are known to according to the provisions of the Declaration of Helsinki. modify sickle cell diseases inciude: associated hemoglobin (?-chain (19, 40) and P-chain (4. 23. 24) structural abnormalities, thalas- semias and thalassemia-like disorders (1, 28. 35. 46): variability in R ESU L.TS the production of fetal hemoglobin (2. 17): and the presence of other associated hematologic disorders (22, 38). CASE REPORTS In this report we describe the clinical and hematologic findings of a child having an apparently modified form of hemoglobin SC The patient, RC', a 7-year-old girl, was referred for hematologic disease related to the association of tr-thalassemia. evaluation in preparation for an adenoidectomy. She had had multiple episodes of otitis media. and had developed severe METHODS nasopharyngeal obstruction with persistant mouth-breathing. She was noted to he anemic. and was found from a hemoglobin HEMATOL.OGIC AND Ht.MOGLOBIN STUDlhS electrophoresis stud! to have hemoglohins S and C. Apart from Hematologic measurements were made with a Coulter model S the problems attributable to her tonsillar hypertrophy, the child electronic cell counter which was standardized daily using a had generallj, been health!.. She hird never been noted to have commercial standard. Other determinations were performed by jaundice, fatigue, pain crisis, or other clinical manifestation of standard methods (5). Osmotic fragility tests were carried out with sickle cell disease. She appeared somewhat pale and her spleen was fresh samples of defibrinated blood as described bk Dncie (8). enlarged extending 2 cm below the costal margin. Blood samples for hemoglobin analysis were collected in EDTA. After completion of the hematologic evaluation the child was The erythrocytes were isolatetl by centrifugation. washed three given a transfusion of packed red cells sufficient to increase her times in isotonic saline, and lysed with 2-3 volumes of water blood hemoglobin concentration to I I .O g/dl. An adenoidectomy without the addition of an organic solvent. Stroma-free lysates under general aneqthesia was performed without con~plication.and were prepared and subjected to electrophoresis on cellulose-acetate her recovery and subsequent course were uneventful. strips in Tris-EDTA-borate buffer at pH 8.6. For quantitative The parents of the child and her 9-year-old sister had been in estimation of individual hemoglobins. the bands were excised after good health, and had not been known to have anemit1 or other electrophoresis and the hemoglobins eluted and me~~suredas hematologic abnornial~t~.During the time that the evaluation of previously described (15). Each indicated value represents the the patient mas in progress. her mother gave birth to a I'ull term mean of determinations done at least in triplicate. Alk:~li-resistant infant after an uneventful pregnancy. The infant did well during hemoglobin was estimated according to the procedure of Betke rr the neonatal period and showed no evidence of anemia orjaundice. al. (3). DEAE-Sephadex column chromatography of carbon Her subsequent course was without apparent complication. Hema- monoxide-saturated hemoglohins was performed as described by tologic studies of the infant were performed at birth and subse- Dozy and coworkers (9). quently at the age of 5 months. A faniil) pedigree. indicating the diagnosis ultirnatel) estab- lished for each of the f:~mily memberh. is shown in Figure I. Blood samples obtained for the globin synthesis determinations HtM.AIOI.O~iIC'AND ti1 MO(;I OBIW l'IN[)IN(;S were collected in hepar~nand kept at ice tcmpcrature before the incubations. Washed cells. 1.0-ml packed cell volume. were The results of hematologic studies of the patient and her family incubated for 2 4 hr in niediuni containing 10 clC'i of L-['"]leu- are presented In Tahle 1. The patient (11-2)was moderi~tclyanemic cine (specific activit! 250 mCi/mmol). Thc incuhat~onprocedure with a persistently elevated reticulocyte count. Her mean corpus- was as previously described (16). Globin chromatography on cular volume (MCV) and hemoglobin (MCH) indice> were carboxymethyl cellulose columns was performed b) thc method of markedly reduced in splte of a normill serum iron level and total Clegg and coworkers (7). Equal itliquots of the chromaiugral)h~c iron-binding capacity. Her blood sme:tr (Fig. 2C') demon5trated effluent fractions were taken li~rdeterminations of incorporated many of the features typical of hen~oglobinSC disease erythro- radioactivit) (16): tu/O + 7 radioactivity ratios were calculated cytes, including the presence of target cells and "safety-pin" using the total incorporated radioactivlt! recovered from the forms. The cclls appeared more flattened, however, and thin. chromatographic fractions corresponding to each of the glohin symmetrical-appe~~ringtarget f;)rnia made up a majority of chains. OXYGtN AFt INIIY AND SICKLlN(; S1lll)ltS Oxygen equilibrium curves were determined with a model 217 blood gas analyzer from Instrumentation Laboratories Inc., Lexington, Mass. Blood samples were equilibrated in the tonome- ter with varying mixtures of 5.60% CO, in air and 5.60'; CO, In nitrogen. After equilibration with each gas mixture. aliquots of the blood were withdrawn for measurement of the PO,, pH, and percentage of oxygen saturation. A normal alkaline Bohr factor was applied whenever neces5ary fbr correction of pO, values to correspond to pH 7.40. Determinations of 2,3-DPG were performed using protcin-free trichloroacetic acid filtrates prepared from whole blood samples. A spectrophoiometric assay procedure was employed as described by Keitt (18). Fig. I. Pedigree dlagram of the l'arn11j Indicating hemoglobin hndings. For enumeration of the percentages of sickled erythrocytes in The proband is represented as 11-2. a-7'hal.: a-thalasseniia: Hh: hemoglo- relation to oxygen saturation, aliquots of equilibrated blood bin. Tahle I. Hrn~arologic~'nlut.\ ' Total iron Packed Serum binding Pedigree Hemoglobin, cell KHC/pI. iron. eapacit). no. Aec. )r g/dl volume, '4 x MCV, I1 MCH, pg MCHC, 'i: Reticulocytes, '7 pg/dl pg/dl I- I 28 13.3 30.2 5.67 7 1 23.8 33.6 1.6 65 297 1-2 28 9.1 28.6 5.01 57 18.1 31.6 4.0 109 358 11-1 9 11.0 31.1 4.27 73 25.8 35.2 0.3 59 398 11-2 7 7.9 25.8 4.9 1 53 16.2 30.6 7.2 98 302 11-3 Newborn 16.9 57.1 6.91 83 24.3 29.5 11.2 5 months 8.5 26.5 5.05 53 16.6 3 1.5 4.6 ------ I MCV: mean corpuscular volume: MCtI: mean corpu5cular hen~oglob~n:MCtIC. mean cc)rpuscular hemoglobin conccntrcltion the erythrocytes seen in stained smears of her blood. Contracted. with brilliant cres!l blue for 1 hr and ;I careful search made for densely staining cells that resembled spherocytes were also regu- erythrocyte inclusion bodies, none were found. A similar study larly seen, but sickle forms were never observed. performed when the infant was 5 months old demonstrated large Hemoglobin electrophoresis of stroma-free hernol!sates from numbers of tjpical hemoglobin H erythrocyte inclusions. the patient demonstrated approximatelg equal quantities of hemo- globins S and C (Table 2). There nas no significant elevation ofthe level of her alkali-resistant hemoglobin. Both parents of the patlent and her older sister (11-1) also \+ere To obtain ;i quantitatiix assessment of the balance bet~beenthe mild11 anemic with niicrocytic erythroclte indices in the absence of synthesi\ of n and non-a-globin proterns. globin synthesis studies demonstrable iron deficiency. Blood smear5 from the fcrthcr (Fig. were performed with peripher~ll hlood red cells from each of the 2A) and the sister (11-1) demonstrated microcytosis but generally famil! members. unremarkable erythrocyte rnorpholopy otherwise. The hlood Cilobin synthesis by blood reticulocytes from the mother (I-.?) srnear of the mother shoned an~soc!tosis. poikilocytosis. target uas signlficantlq deficient in fi chain s)nthesis as compared uith and "pencil" form\, and "safet~-pin" cells (Fig. 1B). the synthesis of 8 chains (Fig. 4). The globin chromatogram froni Electrophoresis of hemoglobin I'rom the firther demonstrated :in this study also demonstrated a relativel! srnall quantit! of 8' AS 1~1ttcrnaith HbS representing 26.05 of the total. Hls Hh.?\ globin in relation to that of$\ recovered from the chromatography and alkali-rwistant hemoglobin fractions were norrnal (Table 1). column. This finding substantiates the relatively small percentage The rnother's hernoglobin electrophoresis stud! denionstratcd of HhC (14'i) that was found in the rnother's blood. its compared hemoglobins :I and C. Hemoglobins I\, + C made up 24'; of her with the range of 35 456 HbC that is usuall) seen in hemolysates total hemogloh~n:her alkali-re.;istant fraction was also uithin the ol' hemoglobin C-tralt individuals. A ver! similar relationship nas normal ranee. Thc older sister's hcnioglobin clectrophores~s seen in the globin s~nthesisstud! of the father with regard to the pattern was normal, as were the levels of HbA, and alkali-resistant synthesis of the tu and non-tu-glohins. as \+ell as the quantitative hemoglobin. representation of the d chains of hemoglobin5 A and S. Frejhl! draibn hlood samples I'rom the patient, her parents. and 'Phe glohin syntliesis studq performed wrth blood from the her older sister were incubated uith brilliant cresbl blue, and :i patient (11-2) (Fig. 5) also demonstrated a relative deficiency of careful search nas made for er~throc!tc inclu\ion bodies. but none L-['4C]leucine incorporation into the ru-globin fraction as corn- were idcntificd. pared with the incorporation into @' t $". The glohin synthesis The p:~tienr'solln2er \rster (11-3). who ua5 horn dur~ngthe stud! of the infant (11-3) denionstrated :I reduced synthcsi~of (! course of the familk btudq. was not anemic at birth (Table I), but chains as compared with that of the @.I+ y chains. both when she her MC'V and MC'H indices were s~gnil'icantl! reduced (normal was studied at birth and at 5 months ol age. values at birth. MCV 104 118 fl;MCH 33.5 41.4 pg). When she The (I/@ r -, synthesis ratios froni each of the famil) members was restudied at agc 5 nionths shc was I'ound to have anemia with arc shown in Tahle 1. In every casc the ratio \+as s~gnil'icantl! less an elevated reticuloc! te count and microc!to\is. Her stained hlovd than those of nonthalc15srrnic control subjects ( I .O4 -. 0.05). I-:ram smear (I-ig. 11)) demonstrated hqpochromia, an~socytosis,poiki- these findings. in the absence of dernonstr:ible Iron deficiency. ue locyto5is. and many target cells. conclude that e:~chmember ofthe famil! h~idn-thalasscriii:~. The\e -.-i',.--- ..J .,+ h.-". ,,-A ,. .I. .- .-..L --.. :.1 : [eiiiopliihin jtii.iis of the inf..r+'1 ll ..\>CIC [lCllUllllCU 'll ,111111 I ' \'b*(- - 1 3~= WJF-/l%2 0 --.. r 3b 0+ c.. -,'I-6 c) YO c2 da +- ='c:>7'~C <'Q-l t/ 1 *q"7J /&Jtf >C\< -. f. ----, 7 b0C) ('Jf-k -$-J /- p. 7'Coo r=-,d u#,.--+ Fig. 2. Photom~crographsoi peripheral biood smears of selecred famiiy rr~c~r~i~c~~.A. ;-1 iPt:<-ct-ih~lci~~i-miri): 19: I-2 iAC-tc-:ha!ii~:icmia); C: 11-17 (SC-a-thalassemia); D: 11-3 (hemoglobin H disease). Table 2. Henloglohitz r~a1ue.satld rr.r~t1t.so/ globit1 sr~t1rht~si.r.rrlidirs Pedigree resistant "(~-["c]Leu) no. Age, yr HbA, %) HbS. "/o tlbC, % HhAz, '81 hemoglobin,'# Hby,, '#I Hb~j,.% 0 + y I- I 28 69.1 26.9 3.5 0.5 0.68 1-2 28 75.0 24.0 I .O 0.77 II-I Y 97.5 2.3 0.2 0.80 11-2 7 50.5 47.7 1.8 0.68 11-3 Newborn 23.0 0. I 50.6 26.3 0.61 5 months 83.1 0.7 9.7 1.5 6.5 0.69 HEMOGLOBIN SC-tr-THALASSEMIA 6 17 experiments performed in a similar manner with blood samples @-chain substitutions. These have included hemoglobins S (39,46), from five patients with HbSC disease. The level of 2.3-DPG in C (36), E (43), and J (41). To our knowledge the patient described fresh blood from the patient was 18.5 i 1.1 pmol/g of hemoglo- in this report represents the first known example of cu-thalassemia bin. The level of 2.3-DPG in a group of HbSC patients was 16.9 + occurring in combination with hemoglobins S and C. 1.9 pmol/g Hb, and in a group of normal controls was 15.0 + 2.4 The asymptomatic clinical course of this patient may have been clmol/g Hb. related to an ameliorative effect of the cu-thalassemia on her In an effort to evaluate the sickling ability of the patient's hemoglobinopathy: however, it has not been uncommon in our erythrocytes, samples of her blood were equilibrated with gas own experience for children with HbSC disease to be free of any mixtures of varying oxygen content to achieve a range of oxygen symptom attrihutahle to sickle cell disease over a period of many saturation values. and at each ~ointthe number of sickled cells years of observation. On the other hand, this patient demonstrated present was determined. The results of this study are shown in a more severe degree of anemia than we have usually encountered Figure 8 together with those obtained with blood samples from five in children with HbSC disease, and her persistently elevated patients with HbSC disease. At oxygen saturation levels of greater reticulocyte counts suggest a more hemolytic process than is than 50% both the patient and the control subjects exhibited less typical of hemoglobin SC disease. than 20% of sickle cell forms. At saturation levels of less than 503, The oxygen equilibrium curve of the patient's blood was the HbSC blood samples showed a nearly linear increase in the indistinguishable from that seen in HbSC disease, indicating a percentage of sickle cells as the oxygen saturation decreased, and comparable degree of hemoglobin oxygen saturation in the two reached more than 90% as total desaturation was approached. At conditions at the same partial pressure of oxygen. However a oxygen saturation levels below 30%, the percentages of sickle cells direct enumeration of the numbers of sickled cells at varying in the HbSC-a thalassemia patient's blood were significantly less oxygen tensions demonstrated a significantly reduced ability of the than those at corresponding oxygen saturation levels in the control patient's erythrocytes to undergo sickle transformation. The subjects. Moreover, at the lowest oxygen saturation level achieved apparent beneficial effect of the a-thalassemia in this patient is (about 4%) the patient's blood contained less than 35% sickle cell consistent with previous observations (39. 46) that cu-thalassemia forms. reduced the severity of the clinical course of patients with sickle cell anemia. The reduced ability of the child's erythrocytes to DISCUSSION sickle may be related to the diminished concentration of hemoglo- bin in her cells, in accordance with the observation of Seakins et a/. Several examples have previously been described of cu-thalass- (33) that sickling is strongly influenced by the intracellular emia occurring in combination with abnormal hemoglobins with concentration of sickle hemoglobin. It is difficult to provide an explanation for the apparently more severe hemolytic process in this child in the presence of diminished erythrocyte sickling. Conceivably, the increased hemolysis is primarily related to some other cause. From available evidence, a-thalassemia appears to occur with high frequency in American blacks. Cord blood electrophoresis studies of black infants have suggested an incidence of a-thalas- semia trait of between 2 and 7% (12. 25. 44). These studies all were based on the association between a-thalassemia and the presence of elevated levels (greater than 2%,) of Bart's (y,) hemoglobin detected at or near the time of birth. Although an increased level of Bart's hemoglobin in the neonatal period has been shown to correlate well with the presence of a-thalassemia, both by hemato- logic and globin biosynthesis criteria (12). recent studies by Esan (10, 11) did not support an association of this kind in black infants in Nigeria. Most importantly, the author observed several pairs of monozygotic twins in which only one twin had an elevated level of Bart's hemoglobin. A similar example of hemoglobin Bart's disparity in identical twins was described by Pembrey and coworkers (30) from a study of a Saudi Arabian population. These FRACTION NO. observations suggest that at least in some cases elevated levels of Fig. 3. DEAE-Sephadex chromatography of hemoglobin prepared from Bart's hemoglobin at birth may be related to other, nongenetic cord blood erythrocytes of 11-3 (hemoglobin H disease). factors. FRACTION NO. Fig. 4. Chromatogram of globin from red cells of 1-2 (Ac-a-thalassemia). @---a: absorbance at 280 nm: 0... .O: incorporated L-["Clleucine HONIG ETAL as from her father, and cu-thalassemia from one parent. The hematologic findings of the proband, the older sister, and both of her parents are characteristic of cu-thalassemia trait, although certain differences are apparent that can be related to the association with the different hemoglobin @-chain abnormalities present in each of them. The hematologic findings of the infant (It-.?), particularly from the study performed when she was 5 months of age, are diagnostic of hemoglobin H disease, a more severe form of cu-thalassemia. Hemoglobin H disease is characteri~edb) anemia, rnicrocytosis. and erythrocyte inclusions. and represents an tu-thalassemia ahnor- mality in which the tu-chain biosynthetic defect is of sufficient magnitude to produce a gross imbalance between n-chain and @-chain synthesis. with the formation of n~easurablequantities of @-chain tetramer molecules (hemoglobin H) and intracellular FRACTION NO. precipitates composed of @-chain protein. This form of cu-thalas- semia has been identified in many parts of the world with the Fig. 5. Chromatogr:~m of globin from red cells of the proband, If-? highest frequencies reported in Mediterranean, Middle Eastern, (SC-a-thalassemia). @-e: absorbance at 280 nni: 0.. . .O:incorporated and South East Asian populations it appears to occur quite L-["C]leucine. (45): uncommonly in black populations in Africa and in the United States. Hemoglobin H disease in American blacks has been studied in detail by Schwartr and Atwater (32). who found that although the disorder shared most ofthe features ofhemoglobin H Fig. 6. Osmotic frag~lityof fresh blood samples from normal controls, subjects with hemoglobin SC disease, and the patient with hernoglob~n SC-n-thalassemia. Thc rcsults shown from the patlent represent separate determinations performed several ueeks apart. Beyond the neonatal period heterozygous forms of cu-thalass- ernia are particularly difficult to identify, and consequently few attempts have been made to determine the frequency of this disorder in older children and adults. Whereas elevated levels of hemoglobin A, and F serr,e to identif!: most heterorygous Sorms of FIF. 7. Oxygeri equ~libriunl culve of I'rcsh whole blood from the @-thalassemia, these hemoglobins are characteristicallq present in SC-n-thalns\emia patlent. The deter~ii~n:~t~onswere performed at 37". ptl normal amounts in a-thalassernia heterozygotes. tven microcqto- 7.4. The solid 11ne (--) rcprescnt\ the \\hole I,luod curve of nnrni;ll sis, a feature shared by most forms of thalassemia, seems not to he henloglohin A subjects. reliable criterion for the identification of cu-thalassemiir trait. In a Greek population indiv~dualswith hematologic and genetic I'ea- tures of n-thalassemia consistently had reduced MCV values (29), but studies of black infants (IZ), children (6). and adults (37) with n-thalassemia showed that the iviCV indices may obcrial~cur~siilcr- ably with the range of normal values. When cu-thalassemia occurs in combination with sickle cell trait or HbC trait, however, the percentage of HbS or HbC becomes significantly reduced in comparison with the usual amounts (35-45%) seen in hetero~ygousindividuals (6. 37). In the absence of iron deficiency (21) or megaloblastic disease (13), th~sfinding provides a reliable means for establishing a diagnosis of n-thalas- semia in adult life. The lower than usual percentages of hemo- globins S and C found in the parents of the proband in this study provide further confirmation ofthis relationship, and together with the globin synthesis determinations established in both of them the % 0, SATURATION presence of cu-thalassemia. Fig. 8. Sickling of the SC-n-thalassemia patient's erythrocytes in The proband, with HbSC-cu thalassemia, apparently inherited relation to oxygen saturation of the blood. 0-0: results ohta~nrdwith the gene for hemoglobin S from her father, henioglob~nC from her blood from the patlcnt. The slzadrd circa indicates the mean and standard mother, and cu-thalassemia from one of her parents. The older deviation of the curves obtained from five sub~ectsh~th hernogloh~n SC sister (11-1)derived a normal P-chain gene from her mother as well disease. HEMOGLOBIN SC-n-THALASSEM IA 619 disease as it occurs in other populations, it is a significantly milder expression of an intermediate fraction of the a-chain genes as N- condition, both clinically and in terms of the a-chain biosynthetic thalassemia. defect, as determined by globin synthesis studies in vitro. The genetics of cu-thalassemia in blacks remains unclear, but In Far Eastern populations in which cu-thalassemia occurs with from available evidence seems to follow a pattern similar to that high frequency, four hematologically distinct forms of a-thalas- observed in the Yemenite and Iraqui Jews (47). with cu-thalassemia semia have been identified. These include a "silent-carrier" state, trait and HbH disease apparently being the only clearly discerna- characterized by mild microcytosis but otherwise normal findings; ble forms of a-thalassemia in this population. The findings of a cu-thalassemia trait, a condition characterized by abnormal eryth- detailed study of black families with HbH disease, as reported by rocyte morphology and hematologic features of mild thalassemia; Schwartz and Atwater (32), were consistent with this genetic hemoglobin H disease: and cu-thalassemia hydrops fetalis, a pattern, but because of the mild degree of abnormality found in the disorder that has been uniformly fatal in the newborn period and is globin synthesis studies of heterozygotes, rigorous proof of this accompanied by severe anemia and a total absence of hemoglobins hypothesis was lacking. An attempt has also been made to A and F, with Bart's hemoglobin and HbH making up all of the distinguish between mild and more severe forms of cu-thalassemia hemoglobin present in the blood (45). Family studies of individuals trait in blacks (34), but a distinction of this kind remains with hemoglobin H disease in these populations (42) have shown unconvincing with the limited family studies that are available. that in most instances one parent has tu-thalassemia trait and the The difficulty in identifying heterozygotes in adult life has been a other the n-thalassemia silent carrier state. On the other hand, the major obstacle to the study of this condition and has caused a parents of infants with the hydrops fetalis form of cu-thalassemia heavy reliance to be placed on the analysis of Bart's hemoglobin in both were shown to have the hematologic features of cu-thalasseniia the newborn period. The findings described by Esan (10, 1 I) raise trait (41). doubts about the total validity of this method as a means for the These observations led to the conclusion (41) that two kinds of identification of cu-thalassemia in black populations and emphasize a-thalassemia genes exist in Far Eastern populations: a severe gene the need for confirming the diagnosis of n-thalassemia by other (cu-thal,) and one causing a lesser degree of impairment of cu-chain methods whenever possible. synthesis (tu-thal,). The hydrops fetalis syndrome was envisioned Both of the parents of the infant with HbH disease described in as representing the homozygous form of n-thal,. Inasmuch as this report appeared from the globin synthesis studies (Table 2) to infants having the hydrops fetalis form of n-thalassemia have been have a form of cu-thalassemia of a similar degree of severity. This is shown to be totally lacking the gene for hemoglobin cu-chains (27). consistent with each parent being heterozygous for a relative]) it follows that the cu-thal, gene is an tun gene with no discernable mild a-thalassemia gene (32). although it is impossible from the gene product. The form of HbH disease seen in this population is evidence available to exclude a double dose of a still milder accordingly thought to represent double heterozygosity for cu-thal, cu-thalassemia gene in either or both parents. and cu-thal, (27), the latter being an tu+ gene that produces only The limited genetic information now available about HbH partial suppression of cu-chain synthesis. disease in blacks, including the present case, seem clearly not to be The foregoing interpretation of genetic inforniation was based explainable by the genetic mechanisms established in Far Eastern on a single-locus model which assumes the existence of single pair populations (41, 42). Two possible mechanisms that are consistent of n-chain genes. Recent evidence has been presented in support of with the available genetic information are: (I) the presence of three the existence of two pairs of n-chain loci (14), at least in some or more n-chain loci in this population, with n-thalassemia genes populations, and using a four gene model, Lehmann (20) suggested of an no type occupying an intermediate fraction of the total cu-loci an alternative interpretation of the tu-thalassemia syndromes. His in HbH individuals, to produce a thalassemic disorder less severe hypothesis assumed a single type of cu-thalassemia gene, which than that of HbH disease as seen in the Far East (30): or (2) the presumably caused complete suppression of a-chain synthesis. presence of a milder (N-)type of n-thalassemia gene which does Using this model, the presence of I. 2, 3, or 4 cu-thalassemia genes not produce complete suppression of cu-chain synthesis. The latter corresponds, respectively, to the silent carrier, cu-thalassemia trait, possibility does not necessarily exclude the existence of more than HbH disease, and the hydrops fetalis forms of the disease. one type of cu-thalassemia gene (with respect to severity) in this Either of the genetic models described appears adequate to population, and is compatible with either a 2-locus or 4-locus account for the expression of tu-thalassemia as seen in Thai and model. Inasmuch as P-thalassemia in blacks in the United States is other Far Eastern populations, but it has not been possible to mainly of the /3+ variety, cu-thalassem~;i may likewise cause account for the forms of tu-thalassemia that have been described in incomplete suppression of cu-chain synthesis in this population. other population group<. In American blacks, Yemenite and In spite of the unquestionably milder expression of hemoglobin Iraqui Jews (47)- and in Arabs in Saudi Arabia (30. 46). the gene H disease in blacks as compared with other populations, it is of frequency of tu-thalassemia is substantial and HbH disease has interest that the infant with HbH disease described in this report been described: however, the hydrops fetalis a-thalassemia syn- had a level of Bart's hemoglobin at birth that was within the rangc drome has never been identified. of values seen in newborns with HbH disease in the Far East (36. In family studies of Yemenite and Iraqui .lewish families with 41). To our knowledge this infant represents the only knwn HbH dlsease, ~nvolvingdeterminations of both hemaroiogic i'ea- example oi' i~crriugioliin H disease in a blsck infan: ;;.hi: I,slLIJ .,- tures and globin synthesis (47). an attempt was made to ident~fy studied at birth. mild and severe cu-thalassemia genes. The experimental findings Hemoglobin H disease in American blacks was only first did not, however, support the existence of more than a single type described in 1972 (32, 34). The present case brings the total of cu-thalassemia gene and suggested that only two forms of number of documented cases to nine. The apparent rarity of this a-thalassemia could be identified in this population, viz. a-thalas- condition seems surprising in view of evidence that the incidence of semia trait and HbH disease. It was proposed that these disorders cu-thalassemia among blacks is substantial, but several characteris- could most readily be explained as representing, respectively, the tics of the disorder as it occurs in the black population may serve to heterozygous and homozygous expression of a single cu-thalas- obscure its presence. Anemia is seldom severe in this form of HbH semia gene. disease, and hemoglobin values may fall well within the range of Pembrey and coworkers (30), in their study of Arabs in Saudi normal values in affected individuals (32. 34), allowing its presence Arabia, concluded that the findings in this population represented to be overlooked. The now widespread application of hemoglobin a form of cu-thalassemia that is expressed as a disorder of a severity electrophoresis testing for the detection of sickling disorders intermediate between that of cu-thalassemia trait and HbH disease among blacks in the United States might also be expected to as they are known to occur in the Far East. As a possible demonstrate the fast moving hemoglobin H fraction in otherwise explanation for these observations the authors postulated that six asymptomatic and nonanemic individuals, but this finding is also a-chain loci might be present in this population, allowing the likely to be missed; the preparation of hemolysates for electropho- resis testing in most hemoglobin screening laboratories includes NH, in hemoglobin Memphis. Hemoglohln MemphisIS, a new variant of the addition of toluene or chloroform to aid in the removal of molecular dlsease. Blochem~stry,5: 3701 (1966). 20. Lehmann, H.: Different types of alpha-thalassaemia and s~gnif~canceof haemo- stromal material. but these chemicals have the unfortunate globln Bart's in neonates. Lancet. ri: 78 ( 1970). property of quantitatively precipitating hemoglobin H and thereby 21. Levere. R. D., Lichtman, H.C.. and Lev~ne,J. 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C;.: Unequal synthe\~a of Ferenc 15 Ch~ldrcn'sMemorial tlosp~tal.23OU Ch~ldren'sI'la/:~. Ch~cago.Ill. complementary globin chain\ of human fetal hcmoglohln hy the effect of 60614 (USA) L-0-methylthreon~ne..I. Biol. Chem., 244: 2027 (1960). 50. The present addrcs\ of Dr. U Gun;~) I\: I\tanhul Un~\cr\~t!.Hur\a Medic:~l 17. Jacob. (;. F.. and Raper, A B. llereditary pcrslstiincc of foetal haemoglohin School, Burja, 'Turkey product~onend its interaction w~ththe s~ckle-celltralt. Br~t..I. Haematol.. 4. 51. This research was supported by Coniprchens~ve Slcklc Center Grant no. 138 (1958). HL-15168 and Research Grant no, AM-I2895 from the N;~tion;~lInstitutes of 18. Keitt. S Reduced nicotinam~dcaden~ne d~nucleot~de-l~nked annlyc~c of 2.3- Health, lln~tcdStates Public He:~lthService. dipho\phoglyceric acid. Spectrophotometric and fluorometr~cprocedure\. J 52. Requests for reprlnts should be addreshed to: G. R. Hon~g.M.D.. Ch~ldren'\ Lab. ('lln. .Med , 77. 470 (1971). Memorial Ho\pital. 2300 Children's PI:l/n. Chicago, Ill 60614 (USA). 19. Kraus. L. M., Miyaj~,T , luchi. I.. and Kraus, A. P: Character~ratlonofa" glu 53 Accepted for puhlication December IX. 1075. Copyright O 1976 International Pedi;~tr~cResearch Found:~tion, Inc. t'rrn~~dit1 Cr..S'.A